SMART MODE:

NORMAL GENOMIC

• Gharibi T et al.
• Investigation of IL-21 gene polymorphisms (rs2221903, rs2055979) in cases with multiple sclerosis of Azerbaijan, Northwest Iran.
• Am J Clin Exp Immunol. 2015; 4: 7-14
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• BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the Central Nervous System that is immunologically mediated in genetically susceptible individuals. IL-21, a cytokine produced by TCD4(+) cells, particularly by Th-17 cells, is believed to play an important role in the MS pathogenesis. OBJECTIVE: This study was performed to investigate the impact of genetic polymorphisms in IL-21 gene on MS susceptibility and clinical profiles. METHODS: Seventy Iranian patients with clinically definite relapsing-remitting MS and 110 age, sex and ethic matched controls were genotyped for IL-21 gene polymorphisms using PCR-RFLP method. RESULTS: Our results showed that the IL-21 rs2221903 SNP is not polymorphic in our population. Also, the allelic and genotypic frequencies of the IL-21 rs2055979 did not differ significantly between the MS patients and controls (P = 0.413 and P = 0.565 respectively, and OR = 1.122, 95% CI = 0.79-1.87 for T allele). However, our results showed that IL-21 rs2055979 (G/T) T allele positive (TT+GT) MS patients had lower (PI

• Pagliari D et al.
• The Interaction among Microbiota, Immunity, and Genetic and Dietary Factors Is the Condicio Sine Qua Non Celiac Disease Can Develop.
• J Immunol Res. 2015; 2015: 123653-123653
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• Celiac disease (CD) is an immune-mediated enteropathy, triggered by dietary wheat gluten and similar proteins of barley and rye in genetically susceptible individuals. This is a complex disorder involving both environmental and immune-genetic factors. The major genetic risk factor for CD is determined by HLA-DQ genes. Dysfunction of the innate and adaptive immune systems can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes. Exposure to gluten is the main environmental trigger responsible for the signs and symptoms of the disease, but exposure to gluten does not fully explain the manifestation of CD. Thus, both genetic determination and environmental exposure to gluten are necessary for the full manifestation of CD; neither of them is sufficient alone. Epidemiological and clinical data suggest that other environmental factors, including infections, alterations in the intestinal microbiota composition, and early feeding practices, might also play a role in disease development. Thus, this interaction is the condicio sine qua non celiac disease can develop. The breakdown of the interaction among microbiota, innate immunity, and genetic and dietary factors leads to disruption of homeostasis and inflammation; and tissue damage occurs. Focusing attention on this interaction and its breakdown may allow a better understanding of the CD pathogenesis and lead to novel translational avenues for preventing and treating this widespread disease.

• Plugis NM, Khosla C
• Therapeutic approaches for celiac disease.
• Best Pract Res Clin Gastroenterol. 2015; 29: 503-521
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• Celiac disease is a common, lifelong autoimmune disorder for which dietary control is the only accepted form of therapy. A strict gluten-free diet is burdensome to patients and can be limited in efficacy, indicating there is an unmet need for novel therapeutic approaches to supplement or supplant dietary therapy. Many molecular events required for disease pathogenesis have been recently characterized and inspire most current and emerging drug-discovery efforts. Genome-wide association studies (GWAS) confirm the importance of human leukocyte antigen genes in our pathogenic model and identify a number of new risk loci in this complex disease. Here, we review the status of both emerging and potential therapeutic strategies in the context of disease pathophysiology. We conclude with a discussion of how genes identified during GWAS and follow-up studies that enhance susceptibility may offer insight into developing novel therapies.

• Torre S et al.
• THEMIS is required for pathogenesis of cerebral malaria and protection against pulmonary tuberculosis.
• Infect Immun. 2015; 83: 759-68
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• We identify an N-ethyl-N-nitrosourea (ENU)-induced I23N mutation in the THEMIS protein that causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Themis(I23N) homozygous mice show reduced CD4(+) and CD8(+) T lymphocyte numbers. ECM resistance in P. berghei ANKA-infected Themis(I23N) mice is associated with decreased cerebral cellular infiltration, retention of blood-brain barrier integrity, and reduced proinflammatory cytokine production. THEMIS(I23N) protein expression is absent from mutant mice, concurrent with the decreased THEMIS(I23N) stability observed in vitro. Biochemical studies in vitro and functional complementation in vivo in Themis(I23N/+):Lck(-/+) doubly heterozygous mice demonstrate that functional coupling of THEMIS to LCK tyrosine kinase is required for ECM pathogenesis. Damping of proinflammatory responses in Themis(I23N) mice causes susceptibility to pulmonary tuberculosis. Thus, THEMIS is required for the development and ultimately the function of proinflammatory T cells. Themis(I23N) mice can be used to study the newly discovered association of THEMIS (6p22.33) with inflammatory bowel disease and multiple sclerosis.

• Yang X et al.
• KIAA1199 as a potential diagnostic biomarker of rheumatoid arthritis related to angiogenesis.
• Arthritis Res Ther. 2015; 17: 140-140
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• INTRODUCTION: Our previous proteomic study on fibroblast-like synoviocytes (FLSs) derived from the synovial tissues found that the expression of KIAA1199 was higher in rheumatoid arthritis (RA) patients than in healthy controls. The aim of this study was to examine the biological function of KIAA1199 and evaluate its clinical diagnosis value in RA. METHODS: The over-expression of KIAA1199 was verified by quantitative real-time polymerase chain reaction (qPCR), Immunohistochemistry, Immunofluorescence and enzyme linked immunosorbent assay (ELISA) in inactive and active RA patients and healthy controls. The effect of KIAA1199 expression on FLSs proliferation, angiogenesis and related pathway were analyzed by MTT, cell migration, tube formation, chorioallantoic membrane (CAM) assay, qPCR and western-blotting after KIAA1199 knockdown and over-expression. RESULTS: The verification results show the up-regulation of KIAA1199 in RA patients at mRNA and protein level as compared to that in healthy controls. ELISA and receiver operator characteristic (ROC) analysis shows that KIAA1199 concentration in serum, synovial fluid and synovial tissues could be used as dependable biomarkers for the diagnosis of active RA, provided an area under roc curve (AUC) of 0.83, 0.92 and 0.92. Sensitivity and specificity, which were determined by cut-off points, reached 72% 84% and 80% in sensitivity and 80%, 93.3%, 93.3% in specificity, respectively. Moreover, KIAA1199 also enhance the proliferation and angiogenesis of synovial membrane, and KIAA1199/ PLXNB3/ SEMA5A/CTGF axis may be a newly found pathway enhancing cell proliferation and angiogenesis. CONCLUSION: KIAA1199 may be a potential diagnostic biomarker of RA related to angiogenesis.

• Ferreira RC et al.
• IL-21 production by CD4+ effector T cells and frequency of circulating follicular helper T cells are increased in type 1 diabetes patients.
• Diabetologia. 2015; 58: 781-90
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• AIMS/HYPOTHESIS: Type 1 diabetes results from the autoimmune destruction of insulin-secreting pancreatic beta cells by T cells. Despite the established role of T cells in the pathogenesis of the disease, to date, with the exception of the identification of islet-specific T effector (Teff) cells, studies have mostly failed to identify reproducible alterations in the frequency or function of T cell subsets in peripheral blood from patients with type 1 diabetes. METHODS: We assessed the production of the proinflammatory cytokines IL-21, IFN-gamma and IL-17 in peripheral blood mononuclear cells from 69 patients with type 1 diabetes and 61 healthy donors. In an additional cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. IL-21 and IL-17 production was also measured in peripheral blood mononuclear cells (PBMCs) from a subset of 46 of the 62 donors immunophenotyped for Tfh. RESULTS: We found a 21.9% (95% CI 5.8, 40.2; p = 3.9 x 10(-3)) higher frequency of IL-21(+) CD45RA(-) memory CD4(+) Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). Consistent with this finding, we found a 14.9% increase in circulating Tfh cells in the patients (95% CI 2.9, 26.9; p = 0.016). CONCLUSIONS/INTERPRETATION: These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.

• Lopez-Contreras AJ et al.
• Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice.
• Genes Dev. 2015; 29: 690-5
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• In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.

• Rossi E et al.
• TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children.
• PLoS One. 2015; 10: 123244-123244
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• BACKGROUND: TNF-alpha and IFN-gamma play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-alpha receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner. METHODS: 511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence). RESULTS: Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations. CONCLUSION: TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.

• Campbell AL et al.
• IL-21 receptor expression in human tendinopathy.
• Mediators Inflamm. 2014; 2014: 481206-481206
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• The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFalpha/IL-1beta) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy.

• Garner C et al.
• Genome-wide association study of celiac disease in North America confirms FRMD4B as new celiac locus.
• PLoS One. 2014; 9: 101428-101428
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• We performed a genome-wide association study (GWAS) of 1550 North American celiac disease cases and 3084 controls. Twelve SNPs, distributed across four regions (3p21.31, 4q27, 6q15, 6q25), were significantly associated with disease (p-value <1.0x10-7), and a further seven SNPs, across four additional regions (1q24.3, 10p15.1, 6q22.31, 17q21.32) had suggestive evidence (1.0x10-7 < p-value < 1.0x10-6). This study replicated a previous suggestive association within FRMD4B (3p14.1), confirming it as a celiac disease locus. All four regions with significant associations and two regions with suggestive results (1q24.3, 10p15.1) were known disease loci. The 6q22.31 and 10p11.23 regions were not replicated. A total of 410 SNPs distributed across the eight significant and suggestive regions were tested for association with dermatitis herpetiformis and microscopic colitis. Preliminary, suggestive statistical evidence for association with the two traits was found at chromosomes 3p21.31, 6q15, 6q25, 1q24.3 and 10p11.23, with future studies being required to validate the reported associations.

• Bondar C et al.
• THEMIS and PTPRK in celiac intestinal mucosa: coexpression in disease and after in vitro gliadin challenge.
• Eur J Hum Genet. 2014; 22: 358-62
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• Celiac disease (CD) is an immune mediated, polygenic disorder, where HLA-DQ2/DQ8 alleles contribute around 35% to genetic risk, but several other genes are also involved. Genome-wide association studies (GWASs) and the more recent immunochip genotyping projects have fine-mapped 39 regions of genetic susceptibility to the disease, most of which harbor candidate genes that could participate in this disease process. We focused our attention to the GWAS peak on chr6: 127.99-128.38 Mb, a region including two genes, thymocyte-expressed molecule involved in selection (THEMIS) and protein tyrosine phosphatase, receptor type, kappa (PTPRK), both of which have immune-related functions. The aim of this work was to evaluate the expression levels of these two genes in duodenal mucosa of active and treated CD patients and in controls, and to determine whether SNPs (rs802734, rs55743914, rs72975916, rs10484718 and rs9491896) associated with CD have any influence on gene expression. THEMIS showed higher expression in active CD compared with treated patients and controls, whereas PTPRK showed lower expression. Our study confirmed the association of this region with CD in our population, but only the genotype of rs802734 showed some influence in the expression of THEMIS. On the other hand, we found a significant positive correlation between THEMIS and PTPRK mRNA levels in CD patients but not in controls. Our results suggest a possible role for both candidate genes in CD pathogenesis and the existence of complex, regulatory relationships that reside in the vast non-coding, functional intergenic regions of the genome. Further investigation is needed to clarify the impact of the disease-associated SNPs on gene function.

• Ubeda-Manzanaro M, Merlo MA, Ortiz-Delgado JB, Rebordinos L, Sarasquete C
• Expression profiling of the sex-related gene Dmrt1 in adults of the Lusitanian toadfish Halobatrachus didactylus (Bloch and Schneider, 1801).
• Gene. 2014; 535: 255-65
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• Doublesex and mab-3 related transcription factor 1 (Dmrt1) gene is a widely conserved gene involved in sex determination and differentiation across phyla. To gain insights on Dmrt1 implication for fish gonad cell differentiation and gametogenesis development, its mRNA was isolated from testis and ovary from the Lusitanian toadfish (Halobatrachus didactylus). The cDNA from Dmrt1 was synthesized and cloned, whereas its quantitative and qualitative gene expression, as well as its protein immunolocalization, were analyzed. A main product of 1.38 kb, which encodes a protein of 295 aa, was reported, but other minority Dmrt1 products were also identified by RACE-PCR. This gene is predominantly expressed in testis (about 20 times more than in other organs or tissues), specially in spermatogonia, spermatocytes and spermatids, as well as in somatic Sertoli cells, indicating that Dmrt1 plays an important role in spermatogenesis. Although Dmrt1 transcripts also seem to be involved in oogenesis development, and it cannot be excluded that toadfish Dmrt1 could be functionally involved in other processes not related to sex.

• Kiryluk K et al.
• Discovery of new risk loci for IgA nephropathy implicates genes involved in immunity against intestinal pathogens.
• Nat Genet. 2014; 46: 1187-96
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• We performed a genome-wide association study (GWAS) of IgA nephropathy (IgAN), the most common form of glomerulonephritis, with discovery and follow-up in 20,612 individuals of European and East Asian ancestry. We identified six new genome-wide significant associations, four in ITGAM-ITGAX, VAV3 and CARD9 and two new independent signals at HLA-DQB1 and DEFA. We replicated the nine previously reported signals, including known SNPs in the HLA-DQB1 and DEFA loci. The cumulative burden of risk alleles is strongly associated with age at disease onset. Most loci are either directly associated with risk of inflammatory bowel disease (IBD) or maintenance of the intestinal epithelial barrier and response to mucosal pathogens. The geospatial distribution of risk alleles is highly suggestive of multi-locus adaptation, and genetic risk correlates strongly with variation in local pathogens, particularly helminth diversity, suggesting a possible role for host-intestinal pathogen interactions in shaping the genetic landscape of IgAN.

• Bragde H, Jansson U, Fredrikson M, Grodzinsky E, Soderman J
• Potential blood-based markers of celiac disease.
• BMC Gastroenterol. 2014; 14: 176-176
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• BACKGROUND: Blood-based diagnostics has the potential to simplify the process of diagnosing celiac disease (CD). Although high levels of autoantibodies against tissue transglutaminase (anti-TG2) are strongly indicative of active CD, several other scenarios involve a need for additional blood-based CD markers. METHODS: We investigated the levels of messenger RNA (mRNA) in whole blood (n = 49) and protein in plasma (n = 22) from cases with active CD (n = 20), with confirmed CD and normalized histology (n = 15), and without a CD diagnosis (n = 14). Group differences were analyzed using Kruskal-Wallis one-way analysis of variance by ranks. We also investigated correlations between levels of potential markers, histopathology according to the modified Marsh scale, and CD risk gradient based on HLA type, using Spearman rank correlation. The relation between HLA-DQ2 gene dose effect and the expression levels of selected blood-based markers was investigated using the Mann-Whitney U test. Finally, the diagnostic performance of anti-TG2, potential blood-based CD markers, and logistic regression models of combined markers was evaluated using receiver operating characteristic (ROC) curve analysis. RESULTS: CXCL11 protein levels and TNFRSF9 and TNFSF13B mRNA levels were identified as potential CD markers. These are all affected by or involved in the regulation of the NF-kappaB complex. CXCL11 protein levels and IL21 and IL15 mRNA levels were correlated with histopathology according to the modified Marsh scale, as were the established CD markers. HLA genotype risk and HLA-DQ2 gene dose effect did not show any significant relations with either the potential CD markers or the established CD markers. ROC curve analysis revealed a slight, non-significant increase in the area under the curve for the combined use of anti-TG2 and different constellations of potential blood-based CD markers compared to anti-TG2 alone. CONCLUSIONS: The CD markers identified in this study further emphasize the significance of components related to NF-kappaB regulation in relation to CD. However, the relevance of CXCL11, TNFSF13B, TNFRSF9, and other NF-kappaB interacting proteins recognized by pathway analysis, needs to be further investigated in relation to diagnosis and monitoring of CD.

• Shostak K et al.
• NF-kappaB-induced KIAA1199 promotes survival through EGFR signalling.
• Nat Commun. 2014; 5: 5232-5232
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• Constitutive activation of EGFR- and NF-kappaB-dependent pathways is a hallmark of cancer, yet signalling proteins that connect both oncogenic cascades are poorly characterized. Here we define KIAA1199 as a BCL-3- and p65-dependent gene in transformed keratinocytes. KIAA1199 expression is enhanced on human papillomavirus (HPV) infection and is aberrantly expressed in clinical cases of cervical (pre)neoplastic lesions. Mechanistically, KIAA1199 binds Plexin A2 and protects from Semaphorin 3A-mediated cell death by promoting EGFR stability and signalling. Moreover, KIAA1199 is an EGFR-binding protein and KIAA1199 deficiency impairs EGF-dependent Src, MEK1 and ERK1/2 phosphorylations. Therefore, EGFR stability and signalling to downstream kinases requires KIAA1199. As such, KIAA1199 promotes EGF-mediated epithelial-mesenchymal transition (EMT). Taken together, our data define KIAA1199 as an oncogenic protein induced by HPV infection and constitutive NF-kappaB activity that transmits pro-survival and invasive signals through EGFR signalling.

• Lange K, Papp JC, Sinsheimer JS, Sobel EM
• Next Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data.
• Annu Rev Stat Appl. 2014; 1: 279-300
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• Statistical genetics is undergoing the same transition to big data that all branches of applied statistics are experiencing. With the advent of inexpensive DNA sequencing, the transition is only accelerating. This brief review highlights some modern techniques with recent successes in statistical genetics. These include: (a) lasso penalized regression and association mapping, (b) ethnic admixture estimation, (c) matrix completion for genotype and sequence data, (d) the fused lasso and copy number variation, (e) haplotyping, (f) estimation of relatedness, (g) variance components models, and (h) rare variant testing. For more than a century, genetics has been both a driver and beneficiary of statistical theory and practice. This symbiotic relationship will persist for the foreseeable future.

• Lebwohl B, Ludvigsson JF, Green PH
• The unfolding story of celiac disease risk factors.
• Clin Gastroenterol Hepatol. 2014; 12: 632-5

• Nongpiur ME et al.
• ABCC5, a gene that influences the anterior chamber depth, is associated with primary angle closure glaucoma.
• PLoS Genet. 2014; 10: 1004089-1004089
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• Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size = -0.045 mm, P = 8.17 x 10(-9)). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06-1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45 x 10(-9); 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions.

• Almeida R et al.
• Fine mapping of the celiac disease-associated LPP locus reveals a potential functional variant.
• Hum Mol Genet. 2014; 23: 2481-9
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• Using the Immunochip for genotyping, we identified 39 non-human leukocyte antigen (non-HLA) loci associated to celiac disease (CeD), an immune-mediated disease with a worldwide frequency of approximately 1%. The most significant non-HLA signal mapped to the intronic region of 70 kb in the LPP gene. Our aim was to fine map and identify possible functional variants in the LPP locus. We performed a meta-analysis in a cohort of 25 169 individuals from six different populations previously genotyped using Immunochip. Imputation using data from the Genome of the Netherlands and 1000 Genomes projects, followed by meta-analysis, confirmed the strong association signal on the LPP locus (rs2030519, P = 1.79 x 10(-49)), without any novel associations. The conditional analysis on this top SNP-indicated association to a single common haplotype. By performing haplotype analyses in each population separately, as well as in a combined group of the four populations that reach the significant threshold after correction (P < 0.008), we narrowed down the CeD-associated region from 70 to 2.8 kb (P = 1.35 x 10(-44)). By intersecting regulatory data from the ENCODE project, we found a functional SNP, rs4686484 (P = 3.12 x 10(-49)), that maps to several B-cell enhancer elements and a highly conserved region. This SNP was also predicted to change the binding motif of the transcription factors IRF4, IRF11, Nkx2.7 and Nkx2.9, suggesting its role in transcriptional regulation. We later found significantly low levels of LPP mRNA in CeD biopsies compared with controls, thus our results suggest that rs4686484 is the functional variant in this locus, while LPP expression is decreased in CeD.

• Calabro A et al.
• A metabolomic perspective on coeliac disease.
• Autoimmune Dis. 2014; 2014: 756138-756138
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• Metabolomics is an "omic" science that is now emerging with the purpose of elaborating a comprehensive analysis of the metabolome, which is the complete set of metabolites (i.e., small molecules intermediates) in an organism, tissue, cell, or biofluid. In the past decade, metabolomics has already proved to be useful for the characterization of several pathological conditions and offers promises as a clinical tool. A metabolomics investigation of coeliac disease (CD) revealed that a metabolic fingerprint for CD can be defined, which accounts for three different but complementary components: malabsorption, energy metabolism, and alterations in gut microflora and/or intestinal permeability. In this review, we will discuss the major advancements in metabolomics of CD, in particular with respect to the role of gut microbiome and energy metabolism.

• Sams A, Hawks J
• Patterns of population differentiation and natural selection on the celiac disease background risk network.
• PLoS One. 2013; 8: 70564-70564
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• Celiac disease is a common small intestinal inflammatory condition induced by wheat gluten and related proteins from rye and barley. Left untreated, the clinical presentation of CD can include failure to thrive, malnutrition, and distension in juveniles. The disease can additionally lead to vitamin deficiencies, anemia, and osteoporosis. Therefore, CD potentially negatively affected fitness in past populations utilizing wheat, barley, and rye. Previous analyses of CD risk variants have uncovered evidence for positive selection on some of these loci. These studies also suggest the possibility that risk for common autoimmune conditions such as CD may be the result of positive selection on immune related loci in the genome to fight infection. Under this evolutionary scenario, disease phenotypes may be a trade-off from positive selection on immunity. If this hypothesis is generally true, we can expect to find a signal of natural selection when we survey across the network of loci known to influence CD risk. This study examines the non-HLA autosomal network of gene loci associated with CD risk in Europe. We reject the null hypothesis of neutrality on this network of CD risk loci. Additionally, we can localize evidence of selection in time and space by adding information from the genome of the Tyrolean Iceman. While we can show significant differentiation between continental regions across the CD network, the pattern of evidence is not consistent with primarily recent (Holocene) selection across this network in Europe. Further localization of ancient selection on this network may illuminate the ecological pressures acting on the immune system during this critically interesting phase of our evolution.

• Ostensson M et al.
• A possible mechanism behind autoimmune disorders discovered by genome-wide linkage and association analysis in celiac disease.
• PLoS One. 2013; 8: 70174-70174
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• Celiac disease is a common autoimmune disorder characterized by an intestinal inflammation triggered by gluten, a storage protein found in wheat, rye and barley. Similar to other autoimmune diseases such as type 1 diabetes, psoriasis and rheumatoid arthritis, celiac disease is the result of an immune response to self-antigens leading to tissue destruction and production of autoantibodies. Common diseases like celiac disease have a complex pattern of inheritance with inputs from both environmental as well as additive and non-additive genetic factors. In the past few years, Genome Wide Association Studies (GWAS) have been successful in finding genetic risk variants behind many common diseases and traits. To complement and add to the previous findings, we performed a GWAS including 206 trios from 97 nuclear Swedish and Norwegian families affected with celiac disease. By stratifying for HLA-DQ, we identified a new genome-wide significant risk locus covering the DUSP10 gene. To further investigate the associations from the GWAS we performed pathway analyses and two-locus interaction analyses. These analyses showed an over-representation of genes involved in type 2 diabetes and identified a set of candidate mechanisms and genes of which some were selected for mRNA expression analysis using small intestinal biopsies from 98 patients. Several genes were expressed differently in the small intestinal mucosa from patients with celiac autoimmunity compared to intestinal mucosa from control patients. From top-scoring regions we identified susceptibility genes in several categories: 1) polarity and epithelial cell functionality; 2) intestinal smooth muscle; 3) growth and energy homeostasis, including proline and glutamine metabolism; and finally 4) innate and adaptive immune system. These genes and pathways, including specific functions of DUSP10, together reveal a new potential biological mechanism that could influence the genesis of celiac disease, and possibly also other chronic disorders with an inflammatory component.

• Smyth AM, Lawrie SM
• The neuroimmunology of schizophrenia.
• Clin Psychopharmacol Neurosci. 2013; 11: 107-17
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• Schizophrenia (SCZ) is a polygenic, multi-factorial disorder and a definitive understanding of its pathophysiology has been lacking since it was first described more than a century ago. The predominant pharmacological approach used to treat SCZ is the use of dopamine receptor antagonists. The fact that many patients remain symptomatic, despite complying with medication regimens, emphasises the need for a more encompassing explanation for both the causes and treatment of SCZ. Recent neuroanatomical, neurobiological, environmental and genetic studies have revived the idea that inflammatory pathways are involved in the pathogenesis of SCZ. These new insights have emerged from multiple lines of evidence, including the levels of inflammatory proteins in the central nervous system of patients with SCZ and animal models. This review focuses on aberrant inflammatory mechanisms present both before and during the onset of the psychotic symptoms that characterise SCZ and discusses recent research into adjunctive immune system modulating therapies for its more effective treatment.

• Fichna M, Zurawek M, Fichna P, Ziolkowska-Suchanek I, Januszkiewicz D, Nowak J
• Polymorphic variant at the IL2 region is associated with type 1 diabetes and may affect serum levels of interleukin-2.
• Mol Biol Rep. 2013; 40: 6957-63
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• Polymorphic variants at the interleukin-2 (IL2) locus affect the risk of several autoimmune disorders. Our aim was to evaluate the association of the four IL2 polymorphisms (rs6822844, rs6534349, rs2069762 and rs3136534) with type 1 diabetes (T1D) in the Polish population, and to correlate them with the serum interleukin-2 levels. 543 unrelated T1D patients and 706 healthy control subjects were enrolled. The minor T allele at rs6822844 was significantly less frequent in T1D compared to controls (p = 0.002; OR 0.71; 95 % CI 0.571-0.880). Likewise, the frequency of the TT genotype was decreased among the affected individuals (p = 0.007). In healthy subjects, stratification according to the rs6822844 genotype revealed significant differences in circulating interleukin-2 (p = 0.037) with the highest levels in TT protective genotypes. Three other IL2 polymorphisms did not display significant differences in allele and genotype distribution. In conclusion, the rs6822844 variant is associated with T1D and may play a functional role, or reflect the influence of another causative genetic variant in linkage disequilibrium.

• Nandiwada SL, Tebo AE
• Testing for antireticulin antibodies in patients with celiac disease is obsolete: a review of recommendations for serologic screening and the literature.
• Clin Vaccine Immunol. 2013; 20: 447-51
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• Celiac disease (CD) is an autoimmune disorder that occurs in genetically susceptible individuals of all ages and is triggered by immune response to gluten and related proteins. The disease is characterized by the presence of HLA-DQ2 and/or -DQ8 haplotypes, diverse clinical manifestations, gluten-sensitive enteropathy, and production of several autoantibodies of which endomysial, tissue transglutaminase, and deamidated gliadin peptide antibodies are considered specific. Although antireticulin antibodies (ARA) have historically been used in the evaluation of CD, these assays lack optimal sensitivities and specificities for routine diagnostic use. This minireview highlights the advances in CD-specific serologic testing and the rationale for eliminating ARA from CD evaluation consistent with recommendations for diagnosis.

• Yoshida H et al.
• KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.
• Proc Natl Acad Sci U S A. 2013; 110: 5612-7
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• Hyaluronan (HA) has an extraordinarily high turnover in physiological tissues, and HA degradation is accelerated in inflammatory and neoplastic diseases. CD44 (a cell surface receptor) and two hyaluronidases (HYAL1 and HYAL2) are thought to be responsible for HA binding and degradation; however, the role of these molecules in HA catabolism remains controversial. Here we show that KIAA1199, a deafness gene of unknown function, plays a central role in HA binding and depolymerization that is independent of CD44 and HYAL enzymes. The specific binding of KIAA1199 to HA was demonstrated in glycosaminoglycan-binding assays. We found that knockdown of KIAA1199 abolished HA degradation by human skin fibroblasts and that transfection of KIAA1199 cDNA into cells conferred the ability to catabolize HA in an endo-beta-N-acetylglucosaminidase-dependent manner via the clathrin-coated pit pathway. Enhanced degradation of HA in synovial fibroblasts from patients with osteoarthritis or rheumatoid arthritis was correlated with increased levels of KIAA1199 expression and was abrogated by knockdown of KIAA1199. The level of KIAA1199 expression in uninflamed synovium was less than in osteoarthritic or rheumatoid synovium. These data suggest that KIAA1199 is a unique hyaladherin with a key role in HA catabolism in the dermis of the skin and arthritic synovium.

• Kiryluk K, Novak J, Gharavi AG
• Pathogenesis of immunoglobulin A nephropathy: recent insight from genetic studies.
• Annu Rev Med. 2013; 64: 339-56
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• Recent genome-wide association studies (GWAS) have identified multiple susceptibility loci for immunoglobulin A nephropathy (IgAN), the most common form of glomerulonephritis, implicating independent defects in adaptive immunity (three loci on chromosome 6p21 in the MHC region), innate immunity (8p23 DEFA locus, 17p23 TNFSF13 locus, 22q12 HORMAD2 locus), and the alternative complement pathway (1q32 CFH/CFHR locus). In geospatial analysis of 85 populations, a genetic risk score based on the replicated GWAS loci is highest in Asians, intermediate in Europeans, and lowest in Africans, capturing the known difference in prevalence among world populations. The genetic risk score also uncovered a previously unsuspected increased prevalence of IgAN-attributable kidney failure in Northern Europe. The IgAN risk alleles have opposing effects on many immune-mediated diseases, suggesting that selection has contributed to variation in risk allele frequencies among different populations. Incorporating genetic, immunologic, and biochemical data, we present a multistep pathogenesis model that provides testable hypotheses for dissecting the mechanisms of disease.

• Marquez A et al.
• IL2/IL21 region polymorphism influences response to rituximab in systemic lupus erythematosus patients.
• Mol Biol Rep. 2013; 40: 4851-6
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• To determine whether the IL2/IL21 region, a general autoimmunity locus, contributes to the observed variation in response to rituximab in patients with systemic lupus erythematosus as well as to analyze its influence in a cohort including other autoimmune diseases. rs6822844 G/T polymorphism at the IL2-IL21 region was analyzed by TaqMan assay in 84 systemic lupus erythematosus (SLE) and 60 different systemic autoimmune diseases Spanish patients receiving rituximab. Six months after the first infusion patients were classified, according to the EULAR criteria, as good responders, partial responders and non-responders. A statistically significant difference was observed in GG genotype frequency between responder (total and partial response) (83.56%) and non-responder (45.45%) SLE patients (p=0.010, odds ratio (OR)=6.10 [1.28-29.06]). No association with the response was evident in the group of patients with autoimmune diseases other than lupus. Furthermore, when both groups of patients were pooled in a meta-analysis, a reduced statistical significance of the association was observed (p=0.024, OR=3.53 [1.06-11.64]). Our results show for a first time that IL2-IL21 region seems to play a role in the response to rituximab in SLE patients but not in other autoimmune diseases.

• Yoshida H, Nagaoka A, Nakamura S, Sugiyama Y, Okada Y, Inoue S
• Murine homologue of the human KIAA1199 is implicated in hyaluronan binding and depolymerization.
• FEBS Open Bio. 2013; 3: 352-6
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• Recently, we have disclosed that human KIAA1199 (hKIAA1199) is a hyaluronan (HA) binding protein implicated in HA depolymerization. Although a murine homologue (mKiaa1199) was previously cloned, no information about the function of the molecule was available. Here, we show that cells transfected with mKiaa1199 cDNA selectively catabolized HA via the clathrin-coated pit pathway. A glycosaminoglycan-binding assay demonstrated the specific binding of mKiaa1199 to HA. These results were similar to our observations with hKIAA1199, although slight differences were found in the peak sizes of the minimum degradates of HA. We conclude that like hKIAA1199, mKiaa1199 is a hyaladherin, leading to HA depolymerization.

• Cenit MC et al.
• Evaluation of the IL2/IL21, IL2RA and IL2RB genetic variants influence on the endogenous non-anterior uveitis genetic predisposition.
• BMC Med Genet. 2013; 14: 52-52
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• BACKGROUND: Recently, different genetic variants located within the IL2/IL21 genetic region as well as within both IL2RA and IL2RB loci have been associated to multiple autoimmune disorders. We aimed to investigate for the first time the potential influence of the IL2/IL21, IL2RA and IL2RB most associated polymorphisms with autoimmunity on the endogenous non-anterior uveitis genetic predisposition. METHODS: A total of 196 patients with endogenous non-anterior uveitis and 760 healthy controls, all of them from Caucasian population, were included in the current study. The IL2/IL21 (rs2069762, rs6822844 and rs907715), IL2RA (2104286, rs11594656 and rs12722495) and IL2RB (rs743777) genetic variants were genotyped using TaqMan(R) allelic discrimination assays. RESULTS: A statistically significant difference was found for the rs6822844 (IL2/IL21 region) minor allele frequency in the group of uveitis patients compared with controls (P(-value)=0.02, OR=0.64 CI 95%=0.43-0.94) although the significance was lost after multiple testing correction. Furthermore, no evidence of association with uveitis was detected for the analyzed genetic variants of the IL2RA or IL2RB loci. CONCLUSION: Our results indicate that analyzed IL2/IL21, IL2RA and IL2RB polymorphisms do not seem to play a significant role on the non-anterior uveitis genetic predisposition although further studies are needed in order to clear up the influence of these loci on the non-anterior uveitis susceptibility.

• Diaz-Gallo LM et al.
• Implication of IL-2/IL-21 region in systemic sclerosis genetic susceptibility.
• Ann Rheum Dis. 2013; 72: 1233-8
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• OBJECTIVE: The interleukin 2 (IL-2) and interleukin 21 (IL-21) locus at chromosome 4q27 has been associated with several autoimmune diseases, and both genes are related to immune system functions. The aim of this study was to evaluate the role of the IL-2/IL-21 locus in systemic sclerosis (SSc). PATIENTS AND METHODS: The case control study included 4493 SSc Caucasian patients and 5856 healthy controls from eight Caucasian populations (Spain, Germany, The Netherlands, USA, Italy, Sweden, UK and Norway). Four single nucleotide polymorphisms (rs2069762, rs6822844, rs6835457 and rs907715) were genotyped using TaqMan allelic discrimination assays. RESULTS: We observed evidence of association of the rs6822844 and rs907715 variants with global SSc (pc=6.6E-4 and pc=7.2E-3, respectively). Similar statistically significant associations were observed for the limited cutaneous form of the disease. The conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs6822844 polymorphism. Consistently, the rs2069762A-rs6822844T-rs6835457G-rs907715T allelic combination showed evidence of association with SSc and limited cutaneous SSc subtype (pc=1.7E-03 and pc=8E-4, respectively). CONCLUSIONS: These results suggested that the IL-2/IL-21 locus influences the genetic susceptibility to SSc. Moreover, this study provided further support for the IL-2/IL-21 locus as a common genetic factor in autoimmune diseases.

• Fowler KE et al.
• Genome wide analysis reveals single nucleotide polymorphisms associated with fatness and putative novel copy number variants in three pig breeds.
• BMC Genomics. 2013; 14: 784-784
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• BACKGROUND: Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). RESULTS: By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARalpha, SLC5A1 and SLC5A4). CONCLUSIONS: Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals.

• Maiti AK, Nath SK
• Gene network analysis of small molecules with autoimmune disease associated genes predicts a novel strategy for drug efficacy.
• Autoimmun Rev. 2013; 12: 510-22
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• Numerous genes/SNPs in autoimmune diseases (ADs) are identified through genome-wide association studies (GWAS) and likely to contribute in developing autoimmune phenotypes. Constructions of biologically meaningful pathways are necessary to determine how these genes interact with each other and with other small molecules to develop various complex AD phenotypes prior to beginning time-consuming rigorous experimentation. We have constructed biological pathways with genetically identified genes leading to shared AD phenotypes. Various environmental and endogenous factors interact with these AD associated genes suggesting their critical role in developing diseases and further association studies could be designed for assessing the role of these factors with risk allele in a specific gene. Additionally, existing drugs that have been used long before the identification of these genetically associated genes also interact with these newly associated genes. Thus advanced therapeutic strategies could be designed by grouping patients with risk allele(s) in particular genes that directly or closely interact with the specified drugs. This drug-susceptible gene network will not only increase our understanding about the additional molecular basis for effectiveness against these diseases but also indicate which drug could be more effective for those patients carrying risk allele(s) in that gene. Additionally, we have also identified several interlinking genes in the pathways that could be used for designing future association studies.

• Arac D et al.
• A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
• EMBO J. 2012; 31: 1364-78
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• The G protein-coupled receptor (GPCR) Proteolysis Site (GPS) of cell-adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the approximately 40-residue GPS motif represents an integral part of a much larger approximately 320-residue domain that we termed GPCR-Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell-adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five beta-strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell-adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine-tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

• Fesel C et al.
• Compensatory T-cell regulation in unaffected relatives of SLE patients, and opposite IL-2/CD25-mediated effects suggested by coreferentiality modeling.
• PLoS One. 2012; 7: 33992-33992
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• In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

• Kumar V, Wijmenga C, Withoff S
• From genome-wide association studies to disease mechanisms: celiac disease as a model for autoimmune diseases.
• Semin Immunopathol. 2012; 34: 567-80
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• Celiac disease is characterized by a chronic inflammatory reaction in the intestine and is triggered by gluten, a constituent derived from grains which is present in the common daily diet in the Western world. Despite decades of research, the mechanisms behind celiac disease etiology are still not fully understood, although it is clear that both genetic and environmental factors are involved. To improve the understanding of the disease, the genetic component has been extensively studied by genome-wide association studies. These have uncovered a wealth of information that still needs further investigation to clarify its importance. In this review, we summarize and discuss the results of the genetic studies in celiac disease, focusing on the "non-HLA" genes. We also present novel approaches to identifying the causal variants in complex susceptibility loci and disease mechanisms.

• Medrano LM et al.
• HLA and celiac disease susceptibility: new genetic factors bring open questions about the HLA influence and gene-dosage effects.
• PLoS One. 2012; 7: 48403-48403
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• Celiac disease (CD) is a chronic inflammatory disorder triggered after gluten ingestion in genetically susceptible individuals. The major genetic determinants are HLA-DQA1*05 and HLA-DQB1*02, which encode the DQ2 heterodimer. These alleles are commonly inherited in cis with DRB1*03ratio01, which is associated with numerous immune-related disorders, in some cases contributing with a different amount of risk depending on the haplotype context. We aimed at investigating those possible differences involving DRB1*03ratio01-carrying haplotypes in CD susceptibility. A family (274 trios) and a case-control sample (369 CD cases/461 controls) were analyzed. DRB1*03ratio01-carrying individuals were classified according to the haplotype present (ancestral haplotype (AH) 8.1, AH 18.2 or non-conserved haplotype) after genotyping of HLA-DRB1, -DQA1, -DQB1, -B8, TNF -308, TNF -376 and the TNFa and TNFb microsatellites. We observe that the AH 8.1 confers higher risk than the remaining DRB1*03ratio01-carrying haplotypes, and this effect only involves individuals possessing a single copy of DQB1*02. CD risk for these individuals is similar to the one conferred by inherit DQA1*05 and DQB1*02 in trans. It seems that an additional CD susceptibility factor is present in the AH 8.1 but not in other DRB1*03ratio01-carrying haplotypes. This factor could be shared with individuals possessing DQ2.5 trans, according to the similar risk observed in those two groups of individuals.

• Manji SS, Miller KA, Williams LH, Dahl HH
• Identification of three novel hearing loss mouse strains with mutations in the Tmc1 gene.
• Am J Pathol. 2012; 180: 1560-9
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• We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.

• Leffler DA et al.
• A randomized, double-blind study of larazotide acetate to prevent the activation of celiac disease during gluten challenge.
• Am J Gastroenterol. 2012; 107: 1554-62
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• OBJECTIVES: In patients with celiac disease, enteropathy is caused by the entry of gluten peptides into the lamina propria of the intestine, in which their immunogenicity is potentiated by tissue transglutaminase (tTG) and T-helper type 1-mediated immune responses are triggered. Tight junction disassembly and paracellular permeability are believed to have an important role in the transport of gluten peptides to the lamina propria. Larazotide acetate is a tight-junction regulator peptide that, in vitro, prevents the opening of intestinal epithelial tight junctions. The aim of this study was to evaluate the efficacy and tolerability of larazotide acetate in protecting against gluten-induced intestinal permeability and gastrointestinal symptom severity in patients with celiac disease. METHODS: In this dose-ranging, placebo-controlled study, 86 patients with celiac disease controlled through diet were randomly assigned to larazotide acetate (0.25, 1, 4, or 8 mg) or placebo three times per day with or without gluten challenge (2.4 g/day) for 14 days. The primary efficacy outcome was the urinary lactulose/mannitol (LAMA) fractional excretion ratio. Secondary endpoints included gastrointestinal symptom severity, quality-of-life measures, and antibodies to tTG. RESULTS: LAMA measurements were highly variable in the outpatient setting. The increase in LAMA ratio associated with the gluten challenge was not statistically significantly greater than the increase in the gluten-free control. Among patients receiving the gluten challenge, the difference in the LAMA ratios for the larazotide acetate and placebo groups was not statistically significant. However, larazotide acetate appeared to limit gluten-induced worsening of gastrointestinal symptom severity as measured by the Gastrointestinal Symptom Rating Scale at some lower doses but not at the higher dose. Symptoms worsened significantly in the gluten challenge-placebo arm compared with the placebo-placebo arm, suggesting that 2.4 g of gluten per day is sufficient to induce reproducible gluten toxicity. Larazotide acetate was generally well tolerated. No serious adverse events were observed. The most common adverse events were headache and urinary tract infection. CONCLUSIONS: LAMA variability in the outpatient setting precluded accurate assessment of the effect of larazotide acetate on intestinal permeability. However, some lower doses of larazotide acetate appeared to prevent the increase in gastrointestinal symptom severity induced by gluten challenge.

• Sapone A et al.
• Spectrum of gluten-related disorders: consensus on new nomenclature and classification.
• BMC Med. 2012; 10: 13-13
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• A decade ago celiac disease was considered extremely rare outside Europe and, therefore, was almost completely ignored by health care professionals. In only 10 years, key milestones have moved celiac disease from obscurity into the popular spotlight worldwide. Now we are observing another interesting phenomenon that is generating great confusion among health care professionals. The number of individuals embracing a gluten-free diet (GFD) appears much higher than the projected number of celiac disease patients, fueling a global market of gluten-free products approaching $2.5 billion (US) in global sales in 2010. This trend is supported by the notion that, along with celiac disease, other conditions related to the ingestion of gluten have emerged as health care concerns. This review will summarize our current knowledge about the three main forms of gluten reactions: allergic (wheat allergy), autoimmune (celiac disease, dermatitis herpetiformis and gluten ataxia) and possibly immune-mediated (gluten sensitivity), and also outline pathogenic, clinical and epidemiological differences and propose new nomenclature and classifications.

• Larussa T et al.
• No evidence of circulating autoantibodies against osteoprotegerin in patients with celiac disease.
• World J Gastroenterol. 2012; 18: 1622-7
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• AIM: To investigate risk factors for low bone mineral density (BMD) in celiac disease (CD) patients, focusing on circulating autoantibodies against osteoprotegerin (OPG). METHODS: Seventy asymptomatic CD adult patients on gluten-free diet (GFD) and harbouring persistent negative CD-related serology were recruited. Conventional risk factors for osteoporosis (e.g., age, sex, menopausal status, history of fractures, smoke, and body mass index) were checked and BMD was assessed by dual energy X ray absorptiometry. Serum calcium and parathyroid hormone (PTH) levels were evaluated. Thirty-eight patients underwent repeat duodenal biopsy. Serum samples from a selected sub-group of 30 patients, who were also typed for human leukocyte antigen (HLA) DQ2 and DQ8 haplotype, were incubated with homodimeric recombinant human OPG and tested by western blotting with an anti-OPG antibody after immunoprecipitation. RESULTS: Despite persistent negative CD-related serology and strict adherence to GFD, 49 out of the 70 (74%) patients displayed low BMD. Among these patients, 13 (24%) showed osteoporosis and 36 (76%) osteopenia. With the exception of age, conventional risk factors for osteoporosis did not differ between patients with normal and low BMD. Circulating serum calcium and PTH levels were normal in all patients. Duodenal mucosa healing was found in 31 (82%) out of 38 patients who underwent repeat duodenal biopsy with 20 (64%) still displaying low BMD. The remaining 7 patients had an incomplete normalization of duodenal mucosa with 6 (84%) showing low BMD. No evidence of circulating antibodies against OPG was found in the serum of 30 celiac patients who were tested for, independent of BMD, duodenal histology, and HLA status. CONCLUSION: If any, the role of circulating autoantibodies against OPG in the pathogenesis of bone derangement in patients with CD is not a major one.

• Low SK et al.
• Genome-wide association study for intracranial aneurysm in the Japanese population identifies three candidate susceptible loci and a functional genetic variant at EDNRA.
• Hum Mol Genet. 2012; 21: 2102-10
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• Aneurysmal subarachnoid hemorrhage (aSAH) is the most serious subtype of stroke. Genetic factors have been known to play an important role in the development of intracranial aneurysm (IA), some of which further progress to subarachnoid hemorrhage (SAH). In this study, we conducted a genome-wide association study (GWAS) to identify common genetic variants that are associated with the risk of IA, using 1383 aSAH subjects and 5484 control individuals in the Japanese population. We selected 36 single-nucleotide polymorphisms (SNPs) that showed suggestive association (P <1 x 10(-4)) in the GWAS as well as additional 7 SNPs that were previously reported to be associated with IA, and further genotyped an additional set of 1048 IA cases and 7212 controls. We identified an SNP, rs6842241, near EDNRA at chromosome 4q31.22 (combined P-value = 9.58 x 10(-9); odds ratio = 1.25), which was found to be significantly associated with IA. Additionally, we successfully replicated and validated rs10757272 on CDKN2BAS at chromosome 9p21.3 (combined P-value = 1.55 x 10(-7); odds ratio = 1.21) to be significantly associated with IA as previously reported. Furthermore, we performed functional analysis with the associated genetic variants on EDNRA, and identified two alleles of rs6841581 that have different binding affinities to a nuclear protein(s). The transcriptional activity of the susceptible allele of this variant was significantly lower than the other, suggesting that this functional variant might affect the expression of EDNRA and subsequently result in the IA susceptibility. Identification of genetic variants on EDNRA is of clinical significance probably due to its role in vessel hemodynamic stress. Our findings should contribute to a better understanding of physiopathology of IA.

• Skibola CF et al.
• A meta-analysis of genome-wide association studies of follicular lymphoma.
• BMC Genomics. 2012; 13: 516-516
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• BACKGROUND: B-cell non-Hodgkin lymphoma represents a diverse group of hematological malignancies, of which follicular lymphoma (FL) is one of the most common subtypes. Family and epidemiological studies suggest an important genetic role in the etiology of FL. In recent genome-wide association studies (GWAS) of FL, several genetic susceptibility loci have been identified on chromosome 6p21.33 (rs6457327) and 6p21.32 (rs10484561, rs2647012) in the human leukocyte antigen class I and class II regions. To identify new genetic variants and further elucidate the genetic basis of FL, a meta-analysis was performed of the top 1000 SNPs associated with FL risk from two GWAS in the US, Denmark and Sweden (592 cases, 1541 controls), with independent validation in 107 cases and 681 controls. RESULTS: rs9275517 and rs3117222 in the HLA class II region were validated and inversely associated with FL risk (rs9275517: OR = 0.63, 95% CI = 0.55-0.73, p = 4.03 x 10(-11); rs3117222: OR = 0.66, 95% CI = 0.57-0.77, p = 1.45 x 10(-7)). rs9275517, which is in high linkage disequilibrium with rs2647012 (r2 = 0.9), was no longer associated with FL after conditioning on rs2647012. The rs3117222 association was independent of established FL SNPs, but not of the HLA-DPB1*0301 allele. Using publicly available gene expression profiles with matching genotype information, we found that rs3117222 also was significantly correlated with increased HLA-DPB1 expression. CONCLUSIONS: By performing a meta-analysis of two GWAS of FL, we further validated the relevance of HLA-DPB1*0301 as a protective allele in the pathogenesis of FL. Moreover, the protective rs3117222 A allele correlated with increased levels of HLA-DPB1, suggesting a possible disease mechanism involving HLA-DPB1 expression regulation. Our results add further support to the major role of HLA genetic variation in the pathogenesis of FL.

• Babron MC et al.
• Determination of the real effect of genes identified in GWAS: the example of IL2RA in multiple sclerosis.
• Eur J Hum Genet. 2012; 20: 321-5
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• Genome-wide association studies (GWAS), although efficient to detect genes involved in complex diseases, are not designed to measure the real effect of the genes. This is illustrated here by the example of IL2RA in multiple sclerosis (MS). Association between IL2RA and MS is clearly established, although the functional variation is still unknown: the effect of IL2RA might be better described by several SNPs than by a single one. This study investigates whether a pair of SNPs better explains the observed linkage and association data than a single SNP. In total, 522 trio families and 244 affected sib-pairs were typed for 26 IL2RA SNPs. For each SNP and pairs of SNPs, the phased genotypes of patients and controls were compared to determine the SNP set offering the best risk discrimination. Consistency between the genotype risks provided by the retained set and the identical by descent allele sharing in affected sib-pairs was assessed. After controlling for multiple testing, the set of SNPs rs2256774 and rs3118470, provides the best discrimination between the case and control genotype distributions (P-corrected=0.009). The relative risk between the least and most at-risk genotypes is 3.54 with a 95% confidence interval of [2.14-5.94]. Furthermore, the linkage information provided by the allele sharing between affected sibs is consistent with the retained set (P=0.80) but rejects the SNP reported in the literature (P=0.006). Establishing a valid modeling of a disease gene is essential to test its potential interaction with other genes and to reconstruct the pathophysiological pathways.

• Tollefsen S et al.
• Structural and functional studies of trans-encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) protein molecule.
• J Biol Chem. 2012; 287: 13611-9
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• MHC class II molecules are composed of one alpha-chain and one beta-chain whose membrane distal interface forms the peptide binding groove. Most of the existing knowledge on MHC class II molecules comes from the cis-encoded variants where the alpha- and beta-chain are encoded on the same chromosome. However, trans-encoded class II MHC molecules, where the alpha- and beta-chain are encoded on opposite chromosomes, can also be expressed. We have studied the trans-encoded class II HLA molecule DQ2.3 (DQA1*03:01/DQB1*02:01) that has received particular attention as it may explain the increased risk of certain individuals to type 1 diabetes. We report the x-ray crystal structure of this HLA molecule complexed with a gluten epitope at 3.05 A resolution. The gluten epitope, which is the only known HLA-DQ2.3-restricted epitope, is preferentially recognized in the context of the DQ2.3 molecule by T-cell clones of a DQ8/DQ2.5 heterozygous celiac disease patient. This preferential recognition can be explained by improved HLA binding as the epitope combines the peptide-binding motif of DQ2.5 (negative charge at P4) and DQ8 (negative charge at P1). The analysis of the structure of DQ2.3 together with all other available DQ crystal structures and sequences led us to categorize DQA1 and DQB1 genes into two groups where any alpha-chain and beta-chain belonging to the same group are expected to form a stable heterodimer.

• Pozo-Rubio T et al.
• Immune development and intestinal microbiota in celiac disease.
• Clin Dev Immunol. 2012; 2012: 654143-654143
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• Celiac disease (CD) is an immune-mediated enteropathy, triggered by dietary wheat gluten and similar proteins of barley and rye in genetically susceptible individuals. The etiology of this disorder is complex, involving both environmental and genetic factors. The major genetic risk factor for CD is represented by HLA-DQ genes, which account for approximately 40% of the genetic risk; however, only a small percentage of carriers develop the disease. Gluten is the main environmental factor responsible for the signs and symptoms of the disease, but exposure to gluten does not fully explain the manifestation of CD. Epidemiological and clinical data suggest that environmental factors other than gluten might play a role in disease development, including early feeding practices (e.g., breast milk versus formula and duration of breastfeeding), infections, and alterations in the intestinal microbiota composition. Herein, we review what is known about the influence of dietary factors, exposure to infectious agents, and intestinal microbiota composition, particularly in early life, on the risk of developing CD, as well as the possible dietary strategies to induce or increase gluten tolerance.

• Ahn R et al.
• Association analysis of the extended MHC region in celiac disease implicates multiple independent susceptibility loci.
• PLoS One. 2012; 7: 36926-36926
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• Celiac disease is a common autoimmune disease caused by sensitivity to the dietary protein gluten. Forty loci have been implicated in the disease. All disease loci have been characterized as low-penetrance, with the exception of the high-risk genotypes in the HLA-DQA1 and HLA-DQB1 genes, which are necessary but not sufficient to cause the disease. The very strong effects from the known HLA loci and the genetically complex nature of the major histocompatibility complex (MHC) have precluded a thorough investigation of the region. The purpose of this study was to test the hypothesis that additional celiac disease loci exist within the extended MHC (xMHC). A set of 1898 SNPs was analyzed for association across the 7.6 Mb xMHC region in 1668 confirmed celiac disease cases and 517 unaffected controls. Conditional recursive partitioning was used to create an informative indicator of the known HLA-DQA1 and HLA-DQB1 high-risk genotypes that was included in the association analysis to account for their effects. A linkage disequilibrium-based grouping procedure was utilized to estimate the number of independent celiac disease loci present in the xMHC after accounting for the known effects. There was significant statistical evidence for four new independent celiac disease loci within the classic MHC region. This study is the first comprehensive association analysis of the xMHC in celiac disease that specifically accounts for the known HLA disease genotypes and the genetic complexity of the region.

• van Eerde AM et al.
• Genes in the ureteric budding pathway: association study on vesico-ureteral reflux patients.
• PLoS One. 2012; 7: 31327-31327
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• Vesico-ureteral reflux (VUR) is the retrograde passage of urine from the bladder to the urinary tract and causes 8.5% of end-stage renal disease in children. It is a complex genetic developmental disorder, in which ectopic embryonal ureteric budding is implicated in the pathogenesis. VUR is part of the spectrum of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). We performed an extensive association study for primary VUR using a two-stage, case-control design, investigating 44 candidate genes in the ureteric budding pathway in 409 Dutch VUR patients. The 44 genes were selected from the literature and a set of 567 single nucleotide polymorphisms (SNPs) capturing their genetic variation was genotyped in 207 cases and 554 controls. The 14 SNPs with p<0.005 were included in a follow-up study in 202 cases and 892 controls. Of the total cohort, ~50% showed a clear-cut primary VUR phenotype and ~25% had both a duplex collecting system and VUR. We also looked for association in these two extreme phenotype groups. None of the SNPs reached a significant p-value. Common genetic variants in four genes (GREM1, EYA1, ROBO2 and UPK3A) show a trend towards association with the development of primary VUR (GREM1, EYA1, ROBO2) or duplex collecting system (EYA1 and UPK3A). SNPs in three genes (TGFB1, GNB3 and VEGFA) have been shown to be associated with VUR in other populations. Only the result of rs1800469 in TGFB1 hinted at association in our study. This is the first extensive study of common variants in the genes of the ureteric budding pathway and the genetic susceptibility to primary VUR.

• Abnet CC et al.
• Genotypic variants at 2q33 and risk of esophageal squamous cell carcinoma in China: a meta-analysis of genome-wide association studies.
• Hum Mol Genet. 2012; 21: 2132-41
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• Genome-wide association studies have identified susceptibility loci for esophageal squamous cell carcinoma (ESCC). We conducted a meta-analysis of all single-nucleotide polymorphisms (SNPs) that showed nominally significant P-values in two previously published genome-wide scans that included a total of 2961 ESCC cases and 3400 controls. The meta-analysis revealed five SNPs at 2q33 with P< 5 x 10(-8), and the strongest signal was rs13016963, with a combined odds ratio (95% confidence interval) of 1.29 (1.19-1.40) and P= 7.63 x 10(-10). An imputation analysis of 4304 SNPs at 2q33 suggested a single association signal, and the strongest imputed SNP associations were similar to those from the genotyped SNPs. We conducted an ancestral recombination graph analysis with 53 SNPs to identify one or more haplotypes that harbor the variants directly responsible for the detected association signal. This showed that the five SNPs exist in a single haplotype along with 45 imputed SNPs in strong linkage disequilibrium, and the strongest candidate was rs10201587, one of the genotyped SNPs. Our meta-analysis found genome-wide significant SNPs at 2q33 that map to the CASP8/ALS2CR12/TRAK2 gene region. Variants in CASP8 have been extensively studied across a spectrum of cancers with mixed results. The locus we identified appears to be distinct from the widely studied rs3834129 and rs1045485 SNPs in CASP8. Future studies of esophageal and other cancers should focus on comprehensive sequencing of this 2q33 locus and functional analysis of rs13016963 and rs10201587 and other strongly correlated variants.

• Medrano LM et al.
• Th17-related genes and celiac disease susceptibility.
• PLoS One. 2012; 7: 31244-31244
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• Th17 cells are known to be involved in several autoimmune or inflammatory diseases. In celiac disease (CD), recent studies suggest an implication of those cells in disease pathogenesis. We aimed at studying the role of genes relevant for the Th17 immune response in CD susceptibility. A total of 101 single nucleotide polymorphisms (SNPs), mainly selected to cover most of the variability present in 16 Th17-related genes (IL23R, RORC, IL6R, IL17A, IL17F, CCR6, IL6, JAK2, TNFSF15, IL23A, IL22, STAT3, TBX21, SOCS3, IL12RB1 and IL17RA), were genotyped in 735 CD patients and 549 ethnically matched healthy controls. Case-control comparisons for each SNP and for the haplotypes resulting from the SNPs studied in each gene were performed using chi-square tests. Gene-gene interactions were also evaluated following different methodological approaches. No significant results emerged after performing the appropriate statistical corrections. Our results seem to discard a relevant role of Th17 cells on CD risk.

• Maurer MF et al.
• Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies.
• MAbs. 2012; 4: 69-83
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• Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope "bins" based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.

• Gujral N, Freeman HJ, Thomson AB
• Celiac disease: prevalence, diagnosis, pathogenesis and treatment.
• World J Gastroenterol. 2012; 18: 6036-59
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• Celiac disease (CD) is one of the most common diseases, resulting from both environmental (gluten) and genetic factors [human leukocyte antigen (HLA) and non-HLA genes]. The prevalence of CD has been estimated to approximate 0.5%-1% in different parts of the world. However, the population with diabetes, autoimmune disorder or relatives of CD individuals have even higher risk for the development of CD, at least in part, because of shared HLA typing. Gliadin gains access to the basal surface of the epithelium, and interact directly with the immune system, via both trans- and para-cellular routes. From a diagnostic perspective, symptoms may be viewed as either "typical" or "atypical". In both positive serological screening results suggestive of CD, should lead to small bowel biopsy followed by a favourable clinical and serological response to the gluten-free diet (GFD) to confirm the diagnosis. Positive anti-tissue transglutaminase antibody or anti-endomysial antibody during the clinical course helps to confirm the diagnosis of CD because of their over 99% specificities when small bowel villous atrophy is present on biopsy. Currently, the only treatment available for CD individuals is a strict life-long GFD. A greater understanding of the pathogenesis of CD allows alternative future CD treatments to hydrolyse toxic gliadin peptide, prevent toxic gliadin peptide absorption, blockage of selective deamidation of specific glutamine residues by tissue, restore immune tolerance towards gluten, modulation of immune response to dietary gliadin, and restoration of intestinal architecture.

• Kupfer SS, Jabri B
• Pathophysiology of celiac disease.
• Gastrointest Endosc Clin N Am. 2012; 22: 639-60
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• Celiac disease results from the interplay of genetic, environmental, and immunologic factors. An understanding of the pathophysiology of celiac disease, in which the trigger (wheat, rye, and barley) is known, will undoubtedly reveal basic mechanisms that underlie other autoimmune diseases (eg, type 1 diabetes) that share many common pathogenic perturbations. This review describes seminal findings in each of the 3 domains of the pathogenesis of celiac disease, namely genetics, environmental triggers, and immune dysregulation, with a focus on newer areas of investigation such as non-HLA genetic variants, the intestinal microbiome, and the role of the innate immune system.

• Kinsella M, Bafna V
• Combinatorics of the breakage-fusion-bridge mechanism.
• J Comput Biol. 2012; 19: 662-78
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• The breakage-fusion-bridge (BFB) mechanism was proposed over seven decades ago and is a source of genomic variability and gene amplification in cancer. Here we formally model and analyze the BFB mechanism, to our knowledge the first time this has been undertaken. We show that BFB can be modeled as successive inverted prefix duplications of a string. Using this model, we show that BFB can achieve a surprisingly broad range of amplification patterns. We find that a sequence of BFB operations can be found that nearly fits most patterns of copy number increases along a chromosome. We conclude that this limits the usefulness of methods like array CGH for detecting BFB.

• Trynka G et al.
• Dense genotyping identifies and localizes multiple common and rare variant association signals in celiac disease.
• Nat Genet. 2011; 43: 1193-201
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• Using variants from the 1000 Genomes Project pilot European CEU dataset and data from additional resequencing studies, we densely genotyped 183 non-HLA risk loci previously associated with immune-mediated diseases in 12,041 individuals with celiac disease (cases) and 12,228 controls. We identified 13 new celiac disease risk loci reaching genome-wide significance, bringing the number of known loci (including the HLA locus) to 40. We found multiple independent association signals at over one-third of these loci, a finding that is attributable to a combination of common, low-frequency and rare genetic variants. Compared to previously available data such as those from HapMap3, our dense genotyping in a large sample collection provided a higher resolution of the pattern of linkage disequilibrium and suggested localization of many signals to finer scale regions. In particular, 29 of the 54 fine-mapped signals seemed to be localized to single genes and, in some instances, to gene regulatory elements. Altogether, we define the complex genetic architecture of the risk regions of and refine the risk signals for celiac disease, providing the next step toward uncovering the causal mechanisms of the disease.

• Han J et al.
• Genetic variants of 6q25 and breast cancer susceptibility: a two-stage fine mapping study in a Chinese population.
• Breast Cancer Res Treat. 2011; 129: 901-7
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• A recent genome-wide association study identified a novel single nucleotide polymorphism (SNP), rs2046210, in the 6q25 region as a breast cancer susceptibility locus in Chinese and subsequently replicated in a multicenter study. Further fine-mapping of this region may help identify the potential causative SNPs of breast cancer. We employed a block-based fine mapping analysis to investigate the tagging SNPs in a 41 kb block with the marker-SNP rs2046210 in the 6q25 region, and also extended our study by including two potentially functional SNPs (rs2234693 and rs1801132) within the ESR1 gene by a two-stage case-control study with 1,792 breast cancer cases and 1,867 controls (878 cases and 900 controls in the testing set and 914 cases and 967 controls in the validation set). Significant associations with breast cancer risk were observed for rs1038304, rs6929137, rs2046210, and rs10484919 in the 41 kb block of the 6q25 region in the testing set after controlling multiple testing. Together with the validation set samples, these four SNPs were all significantly associated with increased risk of breast cancer (additive OR from 1.25 to 1.34, additive P from 4.84 x 10(-6) to 7.17 x 10(-9)). After conditional regression and linkage disequilibrium analyses, rs6929137 and rs10484919 tend to be susceptible markers of breast cancer in this region and both of them were located at sites of histone modification according to the UCSC (http://genome.ucsc.edu/) genome database. Our results support that the 6q25 region is an important susceptibility region for breast cancer in Chinese women, and rs6929137 and rs10484919 are causative or marker SNPs for this region.

• Zhernakova A et al.
• Meta-analysis of genome-wide association studies in celiac disease and rheumatoid arthritis identifies fourteen non-HLA shared loci.
• PLoS Genet. 2011; 7: 1002004-1002004
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• Epidemiology and candidate gene studies indicate a shared genetic basis for celiac disease (CD) and rheumatoid arthritis (RA), but the extent of this sharing has not been systematically explored. Previous studies demonstrate that 6 of the established non-HLA CD and RA risk loci (out of 26 loci for each disease) are shared between both diseases. We hypothesized that there are additional shared risk alleles and that combining genome-wide association study (GWAS) data from each disease would increase power to identify these shared risk alleles. We performed a meta-analysis of two published GWAS on CD (4,533 cases and 10,750 controls) and RA (5,539 cases and 17,231 controls). After genotyping the top associated SNPs in 2,169 CD cases and 2,255 controls, and 2,845 RA cases and 4,944 controls, 8 additional SNPs demonstrated P<5 x 10(-8) in a combined analysis of all 50,266 samples, including four SNPs that have not been previously confirmed in either disease: rs10892279 near the DDX6 gene (P(combined) = 1.2 x 10(-12)), rs864537 near CD247 (P(combined) = 2.2 x 10(-11)), rs2298428 near UBE2L3 (P(combined) = 2.5 x 10(-10)), and rs11203203 near UBASH3A (P(combined) = 1.1 x 10(-8)). We also confirmed that 4 gene loci previously established in either CD or RA are associated with the other autoimmune disease at combined P<5 x 10(-8) (SH2B3, 8q24, STAT4, and TRAF1-C5). From the 14 shared gene loci, 7 SNPs showed a genome-wide significant effect on expression of one or more transcripts in the linkage disequilibrium (LD) block around the SNP. These associations implicate antigen presentation and T-cell activation as a shared mechanism of disease pathogenesis and underscore the utility of cross-disease meta-analysis for identification of genetic risk factors with pleiotropic effects between two clinically distinct diseases.

• Cascella NG et al.
• Prevalence of celiac disease and gluten sensitivity in the United States clinical antipsychotic trials of intervention effectiveness study population.
• Schizophr Bull. 2011; 37: 94-100
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• Celiac disease (CD) and schizophrenia have approximately the same prevalence, but epidemiologic data show higher prevalence of CD among schizophrenia patients. The reason for this higher co-occurrence is not known, but the clinical knowledge about the presence of immunologic markers for CD or gluten intolerance in schizophrenia patients may have implications for treatment. Our goal was to evaluate antibody prevalence to gliadin (AGA), transglutaminase (tTG), and endomysium (EMA) in a group of individuals with schizophrenia and a comparison group. AGA, tTG, and EMA antibodies were assayed in 1401 schizophrenia patients who were part of the Clinical Antipsychotic Trials of Intervention Effectiveness study and 900 controls. Psychopathology in schizophrenia patients was assessed using the Positive and Negative Symptoms Scale (PANSS). Logistic regression was used to assess the difference in the frequency of AGA, immunoglobulin A (IgA), and tTG antibodies, adjusting for age, sex, and race. Linear regression was used to predict PANSS scores from AGA and tTG antibodies adjusting for age, gender, and race. Among schizophrenia patients, 23.1% had moderate to high levels of IgA-AGA compared with 3.1% of the comparison group (chi(2) = 1885, df = 2, P < .001.) Moderate to high levels of tTG antibodies were present in 5.4% of schizophrenia patients vs 0.80% of the comparison group (chi(2) = 392.0, df = 2, P < .001). Adjustments for sex, age, and race had trivial effects on the differences. Regression analyses failed to predict PANSS scores from AGA and tTG antibodies. Persons with schizophrenia have higher than expected titers of antibodies related to CD and gluten sensitivity.

• Izzo V et al.
• Improving the estimation of celiac disease sibling risk by non-HLA genes.
• PLoS One. 2011; 6: 26920-26920
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• Celiac Disease (CD) is a polygenic trait, and HLA genes explain less than half of the genetic variation. Through large GWAs more than 40 associated non-HLA genes were identified, but they give a small contribution to the heritability of the disease. The aim of this study is to improve the estimate of the CD risk in siblings, by adding to HLA a small set of non-HLA genes. One-hundred fifty-seven Italian families with a confirmed CD case and at least one other sib and both parents were recruited. Among 249 sibs, 29 developed CD in a 6 year follow-up period. All individuals were typed for HLA and 10 SNPs in non-HLA genes: CCR1/CCR3 (rs6441961), IL12A/SCHIP1 and IL12A (rs17810546 and rs9811792), TAGAP (rs1738074), RGS1 (rs2816316), LPP (rs1464510), OLIG3 (rs2327832), REL (rs842647), IL2/IL21 (rs6822844), SH2B3 (rs3184504). Three associated SNPs (in LPP, REL, and RGS1 genes) were identified through the Transmission Disequilibrium Test and a Bayesian approach was used to assign a score (BS) to each detected HLA+SNPs genotype combination. We then classified CD sibs as at low or at high risk if their BS was respectively < or >/= median BS value within each HLA risk group. A larger number (72%) of CD sibs showed a BS >/= the median value and had a more than two fold higher OR than CD sibs with a BS value < the median (O.R = 2.53, p = 0.047). Our HLA+SNPs genotype classification, showed both a higher predictive negative value (95% vs 91%) and diagnostic sensitivity (79% vs 45%) than the HLA only. In conclusion, the estimate of the CD risk by HLA+SNPs approach, even if not applicable to prevention, could be a precious tool to improve the prediction of the disease in a cohort of first degree relatives, particularly in the low HLA risk groups.

• Cheng G, Yu A, Malek TR
• T-cell tolerance and the multi-functional role of IL-2R signaling in T-regulatory cells.
• Immunol Rev. 2011; 241: 63-76
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• Signaling through the interleukin-2 receptor (IL-2R) contributes to T-cell tolerance by controlling three important aspects of regulatory T-cell (Treg) biology. IL-2 is essential for thymic Treg development and regulates Treg homeostasis and suppressive function. Analogous to activated conventional T lymphocytes, IL-2R signaling also plays an important part in Treg cell growth, survival, and effector differentiation. However, Treg cells somewhat distinctively assimilate IL-2R signaling. In particular, Treg cells require essentially only IL-2-dependent receptor proximal signal transducer and activator of transcription 5 (Stat5) activation, as they contain inhibitory pathways to minimize IL-2R-dependent activation of the phosphatidyinositol 3-kinase/Akt pathway. Moreover, many IL-2R-dependent activities, including full induction of Foxp3 expression, in Treg cells require minimal and transient Stat5 activation. Thus, Treg cells are equipped to sense and then develop and function within biological niches containing minimal IL-2. These distinguishing features of IL-2R signaling provide a mechanistic underpinning for using IL-2 as an agent to selectively target Treg cells in immunotherapy to induce tolerance in autoimmune diseases and in allogeneic transplant recipients.

• Stringer S, Wray NR, Kahn RS, Derks EM
• Underestimated effect sizes in GWAS: fundamental limitations of single SNP analysis for dichotomous phenotypes.
• PLoS One. 2011; 6: 27964-27964
• Display abstract

• Complex diseases are often highly heritable. However, for many complex traits only a small proportion of the heritability can be explained by observed genetic variants in traditional genome-wide association (GWA) studies. Moreover, for some of those traits few significant SNPs have been identified. Single SNP association methods test for association at a single SNP, ignoring the effect of other SNPs. We show using a simple multi-locus odds model of complex disease that moderate to large effect sizes of causal variants may be estimated as relatively small effect sizes in single SNP association testing. This underestimation effect is most severe for diseases influenced by numerous risk variants. We relate the underestimation effect to the concept of non-collapsibility found in the statistics literature. As described, continuous phenotypes generated with linear genetic models are not affected by this underestimation effect. Since many GWA studies apply single SNP analysis to dichotomous phenotypes, previously reported results potentially underestimate true effect sizes, thereby impeding identification of true effect SNPs. Therefore, when a multi-locus model of disease risk is assumed, a multi SNP analysis may be more appropriate.

• Leonardi E, Andreazza S, Vanin S, Busolin G, Nobile C, Tosatto SC
• A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.
• PLoS One. 2011; 6: 18142-18142
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• Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the beta-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

• Espino-Paisan L et al.
• Study of polymorphisms in 4q27, 10p15, and 22q13 regions in autoantibodies stratified type 1 diabetes patients.
• Autoimmunity. 2011; 44: 624-30
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• Type 1 diabetes (T1D) is a multifactorial disease mainly associated with the human leukocyte antigen region. Previous studies suggested the association of interleukin-2 (IL2) gene polymorphisms and its alpha- and beta-chain receptor (IL2RA and IL2RB) variants with different autoimmune diseases such as T1D, celiac disease, multiple sclerosis, and rheumatoid arthritis. All T1D studies were conducted in diabetic patients younger than 17 years at diagnosis. The aim of our study was to replicate these associations not only in pediatric patients, but also in individuals with late onset. We performed a genetic association study of chromosomal regions 4q27, 10p15, and 22q13 containing the IL2, IL2RA, and IL2RB genes in 445 T1D subjects and 828 healthy controls. Seven single nucleotide polymorphisms (SNPs) were selected, previously described as genetic factors related to several autoimmune diseases, and were analyzed by TaqMan assays. The reported association with T1D patients of the IL2RA-rs41295061 located in the 10p15 region was replicated and our data suggest a trend of association of the polymorphisms IL2-rs17388568 and IL2-rs6822844 in 4q27. The effect of these markers was independent of the age at disease onset. Furthermore, the polymorphisms studied in 4q27 were not dependent on the presence of autoantibodies; however, the effect of the associated SNP in 10p15 (IL2RA-rs41295061) was specific of patients sera positive for diabetes antibodies. In conclusion, our results seem to indicate that late-onset and young T1D patients share most genetic factors located in the studied regions, but some markers could correlate with the presence of T1D specific autoantibodies.

• Janse M et al.
• Three ulcerative colitis susceptibility loci are associated with primary sclerosing cholangitis and indicate a role for IL2, REL, and CARD9.
• Hepatology. 2011; 53: 1977-85
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• Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and fibrosis of the bile ducts. Both environmental and genetic factors contribute to its pathogenesis. To further clarify its genetic background, we investigated susceptibility loci recently identified for ulcerative colitis (UC) in a large cohort of 1,186 PSC patients and 1,748 controls. Single nucleotide polymorphisms (SNPs) tagging 13 UC susceptibility loci were initially genotyped in 854 PSC patients and 1,491 controls from Benelux (331 cases, 735 controls), Germany (265 cases, 368 controls), and Scandinavia (258 cases, 388 controls). Subsequently, a joint analysis was performed with an independent second Scandinavian cohort (332 cases, 257 controls). SNPs at chromosomes 2p16 (P-value 4.12 x 10(-4) ), 4q27 (P-value 4.10 x 10(-5) ), and 9q34 (P-value 8.41 x 10(-4) ) were associated with PSC in the joint analysis after correcting for multiple testing. In PSC patients without inflammatory bowel disease (IBD), SNPs at 4q27 and 9q34 were nominally associated (P < 0.05). We applied additional in silico analyses to identify likely candidate genes at PSC susceptibility loci. To identify nonrandom, evidence-based links we used GRAIL (Gene Relationships Across Implicated Loci) analysis showing interconnectivity between genes in six out of in total nine PSC-associated regions. Expression quantitative trait analysis from 1,469 Dutch and UK individuals demonstrated that five out of nine SNPs had an effect on cis-gene expression. These analyses prioritized IL2, CARD9, and REL as novel candidates. CONCLUSION: We have identified three UC susceptibility loci to be associated with PSC, harboring the putative candidate genes REL, IL2, and CARD9. These results add to the scarce knowledge on the genetic background of PSC and imply an important role for both innate and adaptive immunological factors.

• Sollid LM, Khosla C
• Novel therapies for coeliac disease.
• J Intern Med. 2011; 269: 604-13
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• Coeliac disease is a widespread, lifelong disorder for which dietary control represents the only accepted form of therapy. There is an unmet need for nondietary therapies to treat this condition. Most ongoing and emerging drug-discovery programmes are based on the understanding that coeliac disease is caused by an inappropriate T-cell-mediated immune response to dietary gluten proteins. Recent genome-wide association studies lend further support to this pathogenic model. The central role of human leucocyte antigen genes has been validated, and a number of new risk loci have been identified, most of which are related to the biology of T cells and antigen-presenting cells. Here, we review the status of potential nondietary therapies under consideration for coeliac disease. We conclude that future development of novel therapies will be aided considerably by the identification of new, preferably noninvasive, surrogate markers for coeliac disease activity.

• Cinova J et al.
• Role of intestinal bacteria in gliadin-induced changes in intestinal mucosa: study in germ-free rats.
• PLoS One. 2011; 6: 16169-16169
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• BACKGROUND AND AIMS: Celiac disease (CD) is a chronic inflammatory disorder of the small intestine that is induced by dietary wheat gluten proteins (gliadins) in genetically predisposed individuals. The overgrowth of potentially pathogenic bacteria and infections has been suggested to contribute to CD pathogenesis. We aimed to study the effects of gliadin and various intestinal bacterial strains on mucosal barrier integrity, gliadin translocation, and cytokine production. METHODOLOGY/PRINCIPAL FINDINGS: Changes in gut mucosa were assessed in the intestinal loops of inbred Wistar-AVN rats that were reared under germ-free conditions in the presence of various intestinal bacteria (enterobacteria and bifidobacteria isolated from CD patients and healthy children, respectively) and CD-triggering agents (gliadin and IFN-gamma) by histology, scanning electron microscopy, immunofluorescence, and a rat cytokine antibody array. Adhesion of the bacterial strains to the IEC-6 rat cell line was evaluated in vitro. Gliadin fragments alone or together with the proinflammatory cytokine interferon (IFN)-gamma significantly decreased the number of goblet cells in the small intestine; this effect was more pronounced in the presence of Escherichia coli CBL2 and Shigella CBD8. Shigella CBD8 and IFN-gamma induced the highest mucin secretion and greatest impairment in tight junctions and, consequently, translocation of gliadin fragments into the lamina propria. Shigella CBD8 and E. coli CBL2 strongly adhered to IEC-6 epithelial cells. The number of goblet cells in small intestine increased by the simultaneous incubation of Bifidobacterium bifidum IATA-ES2 with gliadin, IFN-gamma and enterobacteria. B. bifidum IATA-ES2 also enhanced the production of chemotactic factors and inhibitors of metalloproteinases, which can contribute to gut mucosal protection. CONCLUSIONS: Our results suggest that the composition of the intestinal microbiota affects the permeability of the intestinal mucosa and, consequently, could be involved in the early stages of CD pathogenesis.

• Hrdlickova B, Westra HJ, Franke L, Wijmenga C
• Celiac disease: moving from genetic associations to causal variants.
• Clin Genet. 2011; 80: 203-313
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• Genome-wide association studies are providing insight into the genetic basis of common complex diseases: more than 1150 genetic loci [2165 unique single nucleotide polymorphisms (SNPs)] have recently been associated to 159 complex diseases. The hunt for genes contributing to immune-related diseases has been particularly successful in celiac disease, for example, with 27 genome-wide significantly associated loci identified so far. One of the current challenges is how to move from a genetic association with a disease to finding disease-associated genes and causal variants, as a step towards understanding the underlying disease process. About 50% of disease-associated SNPs affect the expression of nearby genes (so-called expression quantitative traits loci or eQTLs) and these can provide clues for finding causal variants. Although eQTLs can be useful, fine mapping and sequencing are required to refine the association signal. Ultimately, sophisticated study designs will be needed to find the causal variants involved in complex diseases. In this review, we use celiac disease as an example to describe the different aspects that need to be considered on the path from genetic association to disease-causing variants.

• Freeman HJ, Chopra A, Clandinin MT, Thomson AB
• Recent advances in celiac disease.
• World J Gastroenterol. 2011; 17: 2259-72
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• Celiac disease now affects about one person in a hundred in Europe and North America. In this review, we consider a number of important and exciting recent developments, such as clinical associations, HLA-DQ2 and HLA-DQ8 predispositions, the concept of potential celiac disease, the use of new imaging/endoscopy techniques, and the development of refractory disease. This review will be of use to all internists, pediatricians and gastroenterologists.

• de Heer AM et al.
• Progressive sensorineural hearing loss and normal vestibular function in a Dutch DFNB7/11 family with a novel mutation in TMC1.
• Audiol Neurootol. 2011; 16: 93-105
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• In a Dutch family with autosomal recessive hearing loss, genome-wide single-nucleotide polymorphism analysis mapped the genetic defect to the DFNB7/11 locus. A novel homozygous A-to-G change in the TMC1 gene was detected near the splice donor site of intron 19 (c.1763+3A-->G) segregating with the hearing loss in this family. One of the 6 transmembrane domains and the actual TMC channel domain are predicted to be absent in the mutant protein. The sensorineural hearing impairment in this DFNB7/11 family has a postlingual onset. Audiometric analysis initially showed a steeply downward-sloping threshold configuration. The progressive phenotype in this family resembles the phenotype previously described for families with dominant TMC1 mutations (DFNA36) rather than that of families with recessive TMC1 mutations (DFNB7/11) which invariably cause severe-to-profound prelingual hearing impairment.

• Rodriguez-Rodriguez L et al.
• Role of the rs6822844 gene polymorphism at the IL2-IL21 region in biopsy-proven giant cell arteritis.
• Clin Exp Rheumatol. 2011; 29: 126-126
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• OBJECTIVES: To assess the influence of the interleukin (IL)2-IL21 rs6822844 G/T polymorphism in the susceptibility to biopsy-proven giant cell arteritis (GCA) and in the clinical spectrum of manifestations of this vasculitis. METHODS: Two hundred and seventy-two biopsy-proven GCA patients were included in this study. DNA from patients and matched controls (n=791) was obtained from peripheral blood. Samples were genotyped for the rs6822844 polymorphism using a predesigned TaqMan allele discrimination assay and by polymerase chain reaction amplification. RESULTS: No significant differences in the allele and genotype frequencies between biopsy-proven GCA patients and controls were observed. However, the stratification of GCA patients disclosed some differences according to gender and ischemic manifestations of the disease. In this regard, the frequency of the minor allele T was increased in males (14.8%) compared to females (8.4%) (odds ratio-OR:1.89 (95% confidence interval-CI: 1.09-3.28); p=0.02; Bonferroni adjustment p=0.12). Also, minor allele T frequency was increased in GCA patients with severe ischemic complications (12.8%) compared to those without severe ischemic complications (7.7%) (OR:1.72 (95% CI: 0.97-3.05); p=0.05; Bonferroni adjustment p=0.30), and specifically in patients with jaw claudication (13.7% versus 8.2% in those without jaw claudication; OR:1.76 (95% CI: 1.02-3.04); p=0.04; Bonferroni adjustment p=0.24). CONCLUSIONS: IL2-IL21 rs6822844 polymorphism does not appear to be a genetic risk factor for susceptibility to biopsy-proven GCA. However, this gene polymorphism may contribute to the different phenotypic expression of this vasculitis, in particular in the development of ischemic complications of the disease.

• Totong R et al.
• The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis.
• Development. 2011; 138: 4199-205
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• Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis.

• Nie D, Liu Y, Xiang Y
• Overexpression a novel zebra fish spermatogenesis-associated gene 17 (SPATA17) induces apoptosis in GC-1 cells.
• Mol Biol Rep. 2011; 38: 3945-52
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• The spermatogenesis-associated 17 gene (SPATA17, previously named MSRG-11) was reported to be a candidate spermatocyte apoptosis-related gene which may play a critical role in human spermatogenesis, especially in meiosis. Analysis of SPATA17 expression and regulation in zebra fish may provide insight into the understanding of the complicated process of gonadogenesis and its potential function in spermatocyte cell apoptosis. In this study, we cloned and characterized the SPATA17 gene from zebra fish which consists of nine exons separated by eight introns. The consensus open reading frame (1258 bp) encodes a polypeptide of 357 amino acids which shares 44% identity with the human SPATA17 gene. Bioinformatic analysis reveals that SPATA17 protein contains three short calmodulin-binding motifs (IQ motif) and is considered to play a critical role in interactions with CaM proteins. Multi-tissue RT-PCR and Northern blot results demonstrated that the zebra fish SPATA17 gene was expressed strongly in testis and a slight amount of expression in ovary. Flow cytometry analysis and genomic DNA ladders result showed that the expression of SPATA17 protein in the GC-1 cell line could accelerate cell apoptosis. Analysis of the SPATA17 sequence and its spatial expression pattern indicate that this gene is highly conserved and may play an important role in the process of zebra fish gonadogenesis.

• Khor CC et al.
• Genome-wide association study identifies FCGR2A as a susceptibility locus for Kawasaki disease.
• Nat Genet. 2011; 43: 1241-6
• Display abstract

• Kawasaki disease is a systemic vasculitis of unknown etiology, with clinical observations suggesting a substantial genetic contribution to disease susceptibility. We conducted a genome-wide association study and replication analysis in 2,173 individuals with Kawasaki disease and 9,383 controls from five independent sample collections. Two loci exceeded the formal threshold for genome-wide significance. The first locus is a functional polymorphism in the IgG receptor gene FCGR2A (encoding an H131R substitution) (rs1801274; P = 7.35 x 10(-11), odds ratio (OR) = 1.32), with the A allele (coding for histadine) conferring elevated disease risk. The second locus is at 19q13, (P = 2.51 x 10(-9), OR = 1.42 for the rs2233152 SNP near MIA and RAB4B; P = 1.68 x 10(-12), OR = 1.52 for rs28493229 in ITPKC), which confirms previous findings(1). The involvement of the FCGR2A locus may have implications for understanding immune activation in Kawasaki disease pathogenesis and the mechanism of response to intravenous immunoglobulin, the only proven therapy for this disease.

• Shi J et al.
• Haplotype-based analysis of ulcerative colitis risk loci identifies both IL2 and IL21 as susceptibility genes in Han Chinese.
• Inflamm Bowel Dis. 2011; 17: 2472-9
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• BACKGROUND: The incidence of ulcerative colitis (UC) varies between Western and Eastern ethnicities. A distinct genetic background may play a role in the differences. Until now, very little was known of the UC genetics in Asian populations. Here we performed a haplotype-based analysis of six known UC susceptibility loci in Han Chinese patients. METHODS: In all, 245 UC patients and 300 healthy controls of Han Chinese descent were genotyped for 27 single nucleotide polymorphisms (SNPs), which cover the major haplotypes of the chromosome regions containing IL10, IL2/IL21, MYO9B, ECM1, MST1, and IL23R in Han Chinese. RESULTS: In contrast to the tight linkage disequilibrium (LD) block of the IL2/IL21 region in Caucasians, IL2 and IL21 reside in two independent LD blocks in Han Chinese. The IL2 SNP rs2069762 (P = 7.0 x 10(-4) , odds ratio [OR] = 1.54, 95% confidence interval [CI] 1.20-1.99) and the IL21 SNP rs2055979 (P = 1.2 x 10(-4) , OR = 1.50, 95% CI 1.17-1.92) were independently associated with UC. We identified one risk haplotype in IL2 and another independent risk haplotype in IL21. In addition to the IL2/IL21 locus, we observed association of the TT genotype of SNP rs1545620 in MYO9B with UC (P = 0.0169; OR = 0.29, 95% CI 0.11-0.78) and association of rs17375018 in IL23R with pancolitis in Chinese UC patients (P = 0.002; OR = 2.38, 95% CI 1.41-4.02). CONCLUSIONS: Our study confirmed the association of the IL2/IL21 region with UC in Han Chinese patients, and further implied both IL2 and IL21 as genetic risk factors for UC. Han Chinese UC patients share part of their genetic susceptibility with Caucasian patients.

• Manji SS et al.
• An ENU-induced mutation of Cdh23 causes congenital hearing loss, but no vestibular dysfunction, in mice.
• Am J Pathol. 2011; 179: 903-14
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• Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.

• Chladkova B, Kamanova J, Palova-Jelinkova L, Cinova J, Sebo P, Tuckova L
• Gliadin fragments promote migration of dendritic cells.
• J Cell Mol Med. 2011; 15: 938-48
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• In genetically predisposed individuals, ingestion of wheat gliadin provokes a T-cell-mediated enteropathy, celiac disease. Gliadin fragments were previously reported to induce phenotypic maturation and Th1 cytokine production by human dendritic cells (DCs) and to boost their capacity to stimulate allogeneic T cells. Here, we monitor the effects of gliadin on migratory capacities of DCs. Using transwell assays, we show that gliadin peptic digest stimulates migration of human DCs and their chemotactic responsiveness to the lymph node-homing chemokines CCL19 and CCL21. The gliadin-induced migration is accompanied by extensive alterations of the cytoskeletal organization, with dissolution of adhesion structures, podosomes, as well as up-regulation of the CC chemokine receptor (CCR) 7 on cell surface and induction of cyclooxygenase (COX)-2 enzyme that mediates prostaglandin E2 (PGE(2)) production. Blocking experiments confirmed that gliadin-induced migration is independent of the TLR4 signalling. Moreover, we showed that the alpha-gliadin-derived 31-43 peptide is an active migration-inducing component of the digest. The migration promoted by gliadin fragments or the 31-43 peptide required activation of p38 mitogen-activated protein kinase (MAPK). As revealed using p38 MAPK inhibitor SB203580, this was responsible for DC cytoskeletal transition, CCR7 up-regulation and PGE(2) production in particular. Taken together, this study provides a new insight into pathogenic features of gliadin fragments by demonstrating their ability to promote DC migration, which is a prerequisite for efficient priming of naive T cells, contributing to celiac disease pathology.

• Einarsdottir E et al.
• Genome-wide analysis of extended pedigrees confirms IL2-IL21 linkage and shows additional regions of interest potentially influencing coeliac disease risk.
• Tissue Antigens. 2011; 78: 428-37
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• Coeliac disease is a chronic inflammatory condition of the small intestine, triggered by dietary exposure to gluten in genetically susceptible individuals. Risk alleles at HLA-DQA1 and HLA-DQB1 are necessary for disease development, but are alone not sufficient for disease onset. We aimed to identify novel loci underlying susceptibility to coeliac disease through the use of extended Finnish and Hungarian families with multiple affected individuals. An initial whole-genome linkage approach yielded several loci that were followed up further using the Immunochip custom array. Loci with a parametric logarithm of odds (LOD) score of >1.3 were identified at 4q, 6p [human leukocyte antigen (HLA) region], 6q, 7p, 17p, 17q and at 22p. The 4q and 6q loci have been identified previously in coeliac disease risk, whereas follow-up analyses indicate that the 17p and 22p loci may be novel risk loci for coeliac disease. These loci harbour previously described risk variants for other autoimmune diseases, but their segregation patterns do not explain the linkage to coeliac disease. We followed up the linkage to the 4q region, containing the previously described interleukin (IL)2 and IL21 genes. The risk variants at 4q in the studied pedigrees are most likely distinct from previously described risk variants, indicating that the observed linkage may be due to rare high-risk variants of still unknown nature. The importance of this locus to coeliac disease risk was further shown by the finding that serum levels of IL21 were elevated in both untreated and treated coeliac patients compared to controls.

• Fehrmann RS et al.
• Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA.
• PLoS Genet. 2011; 7: 1002197-1002197
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• For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

• Capuano M et al.
• MicroRNA-449a overexpression, reduced NOTCH1 signals and scarce goblet cells characterize the small intestine of celiac patients.
• PLoS One. 2011; 6: 29094-29094
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• MiRNAs play a relevant role in regulating gene expression in a variety of physiological and pathological conditions including autoimmune disorders. MiRNAs are also important in the differentiation and function of the mouse intestinal epithelium. Our study was aimed to look for miRNA-based modulation of gene expression in celiac small intestine, and particularly for genes involved in cell intestinal differentiation/proliferation mechanisms. A cohort of 40 children (20 with active CD, 9 on a gluten-free diet (GFD), and 11 controls), were recruited at the Paediatrics Department (University of Naples Federico II). The expression of 365 human miRNAs was quantified by TaqMan low-density arrays. We used bioinformatics to predict putative target genes of miRNAs and to select biological pathways. The presence of NOTCH1, HES1, KLF4, MUC-2, Ki67 and beta-catenin proteins in the small intestine of CD and control children was tested by immunohistochemistry. The expression of about 20% of the miRNAs tested differed between CD and control children. We found that high miR-449a levels targeted and reduced both NOTCH1 and KLF4 in HEK-293 cells. NOTCH1, KLF4 signals and the number of goblet cells were lower in small intestine of children with active CD and in those on a GFD than in controls, whereas more nuclear beta-catenin staining, as a sign of the WNT pathway activation, and more Ki67 staining, as sign of proliferation, were present in crypts from CD patients than in controls. In conclusion we first demonstrate a miRNA mediated gene regulation in small intestine of CD patients. We also highlighted a reduced NOTCH1 pathway in our patients, irrespective of whether the disease was active or not. We suggest that NOTCH pathway could be constitutively altered in the celiac small intestine and could drive the increased proliferation and the decreased differentiation of intestinal cells towards the secretory goblet cell lineage.

• Binia A et al.
• Chromosome 17q21 SNP and severe asthma.
• J Hum Genet. 2011; 56: 97-8

• Plaza-Izurieta L, Castellanos-Rubio A, Irastorza I, Fernandez-Jimenez N, Gutierrez G, Bilbao JR
• Revisiting genome wide association studies (GWAS) in coeliac disease: replication study in Spanish population and expression analysis of candidate genes.
• J Med Genet. 2011; 48: 493-6
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• INTRODUCTION: Recent genome wide association studies (GWAS) on coeliac disease (CD) have identified risk loci harbouring genes that fit the accepted pathogenic model and are considered aetiological candidates. METHODS: Using Taqman single nucleotide polymorphism (SNP) and expression assays, the study genotyped 11 SNPs tagging eight GWAS regions (1q31, 2q11-2q12, 3p21, 3q25-3q26, 3q28, 4q27, 6q25 and 12q24) in a Spanish cohort of 1094 CD patients and 540 controls, and performed expression analyses of candidate genes (RGS1, IL18R1/IL18RAP, CCR3, IL12A/SCHIP1, LPP, IL2/IL21-KIAA1109, TAGAP, and SH2B3) in intestinal mucosa from 29 CD children and eight controls. RESULTS: Polymorphisms in 1q31, 2q11-2q12, and 3q25 showed association in our cohort, and also 3q28 and 4q27 when combined with a previous study. Expression levels of IL12A, IL18RAP, IL21, KIAA1109, LPP, SCHIP1, and SH2B3 were affected by disease status, but the correlation between genotype and mRNA levels was observed only in IL12A, LPP, SCHIP1, and SH2B3. CONCLUSIONS: Expression differences between treated CD patients and controls along with SNP expression associations suggest a possible primary role for these four genes and their variants in pathogenesis. The lack of SNP effect in the remaining genes is probably a consequence of arbitrary candidate gene selection within association signals that are not based on functional studies.

• Stallhofer J et al.
• Analysis of IL2/IL21 gene variants in cholestatic liver diseases reveals an association with primary sclerosing cholangitis.
• Digestion. 2011; 84: 29-35
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• BACKGROUND/AIMS: The chromosome 4q27 region harboring IL2 and IL21 is an established risk locus for ulcerative colitis (UC) and various other autoimmune diseases. Considering the strong coincidence of primary sclerosing cholangitis (PSC) with UC and the increased frequency of other autoimmune disorders in patients with primary biliary cirrhosis (PBC), we investigated whether genetic variation in the IL2/IL21 region may also modulate the susceptibility to these two rare cholestatic liver diseases. METHODS: Four strongly UC-associated single nucleotide polymorphisms (SNPs) within the KIAA1109/TENR/IL2/IL21 linkage disequilibrium block were genotyped in 124 PBC and 41 PSC patients. Control allele frequencies from 1,487 healthy, unrelated Caucasians were available from a previous UC association study. RESULTS: The minor alleles of all four markers were associated with a decreased susceptibility to PSC (rs13151961: p = 0.013, odds ratio (OR) 0.34; rs13119723: p = 0.023, OR 0.40; rs6822844: p = 0.031, OR 0.41; rs6840978: p = 0.043, OR 0.46). Moreover, a haplotype consisting of the four minor alleles also had a protective effect on PSC susceptibility (p = 0.0084, OR 0.28). A haplotype of the four major alleles was independently associated with PSC when excluding the patients with concomitant inflammatory bowel disease (p = 0.033, OR 4.18). CONCLUSION: The IL2/IL21 region may be one of the highly suggestive but so far rarely identified shared susceptibility loci for PSC and UC.

• Shugart YY, Wang Y, Jia WH, Zeng YX
• GWAS signals across the HLA regions: revealing a clue for common etiology underlying infectious tumors and other immunity diseases.
• Chin J Cancer. 2011; 30: 226-30
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• Increasing evidence suggests that multiple genes in the human leukocyte antigen(HLA) regions play an important role in development of cancers and immunity disorders. However, the biological mechanisms of the HLA associations are not well understood. We recently conducted a survey of all genome-wide association studies (GWAS) with significant findings in the HLA regions and concluded that diseases such as cancer and immune disorders are more likely to be associated with genetic variants located in the HLA regions than other diseases. This finding is suggestive for testing a hypothesis of a common etiology of infectious tumors and other immunity diseases.

• Sherrill JD, Rothenberg ME
• Genetic dissection of eosinophilic esophagitis provides insight into disease pathogenesis and treatment strategies.
• J Allergy Clin Immunol. 2011; 128: 23-32
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• Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus that is compounded by both genetic predisposition and aberrant responses to environmental antigens, particularly those that are food derived. Data have indicated a unique transcriptional response in vivo that defines EoE and that appears to be partially attributable to the T(H)2 cytokine IL-13. Moreover, a number of genetic risk variants in proinflammatory and epithelial cell genes associate with EoE susceptibility, demonstrating novel heritable mechanisms that contribute to disease risk. Here we discuss recent advances in our understanding of the intrinsic (genetic) and extrinsic (environmental) components that illustrate the complex nature of EoE.

• Arakawa S et al.
• Genome-wide association study identifies two susceptibility loci for exudative age-related macular degeneration in the Japanese population.
• Nat Genet. 2011; 43: 1001-4
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• Age-related macular degeneration (AMD), the leading cause of irreversible blindness in the world, is a complex disease caused by multiple environmental and genetic risk factors. To identify genetic factors that modify the risk of exudative AMD in the Japanese population, we conducted a genome-wide association study and a replication study using a total of 1,536 individuals with exudative AMD and 18,894 controls. In addition to CFH (rs800292, P = 4.23 x 10(-15)) and ARMS2 (rs3750847, P = 8.67 x 10(-29)) loci, we identified two new susceptibility loci for exudative AMD: TNFRSF10A-LOC389641 on chromosome 8p21 (rs13278062, combined P = 1.03 x 10(-12), odds ratio = 0.73) and REST-C4orf14-POLR2B-IGFBP7 on chromosome 4q12 (rs1713985, combined P = 2.34 x 10(-8), odds ratio = 1.30). Fine mapping revealed that rs13278062, which is known to alter TNFRSF10A transcriptional activity, had the most significant association in 8p21 region. Our results provide new insights into the pathophysiology of exudative AMD.

• Ramakrishna BS
• Celiac disease: can we avert the impending epidemic in India?
• Indian J Med Res. 2011; 133: 5-8

• Warren RB et al.
• A systematic investigation of confirmed autoimmune loci in early-onset psoriasis reveals an association with IL2/IL21.
• Br J Dermatol. 2011; 164: 660-4
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• BACKGROUND: Many autoimmune diseases share common susceptibility loci suggesting similar underlying cellular mechanisms involved in disease expression. OBJECTIVES: The purpose of this investigation was to study 21 genetic variants in 14 genes that are confirmed autoimmune loci in a cohort of patients with early-onset psoriasis. METHODS: Patients with early-onset psoriasis (n = 750) and controls (n = 3531) were genotyped using the Sequenom((R)) MassArray iPLEX Gold platform. RESULTS: We found strong evidence of association with two variants in the IL2/IL21 (rs6822844, genotypic P = 3.3 x 10(-4) ; rs2069778, genotypic P = 7.86 x 10(-4)) region. CONCLUSIONS: The findings, although requiring replication, suggest that IL2/IL21 may play a key role in the pathogenesis of psoriasis as well as in other diverse autoimmune diseases.

• Linden M et al.
• No evidence of IL21 association with multiple sclerosis in a Swedish population.
• Tissue Antigens. 2011; 78: 271-4
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• Multiple sclerosis (MS) patients, with a second autoimmune disease after lymphocyte depletion, had elevated serum IL-21 before and after treatment which correlated to IL21 genotypes. In addition, the IL21 gene has been associated to several other autoimmune diseases. However, in a Spanish population there was no association to MS. Here, in a Swedish cohort (2090 MS cases and 1732 controls) 12 single nucleotide polymorphisms (SNPs) tagging IL21 were not associated to disease. There was no interaction with risk alleles of IL21R and HLA-DRB1*15. Lack of genetic association was confirmed in a meta-analysis with pooled data from the present study and the Spanish study. In conclusion, IL21 has not been shown to be a major risk gene for MS.

• Silano M, Agostoni C, Guandalini S
• Effect of the timing of gluten introduction on the development of celiac disease.
• World J Gastroenterol. 2010; 16: 1939-42
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• Celiac disease (CD) is a permanent auto-immune enteropathy, triggered in genetically predisposed individuals by the ingestion of dietary gluten. Gluten is the alcohol-soluble protein component of the cereals wheat, rye and barley. CD is a multifactorial condition, originating from the interplay of genetic and environmental factors. The necessary environmental trigger is gluten, while the genetic predisposition has been identified in the major histocompatibility complex region on chromosome 6p21, with over 90% of CD patients expressing HLA DQ2 and the remaining celiac patients express DQ8. The fact that only about 4% of DQ2/8-positive individuals exposed to gluten develop CD, has led to the recognition that other genetic and environmental factors are also necessary. In the last few years, several epidemiological studies have suggested that the timing of the introduction of gluten, as well as the pattern of breastfeeding, may play an important role in the subsequent development of CD. Here, we present and review the most recent evidences regarding the effect of timing of gluten introduction during weaning, the amount of gluten introduced and simultaneous breastfeeding, on the development of CD.

• Thye T et al.
• Genome-wide association analyses identifies a susceptibility locus for tuberculosis on chromosome 18q11.2.
• Nat Genet. 2010; 42: 739-41
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• We combined two tuberculosis genome-wide association studies from Ghana and The Gambia with subsequent replication in a combined 11,425 individuals. rs4331426, located in a gene-poor region on chromosome 18q11.2, was associated with disease (combined P = 6.8 x 10(-9), odds ratio = 1.19, 95% CI = 1.13-1.27). Our study demonstrates that genome-wide association studies can identify new susceptibility loci for infectious diseases, even in African populations, in which levels of linkage disequilibrium are particularly low.

• Hinks A et al.
• Association of the AFF3 gene and IL2/IL21 gene region with juvenile idiopathic arthritis.
• Genes Immun. 2010; 11: 194-8
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• Recent genetic studies have led to identification of numerous loci that are associated with susceptibility to autoimmune diseases. The strategy of using information from these studies has facilitated the identification of novel juvenile idiopathic arthritis (JIA) susceptibility loci, specifically, PTPN22 and IL2RA. Several novel autoimmune susceptibility loci have recently been identified, and we hypothesise that single-nucleotide polymorphisms (SNPs) within these genes may also be JIA susceptibility loci. Five SNPs within the genes AFF3, IL2/IL21, IL7R, CTLA4 and CD226, previously associated with multiple autoimmune diseases were genotyped, in a large data set of Caucasian JIA patients and controls, and tested for association with JIA. We identified two susceptibility loci for JIA, AFF3 and the IL2/IL21 region and additional weak evidence supporting an association with the CTLA4 and IL7R genes, which warrant further investigation. All results require validation in independent JIA data sets. Further characterisation of the specific causal variants will be required before functional studies can be performed.

• Trynka G, Wijmenga C, van Heel DA
• A genetic perspective on coeliac disease.
• Trends Mol Med. 2010; 16: 537-50
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• Coeliac disease is an inflammatory disorder of the small intestine with an autoimmune component and strong heritability. Genetic studies have confirmed strong association to HLA and identified 39 nonHLA risk genes, mostly immune-related. Over 50% of the disease-associated single nucleotide polymorphisms are correlated with gene expression. Most of the coeliac disease-associated regions are shared with other immune-related diseases, as well as with metabolic, haematological or neurological traits, or cancer. We review recent progress in the genetics of coeliac disease and describe the pathways these genes are in, the functional consequences of the associated markers on gene expression and the genes shared between coeliac disease and other traits.

• Tindall EA et al.
• The 4q27 locus and prostate cancer risk.
• BMC Cancer. 2010; 10: 69-69
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• BACKGROUND: Chronic inflammation is considered to be implicated in the development of prostate cancer. In this study we are the first to investigate a potential association between variants in an autoimmune related region on chromosome 4q27 and prostate cancer risk. This region harbors two cytokine genes IL-2 and the recently described IL-21. METHODS: We genotyped six variants previously associated with autoimmune disease (namely rs13151961, rs13119723, rs17388568, rs3136534, rs6822844 and rs6840978) and one functional IL-2 promoter variant (rs2069762) for possible association with prostate cancer risk using the Australian Risk Factors for Prostate Cancer case-control Study. RESULTS: Overall, our results do not support an association between the seven variants at position 4q27 and prostate cancer risk. Per allele odds ratios (ORs) were not significantly different from 1 (all P-values = 0.06). However, we found suggestive evidence for a significant association between the presence of the rs13119723 variant (located in a protein of unknown function) and men with a family history of prostate cancer in first-degree relatives (P-value for interaction 0.02). The per allele OR associated with this variant was significantly higher than 1 (2.37; 95% C.I. = 1.01-5.57). CONCLUSIONS: We suggest that genetic variation within the chromosome 4q27 locus might be associated with prostate cancer susceptibility in men with a family history of the disease. Furthermore, our study alludes to a potential role of unknown protein KIAA1109 in conferring this risk.

• Hinks A et al.
• Overlap of disease susceptibility loci for rheumatoid arthritis and juvenile idiopathic arthritis.
• Ann Rheum Dis. 2010; 69: 1049-53
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• BACKGROUND: Genome-wide association studies (GWAS) have been extremely successful in the search for susceptibility risk factors for complex genetic autoimmune diseases. As more studies are published, evidence is emerging of considerable overlap of loci between these diseases. In juvenile idiopathic arthritis (JIA), another complex genetic autoimmune disease, the strategy of using information from autoimmune disease GWAS or candidate gene studies to help in the search for novel JIA susceptibility loci has been successful, with confirmed association with two genes, PTPN22 and IL2RA. Rheumatoid arthritis (RA) is an autoimmune disease that shares similar clinical and pathological features with JIA and, therefore, recently identified confirmed RA susceptibility loci are also excellent JIA candidate loci. OBJECTIVE: To determine the overlap of disease susceptibility loci for RA and JIA. METHODS: /st> Fifteen single nucleotide polymorphisms (SNPs) at nine RA-associated loci were genotyped in Caucasian patients with JIA (n=1054) and controls (n=3531) and tested for association with JIA. Allele and genotype frequencies were compared between cases and controls using the genetic analysis software, PLINK. RESULTS: Two JIA susceptibility loci were identified, one of which was a novel JIA association (STAT4) and the second confirmed previously published associations of the TRAF1/C5 locus with JIA. Weak evidence of association of JIA with three additional loci (Chr6q23, KIF5A and PRKCQ) was also obtained, which warrants further investigation. CONCLUSION: All these loci are good candidates in view of the known pathogenesis of JIA, as genes within these regions (TRAF1, STAT4, TNFAIP3, PRKCQ) are known to be involved in T-cell receptor signalling or activation pathways.

• Hollis-Moffatt JE et al.
• Only one independent genetic association with rheumatoid arthritis within the KIAA1109-TENR-IL2-IL21 locus in Caucasian sample sets: confirmation of association of rs6822844 with rheumatoid arthritis at a genome-wide level of significance.
• Arthritis Res Ther. 2010; 12: 116-116
• Display abstract

• INTRODUCTION: The single nucleotide polymorphism (SNP) rs6822844 within the KIAA1109-TENR-IL2-IL21 gene cluster has been associated with rheumatoid arthritis (RA). Other variants within this cluster, including rs17388568 that is not in linkage disequilibrium (LD) with rs6822844, and rs907715 that is in moderate LD with rs6822844 and rs17388568, have been associated with a number of autoimmune phenotypes, including type 1 diabetes (T1D). Here we aimed to: one, confirm at a genome-wide level of significance association of rs6822844 with RA and, two, evaluate whether or not there were effects independent of rs6822844 on RA at the KIAA1109-TENR-IL2-IL21 locus. METHODS: A total of 842 Australasian RA patients and 1,115 controls of European Caucasian ancestry were genotyped for rs6822844, rs17388568 and rs907715. Meta-analysis of these data with published and publicly-available data was conducted using STATA. RESULTS: No statistically significant evidence for association was observed in the Australasian sample set for rs6822844 (odds ratio (OR)=0.95 (0.80 to 1.12), P=0.54), or rs17388568 (OR=1.03 (0.90 to 1.19), P=0.65) or rs907715 (OR=0.98 (0.86 to 1.12), P=0.69). When combined in a meta-analysis using data from a total of 9,772 cases and 10,909 controls there was a genome-wide level of significance supporting association of rs6822844 with RA (OR=0.86 (0.82 to 0.91), P=8.8x10(-8), P=2.1x10(-8) including North American Rheumatoid Arthritis Consortium data). Meta-analysis of rs17388568, using a total of 6,585 cases and 7,528 controls, revealed no significant association with RA (OR=1.03, (0.98 to 1.09); P=0.22) and meta-analysis of rs907715 using a total of 2,689 cases and 4,045 controls revealed a trend towards association (OR=0.93 (0.87 to 1.00), P=0.07). However, this trend was not independent of the association at rs6822844. CONCLUSIONS: The KIAA1109-TENR-IL2-IL21 gene cluster, that encodes an interleukin (IL-21) that plays an important role in Th17 cell biology, is the 20th locus for which there is a genome-wide (P

• Flesher DL, Sun X, Behrens TW, Graham RR, Criswell LA
• Recent advances in the genetics of systemic lupus erythematosus.
• Expert Rev Clin Immunol. 2010; 6: 461-79
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• Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of antinuclear autoantibodies and the inflammatory infiltration of many organ systems. SLE is a complex disorder in which multiple genetic variants, together with environmental and hormonal factors, contribute to disease risk. In this article, we summarize our current understanding of the genetic contribution to SLE in light of recent genome-wide association studies, which have brought the total number of confirmed SLE susceptibility loci to 29. In the second section, we explore the functional implications of these risk loci and, in particular, highlight the role that many of these genes play in the Toll-like receptor and type I interferon signaling pathways. Finally, we discuss the genetic overlap between SLE and other autoimmune and inflammatory conditions as several risk loci are shared among multiple disorders, suggesting common underlying pathogenic mechanisms.

• Zhou H, Liu ZG, Sun ZW, Huang Y, Yu WY
• Generation of stable cell lines by site-specific integration of transgenes into engineered Chinese hamster ovary strains using an FLP-FRT system.
• J Biotechnol. 2010; 147: 122-9
• Display abstract

• Random integration linking genomic amplification is widely used to generate desired cell lines for stable and high-level expressing recombinant proteins. But this technique is laborious, and the expression level is unpredictable due to position effects. After reading many reports on gene amplification, we hypothesized that there should be several loci in the genome of Chinese hamster ovary (CHO) cells that allow not only high-level, but also stable gene expression. Based on this hypothesis, we constructed a plasmid pMCEscan, which introduces a site-specific recombinase-recognition sequence, FRT, for gene targeting into those sites. Another targeting vector, pcDNA5/FRT, has an FRT sequence fused to a promoterless hygromycin-resistance gene that can be expressed only when correct gene targeting occurs. Using the pMCEscan, which is a novel and stringent selection system used to create a few high protein-producing clones, we constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene targeting. We selected 28 CHO strains that expressed a high-level of reporter genes and carried one copy of the pMCEscan in their chromosomes, and we treated these strains with methotrexate (MTX) to evaluate dihydrofolate reductase (DHFR)-mediated gene amplification. Nine clones showed high-level tissue plasminogen activator (tPA) production without amplification. We then targeted other genes (tPA, secreted alkaline phosphatase (SEAP), erythropoietin (EPO)) to test the basal expression ability of nine CHO strains. CHO strains 8-1 and 8-11 consistently expressed high basal levels of the recombinant genes. Using this cell-vector system, we obtained the tPA stable high producers by gene targeting and gene amplification. This system allows for rapid generation of recombinant proteins without cloning and greatly simplifies selection of cell lines for the production of potential therapeutic proteins.

• Simula MP et al.
• PPAR signaling pathway and cancer-related proteins are involved in celiac disease-associated tissue damage.
• Mol Med. 2010; 16: 199-209
• Display abstract

• Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of wheat gliadin and related proteins in genetically predisposed individuals. To find a proteomic CD diagnostic signature and to gain a better understanding of pathogenetic mechanisms associated with CD, we analyzed the intestinal mucosa proteome alterations using two dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF ms) of CD patients with varying degrees of histological abnormalities defined by Marsh criteria and controls. Our results clearly evidenced the presence of two groups of patients: Group A, including controls and Marsh 0-I CD patients; and Group B, consisting of CD subjects with grade II-III Oberhuber-Marsh classification. Differentially expressed proteins were involved mainly in lipid, protein and sugar metabolism. Interestingly, in Group B, several downregulated proteins (FABP1, FABP2, APOC3, HMGCS2, ACADM and PEPCK) were implicated directly in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Moreover, Group B patients presented a deregulation of some proteins involved in apoptosis/survival pathways: phosphatidylethanolamine-binding protein 1 (PEBP1), Ras-related nuclear protein (Ran) and peroxiredoxin 4 (PRDX4). PEBP1 downregulation and RAN and PRDX4 upregulation were associated with more severe tissue damage. Likewise, IgMs were found strongly upregulated in Group B. In conclusion, our results indicate that a downregulation of proteins involved in PPAR signaling and the modulation of several cancer-related proteins are associated with the highest CD histological score according to Oberhuber-Marsh classification.

• Cantor RM, Lange K, Sinsheimer JS
• Prioritizing GWAS results: A review of statistical methods and recommendations for their application.
• Am J Hum Genet. 2010; 86: 6-22
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• Genome-wide association studies (GWAS) have rapidly become a standard method for disease gene discovery. A substantial number of recent GWAS indicate that for most disorders, only a few common variants are implicated and the associated SNPs explain only a small fraction of the genetic risk. This review is written from the viewpoint that findings from the GWAS provide preliminary genetic information that is available for additional analysis by statistical procedures that accumulate evidence, and that these secondary analyses are very likely to provide valuable information that will help prioritize the strongest constellations of results. We review and discuss three analytic methods to combine preliminary GWAS statistics to identify genes, alleles, and pathways for deeper investigations. Meta-analysis seeks to pool information from multiple GWAS to increase the chances of finding true positives among the false positives and provides a way to combine associations across GWAS, even when the original data are unavailable. Testing for epistasis within a single GWAS study can identify the stronger results that are revealed when genes interact. Pathway analysis of GWAS results is used to prioritize genes and pathways within a biological context. Following a GWAS, association results can be assigned to pathways and tested in aggregate with computational tools and pathway databases. Reviews of published methods with recommendations for their application are provided within the framework for each approach.

• Cavanillas ML et al.
• Polymorphisms in the IL2, IL2RA and IL2RB genes in multiple sclerosis risk.
• Eur J Hum Genet. 2010; 18: 794-9
• Display abstract

• Interleukin (IL)-2/IL-2R signalling promotes proliferation and survival of activated T cells and has an essential non-redundant role in the production of regulatory T cells. Associations with different autoimmune diseases of polymorphisms in a linkage disequilibrium block in which the IL2/IL21 genes map (4q27), and also in genes encoding the IL2RA and IL2RB subunits (located in 10p15 and 22q13, respectively), were identified through genome-wide studies. Polymorphisms in these three genes were studied in 430 multiple sclerosis (MS) patients and in 550 ethnically matched controls from Madrid (Spain). Replication and meta-analysis with results from an independent cohort of 771 MS patients and 759 controls from Andalucia (Spain) confirmed the association of polymorphisms in the IL2RA gene (P(Mantel-Haenszel,) odds ratio (OR)(M-H) (95% confidence interval, CI) for rs2104286: 0.0001, 0.75 (0.65-0.87); for rs11594656/rs35285258: 0.004, 1.19 (1.06-1.34); for rs41295061: 0.03, 0.77 (0.60-0.98)); showed a trend for association of the IL2/IL21 rs6822844 (P(M-H)=0.07, OR(M-H) (95% CI)=0.86 (0.73-1.01)), but did not corroborate the association for IL2RB. Regression analyses of the combined Spanish cohort revealed the independence of two IL2RA association signals: rs2104286 and rs11594656/rs35285258. The relevant role of the IL2RA gene on MS susceptibility adds support to its common effect on autoimmune risk and the suggestive association of IL2/IL21 warrants further investigation.

• Kiryluk K et al.
• Genetic studies of IgA nephropathy: past, present, and future.
• Pediatr Nephrol. 2010; 25: 2257-68
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• Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide and an important cause of kidney disease in young adults. Highly variable clinical presentation and outcome of IgAN suggest that this diagnosis may encompass multiple subsets of disease that are not distinguishable by currently available clinical tools. Marked differences in disease prevalence between individuals of European, Asian, and African ancestry suggest the existence of susceptibility genes that are present at variable frequencies in these populations. Familial forms of IgAN have also been reported throughout the world but are probably underrecognized because associated urinary abnormalities are often intermittent in affected family members. Of the many pathogenic mechanisms reported, defects in IgA1 glycosylation that lead to formation of immune complexes have been consistently demonstrated. Recent data indicates that these IgA1 glycosylation defects are inherited and constitute a heritable risk factor for IgAN. Because of the complex genetic architecture of IgAN, the efforts to map disease susceptibility genes have been difficult, and no causative mutations have yet been identified. Linkage-based approaches have been hindered by disease heterogeneity and lack of a reliable noninvasive diagnostic test for screening family members at risk of IgAN. Many candidate-gene association studies have been published, but most suffer from small sample size and methodological problems, and none of the results have been convincingly validated. New genomic approaches, including genome-wide association studies currently under way, offer promising tools for elucidating the genetic basis of IgAN.

• Zhernakova A et al.
• Evolutionary and functional analysis of celiac risk loci reveals SH2B3 as a protective factor against bacterial infection.
• Am J Hum Genet. 2010; 86: 970-7
• Display abstract

• Celiac disease (CD) is an intolerance to dietary proteins of wheat, barley, and rye. CD may have substantial morbidity, yet it is quite common with a prevalence of 1%-2% in Western populations. It is not clear why the CD phenotype is so prevalent despite its negative effects on human health, especially because appropriate treatment in the form of a gluten-free diet has only been available since the 1950s, when dietary gluten was discovered to be the triggering factor. The high prevalence of CD might suggest that genes underlying this disease may have been favored by the process of natural selection. We assessed signatures of selection for ten confirmed CD-associated loci in several genome-wide data sets, comprising 8154 controls from four European populations and 195 individuals from a North African population, by studying haplotype lengths via the integrated haplotype score (iHS) method. Consistent signs of positive selection for CD-associated derived alleles were observed in three loci: IL12A, IL18RAP, and SH2B3. For the SH2B3 risk allele, we also show a difference in allele frequency distribution (Fst) between HapMap phase II populations. Functional investigation of the effect of the SH2B3 genotype in response to lipopolysaccharide and muramyl dipeptide revealed that carriers of the SH2B3 rs3184504*A risk allele showed stronger activation of the NOD2 recognition pathway. This suggests that SH2B3 plays a role in protection against bacteria infection, and it provides a possible explanation for the selective sweep on SH2B3, which occurred sometime between 1200 and 1700 years ago.

• Stahl EA et al.
• Genome-wide association study meta-analysis identifies seven new rheumatoid arthritis risk loci.
• Nat Genet. 2010; 42: 508-14
• Display abstract

• To identify new genetic risk factors for rheumatoid arthritis, we conducted a genome-wide association study meta-analysis of 5,539 autoantibody-positive individuals with rheumatoid arthritis (cases) and 20,169 controls of European descent, followed by replication in an independent set of 6,768 rheumatoid arthritis cases and 8,806 controls. Of 34 SNPs selected for replication, 7 new rheumatoid arthritis risk alleles were identified at genome-wide significance (P < 5 x 10(-8)) in an analysis of all 41,282 samples. The associated SNPs are near genes of known immune function, including IL6ST, SPRED2, RBPJ, CCR6, IRF5 and PXK. We also refined associations at two established rheumatoid arthritis risk loci (IL2RA and CCL21) and confirmed the association at AFF3. These new associations bring the total number of confirmed rheumatoid arthritis risk loci to 31 among individuals of European ancestry. An additional 11 SNPs replicated at P < 0.05, many of which are validated autoimmune risk alleles, suggesting that most represent genuine rheumatoid arthritis risk alleles.

• Tjon JM, van Bergen J, Koning F
• Celiac disease: how complicated can it get?
• Immunogenetics. 2010; 62: 641-51
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• In the small intestine of celiac disease patients, dietary wheat gluten and similar proteins in barley and rye trigger an inflammatory response. While strict adherence to a gluten-free diet induces full recovery in most patients, a small percentage of patients fail to recover. In a subset of these refractory celiac disease patients, an (aberrant) oligoclonal intraepithelial lymphocyte population develops into overt lymphoma. Celiac disease is strongly associated with HLA-DQ2 and/or HLA-DQ8, as both genotypes predispose for disease development. This association can be explained by the fact that gluten peptides can be presented in HLA-DQ2 and HLA-DQ8 molecules on antigen presenting cells. Gluten-specific CD4(+) T cells in the lamina propria respond to these peptides, and this likely enhances cytotoxicity of intraepithelial lymphocytes against the intestinal epithelium. We propose a threshold model for the development of celiac disease, in which the efficiency of gluten presentation to CD4(+) T cells determines the likelihood of developing celiac disease and its complications. Key factors that influence the efficiency of gluten presentation include: (1) the level of gluten intake, (2) the enzyme tissue transglutaminase 2 which modifies gluten into high affinity binding peptides for HLA-DQ2 and HLA-DQ8, (3) the HLA-DQ type, as HLA-DQ2 binds a wider range of gluten peptides than HLA-DQ8, (4) the gene dose of HLA-DQ2 and HLA-DQ8, and finally,(5) additional genetic polymorphisms that may influence T cell reactivity. This threshold model might also help to understand the development of refractory celiac disease and lymphoma.

• Chu X, Nishimura K, Jisaka M, Nagaya T, Shono F, Yokota K
• Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction.
• Prostaglandins Other Lipid Mediat. 2010; 91: 1-9
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• Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE(2) involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE(2) associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE(2) and PGF(2alpha), during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

• Park JY et al.
• Identification and analysis of specific chromosomal region adjacent to exogenous Dhfr-amplified region in Chinese hamster ovary cell genome.
• J Biosci Bioeng. 2010; 109: 504-11
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• Chinese hamster ovary (CHO) cells are widely used for the stable production of recombinant proteins. Gene amplification techniques are frequently used to improve of protein production, and the dihydrofolate reductase (DHFR) gene amplification system is most widely used in the CHO cell line. We previously constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line and one BAC clone (Cg0031N14) containing the CHO genomic DNA sequence adjacent to Dhfr was selected. To identify the specific chromosomal region adjacent to the exogenous Dhfr-amplified region in the CHO cell genome, we performed further screening of BAC clones to obtain other Dhfr-amplified regions in the CHO genome. From the screening by high-density replica filter hybridization using a digoxigenin-labeled pSV2-dhfr/hGM-CSF probe, we obtained 8 new BAC clones containing a Dhfr-amplified region. To define the structures of the 8 BAC clones, Southern blot analysis, BAC end sequencing and fluorescence in situ hybridization (FISH) were performed. These results revealed that all the selected BAC clones contained a large palindrome structure with a small inverted repeat in the junction region. This suggests that the obtained amplicon structure in the Dhfr-amplified region in the CHO genome plays an important role in exogenous gene amplification.

• Akhabir L, Sandford A
• Genetics of interleukin 1 receptor-like 1 in immune and inflammatory diseases.
• Curr Genomics. 2010; 11: 591-606
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• Interleukin 1 receptor-like 1 (IL1RL1) is gaining in recognition due to its involvement in immune/inflammatory disorders. Well-designed animal studies have shown its critical role in experimental allergic inflammation and human in vitro studies have consistently demonstrated its up-regulation in several conditions such as asthma and rheumatoid arthritis. The ligand for IL1RL1 is IL33 which emerged as playing an important role in initiating eosinophilic inflammation and activating other immune cells resulting in an allergic phenotype.An IL1RL1 single nucleotide polymorphism (SNP) was among the most significant results of a genome-wide scan investigating eosinophil counts; in the same study, this SNP associated with asthma in 10 populations.The IL1RL1 gene resides in a region of high linkage disequilibrium containing interleukin 1 receptor genes as well as interleukin 18 receptor and accessory genes. This poses a challenge to researchers interested in deciphering genetic association signals in the region as all of the genes represent interesting candidates for asthma and allergic disease.The IL1RL1 gene and its resulting soluble and receptor proteins have emerged as key regulators of the inflammatory process implicated in a large variety of human pathologies We review the function and expression of the IL1RL1 gene. We also describe the role of IL1RL1 in asthma, allergy, cardiovascular disease, infections, liver disease and kidney disease.

• Plenge RM
• Unlocking the pathogenesis of celiac disease.
• Nat Genet. 2010; 42: 281-2
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• A genome-wide association study reports more than a dozen new susceptibility loci for celiac disease. Analysis of eQTL data from these and previously established risk loci sheds light on the genetic pathways underlying this common autoimmune disease.

• Dubois PC et al.
• Multiple common variants for celiac disease influencing immune gene expression.
• Nat Genet. 2010; 42: 295-302
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• We performed a second-generation genome-wide association study of 4,533 individuals with celiac disease (cases) and 10,750 control subjects. We genotyped 113 selected SNPs with P(GWAS) < 10(-4) and 18 SNPs from 14 known loci in a further 4,918 cases and 5,684 controls. Variants from 13 new regions reached genome-wide significance (P(combined) < 5 x 10(-8)); most contain genes with immune functions (BACH2, CCR4, CD80, CIITA-SOCS1-CLEC16A, ICOSLG and ZMIZ1), with ETS1, RUNX3, THEMIS and TNFRSF14 having key roles in thymic T-cell selection. There was evidence to suggest associations for a further 13 regions. In an expression quantitative trait meta-analysis of 1,469 whole blood samples, 20 of 38 (52.6%) tested loci had celiac risk variants correlated (P < 0.0028, FDR 5%) with cis gene expression.

• Hong H et al.
• Assessing sources of inconsistencies in genotypes and their effects on genome-wide association studies with HapMap samples.
• Pharmacogenomics J. 2010; 10: 364-74
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• The discordance in results of independent genome-wide association studies (GWAS) indicates the potential for Type I and Type II errors. We assessed the repeatibility of current Affymetrix technologies that support GWAS. Reasonable reproducibility was observed for both raw intensity and the genotypes/copy number variants. We also assessed consistencies between different SNP arrays and between genotype calling algorithms. We observed that the inconsistency in genotypes was generally small at the specimen level. To further examine whether the differences from genotyping and genotype calling are possible sources of variation in GWAS results, an association analysis was applied to compare the associated SNPs. We observed that the inconsistency in genotypes not only propagated to the association analysis, but was amplified in the associated SNPs. Our studies show that inconsistencies between SNP arrays and between genotype calling algorithms are potential sources for the lack of reproducibility in GWAS results.

• Zenewicz LA, Abraham C, Flavell RA, Cho JH
• Unraveling the genetics of autoimmunity.
• Cell. 2010; 140: 791-7
• Display abstract

• The chronic autoimmune diseases include multiple complex genetic disorders. Recently, genome-wide association studies (GWAS) have identified a large number of major loci, with many associations shared between various autoimmune diseases. These associations highlight key roles for lymphocyte activation and prioritize specific cytokine pathways and mechanisms of host-microbe recognition. Despite success in identifying loci, comprehensive models of disease pathogenesis are currently lacking. Future efforts comparing association patterns between autoimmune diseases may be particularly illustrative. New genomic technologies applied to classic genetic studies involving twins, early onset cases, and phenotypic extremes may provide key insights into developmental and gene-environment interactions in autoimmunity.

• Amundsen SS et al.
• Four novel coeliac disease regions replicated in an association study of a Swedish-Norwegian family cohort.
• Genes Immun. 2010; 11: 79-86
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• Recent genome-wide association studies have identified 1q31 (RGS1), 2q11-12 (IL18RAP), 3p21 (CCR1/CCR3/CCR2), 3q25-26 (IL12A/SCHIP1), 3q28 (LPP), 4q27 (IL2/IL21), 6q25 (TAGAP) and 12q24 (SH2B3) as susceptibility regions for coeliac disease (CD). We have earlier replicated association with the IL2/IL21 region. This study aimed at replicating the remaining regions in a family cohort using the transmission disequilibrium test, which is not prone to population stratification as a source of false-positive results. Nine single nucleotide polymorphisms (SNPs) within these regions were genotyped in 325 Swedish-Norwegian CD families. We found significant associations with the same alleles in the regions 1q31 (rs2816316; P(nc)=0.0060), 3p21 (rs6441961; P(nc)=0.0006), 3q25-26 (rs17810564; P(nc)=0.0316 and rs9811792; P(nc)=0.0434) and 3q28 (rs1464510; P(nc)=0.0037). Borderline, but non-significant, associations were found for rs917997 (IL18RAP), whereas no evidence for association could be obtained for rs13015714 (IL18RAP) or rs1738074 (TAGAP). The lack of replication of the latter SNPs could be because of limited power. rs3184504 (SH2B3) was not analysed because of assay failure. The most significantly associated region, 3p21 (CCR1/CCR3/CCR2), was further analysed by typing of 30 SNPs, with the aim of identifying the causal variant responsible for the initial association. Several SNPs showed association with CD, but none displayed associations stronger than rs6441961, nor did any of them add to the effect initially marked by rs6441961 in a conditional analysis. However, differential effects of rs6441961(*)C carrying haplotypes were indicated, and we thus cannot exclude the possibility that our inability to obtain evidence for multiple independent effects in the CCR1/CCR3/CCR2 gene region was related to a power issue.

• Plenge R
• GWASs and the age of human as the model organism for autoimmune genetic research.
• Genome Biol. 2010; 11: 212-212
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• Genetic studies have identified more than 150 autoimmune loci, and next-generation sequencing will identify more. Is it time to make human the model organism for autoimmune research?

• Wodarczyk C et al.
• Nephrocystin-1 forms a complex with polycystin-1 via a polyproline motif/SH3 domain interaction and regulates the apoptotic response in mammals.
• PLoS One. 2010; 5: 12719-12719
• Display abstract

• Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confirm the interaction between the endogenous proteins. Finally, we show that NPHP1 trafficking to cilia does not require PC-1 and that PC-1 may require NPHP1 to regulate resistance to apoptosis, but not to regulate cell cycle progression. In line with this, we find high levels of apoptosis in renal specimens of NPHP patients. Our data uncover a link between two different ciliopathies, ADPKD and NPHP, supporting the notion that common pathogenetic defects, possibly involving de-regulated apoptosis, underlie renal cyst formation.

• Sarra M, Monteleone G
• Interleukin-21: a new mediator of inflammation in systemic lupus erythematosus.
• J Biomed Biotechnol. 2010; 2010: 294582-294582
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• Systemic Lupus Erythematosus (SLE) is an autoimmune disorder characterized by excessive production of a variety of autoantibodies and a wide range of clinical manifestations. Pathogenesis of SLE is complex and not fully understood. There is however evidence that B and T cells are critical to the development of disease, and that T cell-derived cytokines are involved in the SLE-associated inflammatory response. One such cytokine seems to be interleukin (IL)-21, the latest identified member of the gamma-chain-related cytokine family. IL-21 has an important role in the control of the growth, survival, differentiation, and function of both T and B cells, and excessive production of IL-21 has been associated with the development of multiple immune-mediated diseases. Here we review data supporting the involvement of IL-21 in the pathogenesis of SLE.

• Pruvot B, Laurens V, Salvadori F, Solary E, Pichon L, Chluba J
• Comparative analysis of nonaspanin protein sequences and expression studies in zebrafish.
• Immunogenetics. 2010; 62: 681-99
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• Nonaspanins constitute a family of proteins, also called TM9SF, characterized by a large non-cytoplasmic domain and nine putative transmembrane domains. This family is highly conserved through evolution and comprises three members in Saccharomyces cerevisiae, Dictyostelium discoideum, and Drosophila melanogaster, and four members are reported in mammals (TM9SF1-TM9SF4). Genetic studies in Dictyostelium and Drosophila have shown that TM9SF members are required for adhesion and phagocytosis in innate immune response, furthermore, human TM9SF1 plays a role in the regulation of autophagy and human TM9SF4 in tumor cannibalism. Here we report that the zebrafish genome encodes five members of this family, TM9SF1-TM9SF5, which show high level of sequence conservation with the previously reported members. Expression analysis in zebrafish showed that all members are maternally expressed and continue to be present throughout embryogenesis to adults. Gene expression could not be regulated by pathogen-associated molecular patterns such as LPS, CpG, or Poly I:C. By bioinformatic analyses of 80 TM9SF protein sequences from yeast, plants, and animals, we confirmed a very conserved protein structure. An evolutionary conserved immunoreceptor tyrosine-based inhibition motif has been detected in the cytoplasmic domain between transmembrane domain (TM) 7 and TM8 in TM9SF1, TM9SF2, TM9SF4 and TM9SF5, and at the extreme C-terminal end of TM9SF4. Finally, a conserved TRAF2 binding domain could also be predicted in the cytoplasmic regions of TM9SF2, TM9SF3, TM9SF4, and TM9SF5. This confirms the hypothesis that TM9SF proteins may play a regulatory role in a specific and ancient cellular mechanism that is involved in innate immunity.

• Fedetz M et al.
• Multiple sclerosis association study with the TENR-IL2-IL21 region in a Spanish population.
• Tissue Antigens. 2009; 74: 244-7
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• Polymorphisms from the TENR-IL2-IL21 block in the 4q27 chromosome were recently associated with type 1 diabetes, celiac disease, rheumatoid arthritis and psoriasis. We undertook this study to investigate the potential role of polymorphisms rs3136534, rs6822844 and rs2069762 (-330 T/G IL2) in multiple sclerosis (MS) (805 patients of Spanish Caucasian origin and 952 health controls). We did not find evidence for association with any single nucleotide polymorphisms (SNPs) tested. Allele and genotype frequencies of the SNPs, which were studied, were similar in DRB1*15-positive or DRB1*15-negative patients. After stratification of MS patients by clinical course, a weak association was observed with rs2069762 G allele and haplotype bearing this allele with secondary progressive MS, although these cases represent 22% of the MS cases. Our results did not show major influence of TENR-IL2-IL21 locus on susceptibility or disease progression in MS. However, we could not exclude completely the effect in MS for this region. Additional studies, using much larger sample sizes and analysis of additional polymorphisms in the gene and its flanking region, will be required to ascertain their contributions to MS susceptibility.

• Wu TT, Chen YF, Hastie T, Sobel E, Lange K
• Genome-wide association analysis by lasso penalized logistic regression.
• Bioinformatics. 2009; 25: 714-21
• Display abstract

• MOTIVATION: In ordinary regression, imposition of a lasso penalty makes continuous model selection straightforward. Lasso penalized regression is particularly advantageous when the number of predictors far exceeds the number of observations. METHOD: The present article evaluates the performance of lasso penalized logistic regression in case-control disease gene mapping with a large number of SNPs (single nucleotide polymorphisms) predictors. The strength of the lasso penalty can be tuned to select a predetermined number of the most relevant SNPs and other predictors. For a given value of the tuning constant, the penalized likelihood is quickly maximized by cyclic coordinate ascent. Once the most potent marginal predictors are identified, their two-way and higher order interactions can also be examined by lasso penalized logistic regression. RESULTS: This strategy is tested on both simulated and real data. Our findings on coeliac disease replicate the previous SNP results and shed light on possible interactions among the SNPs. AVAILABILITY: The software discussed is available in Mendel 9.0 at the UCLA Human Genetics web site. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

• Jones JL et al.
• IL-21 drives secondary autoimmunity in patients with multiple sclerosis, following therapeutic lymphocyte depletion with alemtuzumab (Campath-1H).
• J Clin Invest. 2009; 119: 2052-61
• Display abstract

• Phase II clinical trials revealed that the lymphocyte-depleting humanized monoclonal antibody alemtuzumab (Campath-1H) is highly effective in the treatment of early relapsing-remitting multiple sclerosis. However, 30% of patients develop autoimmunity months to years after pulsed exposure to alemtuzumab, usually targeting the thyroid gland and, more rarely, blood components. In this study, we show that autoimmunity arose in those patients with greater T cell apoptosis and cell cycling in response to alemtuzumab-induced lymphocyte depletion, a phenomenon that is driven by higher levels of IL-21. Before treatment, patients who went on to develop secondary autoimmunity had more than 2-fold greater levels of serum IL-21 than the nonautoimmune group. We suggest that serum IL-21 may, therefore, serve as a biomarker for the risk of developing autoimmunity months to years after alemtuzumab treatment. This has implications for counseling those patients with multiple sclerosis who are considering lymphocyte-depleting therapy with alemtuzumab. Finally, we demonstrate through genotyping that IL-21 expression is genetically predetermined. We propose that, by driving cycles of T cell expansion and apoptosis to excess, IL-21 increases the stochastic opportunities for T cells to encounter self antigen and, hence, for autoimmunity.

• Forabosco P et al.
• Meta-analysis of genome-wide linkage studies in celiac disease.
• Hum Hered. 2009; 68: 223-30
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• OBJECTIVE: A meta-analysis of genome-wide linkage studies allows us to summarize the extensive information available from family-based studies, as the field moves into genome-wide association studies. METHODS: Here we apply the genome scan meta-analysis (GSMA) method, a rank-based, model-free approach, to combine results across eight independent genome-wide linkages performed on celiac disease (CD), including 554 families with over 1,500 affected individuals. We also investigate the agreement between signals we identified from this meta-analysis of linkage studies and those identified from genome-wide association analysis using a hypergeometric distribution. RESULTS: Not surprisingly, the most significant result was obtained in the HLA region. Outside the HLA region, suggestive evidence for linkage was obtained at the telomeric region of chromosome 10 (10q26.12-qter; p = 0.00366), and on chromosome 8 (8q22.2-q24.21; p = 0.00491). Testing signals of association and linkage within bins showed no significant evidence for co-localization of results. CONCLUSION: This meta-analysis allowed us to pool the results from available genome-wide linkage studies and to identify novel regions potentially harboring predisposing genetic variation contributing to CD. This study also shows that linkage and association studies may identify different types of disease-predisposing variants.

• Becker T, Herold C
• Joint analysis of tightly linked SNPs in screening step of genome-wide association studies leads to increased power.
• Eur J Hum Genet. 2009; 17: 1043-9
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• Recent developments in genome-wide association studies (GWAS) have lead to the localization of disease genes for many complex diseases. The scrutiny of the respective publications reveals, first, that statistical analysis is restricted typically to single-marker analysis in the first step, and that, second, the presence of multiple, independently associated SNPs within the same linkage disequilibrium (LD) region is a common phenomenon. Motivated by this observation, we show through a power simulation study that a simultaneous analysis of tightly linked SNPs in the initial GWAS analysis step would lead to increased power, when compared with that in single-marker analysis. This is true for all the three approaches we considered (implementations in BEAGLE, FAMHAP and UNPHASED). The best performance was obtained using a two-marker haplotype analysis. In conclusion, we would expect additional gene findings for re-analyzing successful GWAS with a multi-marker approach.

• Geboes K, Geboes KP
• Diagnosis and treatment of coeliac disease.
• F1000 Med Rep. 2009; 1: 0-0
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• It is now clearly established that coeliac disease is much more common than originally considered. While in the past it was taught that coeliac disease was mainly a disease of children, it is clear now that it may be diagnosed at any age. The clinical presentation of adolescents and adults is, however, less typical. Recent evidence suggests that coeliac disease is a multi-organ disease. The diagnostic techniques involving histology and serologic testing have been improved and the involvement of environmental and genetic factors in the pathogenesis of this immune disorder has been clarified, although the pathogenesis is not yet completely understood. Complications are now better identified, and new treatment strategies are under consideration.

• Teixeira VH et al.
• Testing for the association of the KIAA1109/Tenr/IL2/IL21 gene region with rheumatoid arthritis in a European family-based study.
• Arthritis Res Ther. 2009; 11: 45-45
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• INTRODUCTION: A candidate gene approach, in a large case-control association study in the Dutch population, has shown that a 480 kb block on chromosome 4q27 encompassing KIAA1109/Tenr/IL2/IL21 genes is associated with rheumatoid arthritis. Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure. Therefore, our aim was to test this association in populations of European origin by using a family-based approach. METHODS: A total of 1,302 West European white individuals from 434 trio families were genotyped for the rs4505848, rs11732095, rs6822844, rs4492018 and rs1398553 polymorphisms using the TaqMan Allelic discrimination assay (Applied Biosystems). The genetic association analyses for each SNP and haplotype were performed using the Transmission Disequilibrium Test and the genotype relative risk. RESULTS: We observed evidence for association of the heterozygous rs4505848-AG genotype with rheumatoid arthritis (P = 0.04); however, no significance was found after Bonferroni correction. In concordance with previous findings in the Dutch population, we observed a trend of undertransmission for the rs6822844-T allele and rs6822844-GT genotype to rheumatoid arthritis patients. We further investigated the five SNP haplotypes of the KIAA1109/Tenr/IL2/IL21 gene region. We observed, as described in the Dutch population, a nonsignificant undertransmission of the AATGG haplotype to rheumatoid arthritis patients. CONCLUSIONS: Using a family-based study, we have provided a trend for the association of the KIAA1109/Tenr/IL2/IL21 gene region with rheumatoid arthritis in populations of European descent. Nevertheless, we failed to replicate a significant association of this region in our rheumatoid arthritis family sample. Further investigation of this region, including detection and testing of all variants, is required to confirm rheumatoid arthritis association.

• Coenen MJ et al.
• Common and different genetic background for rheumatoid arthritis and coeliac disease.
• Hum Mol Genet. 2009; 18: 4195-203
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• Recent genome-wide association studies (GWAS) have revealed genetic risk factors in autoimmune and inflammatory disorders. Several of the associated genes and underlying pathways are shared by various autoimmune diseases. Rheumatoid arthritis (RA) and coeliac disease (CD) are two autoimmune disorders which have commonalities in their pathogenesis. We aimed to replicate known RA loci in a Dutch RA population, and to investigate whether the effect of known RA and CD risk factors generalize across the two diseases. We selected all loci associated to either RA or CD in a GWAS and confirmed in an independent cohort, with a combined P-value cut-off P < 5 x 10(-6). We genotyped 11 RA and 11 CD loci in 1368 RA patients, 795 CD patients and 1683 Dutch controls. We combined our results in a meta-analysis with UK GWAS on RA (1860 cases; 2938 controls) and CD (767 cases; 1422 controls). In the Dutch RA cohort, the PTPN22 and IL2/IL21 variants showed convincing association (P = 3.4 x 10(-12) and P = 2.8 x 10(-4), respectively). Association of RA with the known CD risk variant in the SH2B3 was also observed, predominantly in the subgroup of rheumatoid factor-positive RA patients (P = 0.0055). In a meta-analysis of Dutch and UK data sets, shared association with six loci (TNFAIP3, IL2/IL21, SH2B3, LPP, MMEL1/TNFRSF14 and PFKFB3/PRKCQ) was observed in both RA and CD cohorts. We confirmed two known loci and identified four novel ones for shared CD-RA genetic risk. Most of the shared loci further emphasize a role for adaptive and innate immunity in these diseases.

• Kitada K, Yamasaki T, Aikawa S
• Amplification of the ABCB1 region accompanied by a short sequence of 200bp from chromosome 2 in lung cancer cells.
• Cancer Genet Cytogenet. 2009; 194: 4-11
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• Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7. The sequence was identical in the two sublines. A short sequence of 200bp derived from chromosome 2 was incorporated, suggesting translocation between chromosomes 2 and 7. The copy number of the short sequence was comparable to that of the neighboring sequence, suggesting coamplification. The timing of the occurrence of the putative translocation and the initiation of BFB-cycle-driven amplification during the stepwise selection were determined by using the unique junction sequences specific to these events as indicators. The results demonstrated that the translocation occurred at the step of 100 nmol/L treatment and the BFB cycle initiated in the step of 400 nmol/L-treatment. It is likely that the translocation, preceding amplification by several selection steps, activated ABCB1 gene expression. The diversity in amplification profiles between the two sublines was generated by the separately operating BFB cycles, after an initial break-and-fusion that probably occurred in a single cell.

• Rothenberg ME
• Biology and treatment of eosinophilic esophagitis.
• Gastroenterology. 2009; 137: 1238-49
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• Eosinophilic esophagitis is a recently recognized but expanding disorder characterized by antigen-driven eosinophil accumulation in the esophagus. Symptoms frequently mimic those of gastroesophageal reflux disease, but the diseases are distinct in their histopathology, gene expression signature, response to therapy, hereditary risk, and association with allergies. The pathogenesis of eosinophilic esophagitis involves environmental and genetic factors, particularly food antigens and expression level of the eosinophil chemoattractant eotaxin-3, respectively. Analyses of gene expression signatures and animal models have indicated the importance of adaptive T-cell immunity that involves interleukin-5 and interleukin-13-induced esophageal epithelial cell responses. Symptoms, dysregulation of esophageal gene expression, and pathology are largely reversible following reduced exposure to specific food antigens as well as anti-inflammatory therapy, but chronic treatment is necessary to prevent relapse. Therefore, eosinophilic esophagitis is a disease with unique features that include chronic esophagitis, atopy, immune sensitization to oral antigens, reversibility, and familial association.

• Heap GA et al.
• Complex nature of SNP genotype effects on gene expression in primary human leucocytes.
• BMC Med Genomics. 2009; 2: 1-1
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• BACKGROUND: Genome wide association studies have been hugely successful in identifying disease risk variants, yet most variants do not lead to coding changes and how variants influence biological function is usually unknown. METHODS: We correlated gene expression and genetic variation in untouched primary leucocytes (n = 110) from individuals with celiac disease - a common condition with multiple risk variants identified. We compared our observations with an EBV-transformed HapMap B cell line dataset (n = 90), and performed a meta-analysis to increase power to detect non-tissue specific effects. RESULTS: In celiac peripheral blood, 2,315 SNP variants influenced gene expression at 765 different transcripts (< 250 kb from SNP, at FDR = 0.05, cis expression quantitative trait loci, eQTLs). 135 of the detected SNP-probe effects (reflecting 51 unique probes) were also detected in a HapMap B cell line published dataset, all with effects in the same allelic direction. Overall gene expression differences within the two datasets predominantly explain the limited overlap in observed cis-eQTLs. Celiac associated risk variants from two regions, containing genes IL18RAP and CCR3, showed significant cis genotype-expression correlations in the peripheral blood but not in the B cell line datasets. We identified 14 genes where a SNP affected the expression of different probes within the same gene, but in opposite allelic directions. By incorporating genetic variation in co-expression analyses, functional relationships between genes can be more significantly detected. CONCLUSION: In conclusion, the complex nature of genotypic effects in human populations makes the use of a relevant tissue, large datasets, and analysis of different exons essential to enable the identification of the function for many genetic risk variants in common diseases.

• De Nitto D, Monteleone I, Franze E, Pallone F, Monteleone G
• Involvement of interleukin-15 and interleukin-21, two gamma-chain-related cytokines, in celiac disease.
• World J Gastroenterol. 2009; 15: 4609-14
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• Celiac disease (CD), an enteropathy caused by dietary gluten in genetically susceptible individuals, is histologically characterized by villous atrophy, crypt cell hyperplasia, and increased number of intra-epithelial lymphocytes. The nature of CD pathogenesis remains unclear, but recent evidence indicates that both innate and adaptive immune responses are necessary for the phenotypic expression and pathologic changes characteristic of CD. Extensive studies of molecules produced by immune cells in the gut of CD patients have led to identification of two cytokines, namely interleukin (IL)-15 and IL-21, which are thought to play a major role in orchestrating the mucosal inflammatory response in CD. Here we review the current knowledge of the expression and function of IL-15 and IL-21 in CD.

• Garner CP, Murray JA, Ding YC, Tien Z, van Heel DA, Neuhausen SL
• Replication of celiac disease UK genome-wide association study results in a US population.
• Hum Mol Genet. 2009; 18: 4219-25
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• Celiac disease is a common disease with a prevalence of approximately 1%. A recent genome-wide association study (GWAS) and follow-up study identified eight loci significantly associated with celiac disease risk. We genotyped the top 1020 non-HLA single nucleotide polymorphisms (SNPs) from the GWAS study that were genotyped in the previous follow-up study. After quality control assessments, 975 SNPs were analyzed for association with 906 celiac disease cases and 3819 controls, using logistic regression. Additional genotype data were generated by imputation and analyzed across the regions showing the strongest statistical evidence for association. Twenty SNPs were associated with celiac disease with P < 0.01 in the current study as well as in the previous follow-up study, of which 16 had P < 0.001 and 11 had P < 1 x 10(-11). Five of eight regions identified in the follow-up study were strongly associated with celiac disease, including regions on 1q31, 3q25, 3q28, 4q27 and 12q24. The strongest associations were at 4q27, the region most strongly associated in the GWAS and follow-up study and containing IL2 and IL21, and at 3q28 harboring LPP. In addition, we provide new evidence for an association, not previously reported, on 2q31 harboring a strong candidate gene, ITGA4. In conclusion, in this first follow-up study of celiac cases from the USA, we provide additional evidence that five of eight previously identified regions harbor risk alleles for celiac disease, and new evidence for an association on 2q31. The underlying functional mutations responsible for these replicated associations need to be identified.

• Coenen MJ, Gregersen PK
• Rheumatoid arthritis: a view of the current genetic landscape.
• Genes Immun. 2009; 10: 101-11
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• The field of genetics and autoimmune diseases is undergoing a rapid and unprecedented expansion with new genetic findings being reported at an astounding pace. It is now clear that multiple genes contribute to each of the major autoimmune disorders, with significant genetic overlaps among them. Rheumatoid arthritis (RA) is no exception to this, and emerging data are beginning to reveal the outlines of new diagnostic subgroups, complex overlapping relationships with other autoimmune disorders and potential new targets for therapy. This review describes the evolving genetic landscape of RA, with the full knowledge that our current view is far from complete. However, with the first round of genome-wide association scans now completed, it is reasonable to begin to take stock of the direction in which the major common genetic risk factors are leading us.

• Ferwerda B et al.
• Human dectin-1 deficiency and mucocutaneous fungal infections.
• N Engl J Med. 2009; 361: 1760-7
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• Mucocutaneous fungal infections are typically found in patients who have no known immune defects. We describe a family in which four women who were affected by either recurrent vulvovaginal candidiasis or onychomycosis had the early-stop-codon mutation Tyr238X in the beta-glucan receptor dectin-1. The mutated form of dectin-1 was poorly expressed, did not mediate beta-glucan binding, and led to defective production of cytokines (interleukin-17, tumor necrosis factor, and interleukin-6) after stimulation with beta-glucan or Candida albicans. In contrast, fungal phagocytosis and fungal killing were normal in the patients, explaining why dectin-1 deficiency was not associated with invasive fungal infections and highlighting the specific role of dectin-1 in human mucosal antifungal defense.

• Fumagalli M et al.
• Parasites represent a major selective force for interleukin genes and shape the genetic predisposition to autoimmune conditions.
• J Exp Med. 2009; 206: 1395-408
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• Many human genes have adapted to the constant threat of exposure to infectious agents; according to the "hygiene hypothesis," lack of exposure to parasites in modern settings results in immune imbalances, augmenting susceptibility to the development of autoimmune and allergic conditions. Here, by estimating the number of pathogen species/genera in a specific geographic location (pathogen richness) for 52 human populations and analyzing 91 interleukin (IL)/IL receptor genes (IL genes), we show that helminths have been a major selective force on a subset of these genes. A population genetics analysis revealed that five IL genes, including IL7R and IL18RAP, have been a target of balancing selection, a selection process that maintains genetic variability within a population. Previous identification of polymorphisms in some of these loci, and their association with autoimmune conditions, prompted us to investigate the relationship between adaptation and disease. By searching for variants in IL genes identified in genome-wide association studies, we verified that six risk alleles for inflammatory bowel (IBD) or celiac disease are significantly correlated with micropathogen richness. These data support the hygiene hypothesis for IBD and provide a large set of putative targets for susceptibility to helminth infections.

• Elbers CC et al.
• Variants in neuropeptide Y receptor 1 and 5 are associated with nutrient-specific food intake and are under recent selection in Europeans.
• PLoS One. 2009; 4: 7070-7070
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• There is a large variation in caloric intake and macronutrient preference between individuals and between ethnic groups, and these food intake patterns show a strong heritability. The transition to new food sources during the agriculture revolution around 11,000 years ago probably created selective pressure and shaped the genome of modern humans. One major player in energy homeostasis is the appetite-stimulating hormone neuropeptide Y, in which the stimulatory capacity may be mediated by the neuropeptide Y receptors 1, 2 and 5 (NPY1R, NPY2R and NPY5R). We assess association between variants in the NPY1R, NPY2R and NPY5R genes and nutrient intake in a cross-sectional, single-center study of 400 men aged 40 to 80 years, and we examine whether genomic regions containing these genes show signatures of recent selection in 270 HapMap individuals (90 Africans, 90 Asians, and 90 Caucasians) and in 846 Dutch bloodbank controls. Our results show that derived alleles in NPY1R and NPY5R are associated with lower carbohydrate intake, mainly because of a lower consumption of mono- and disaccharides. We also show that carriers of these derived alleles, on average, consume meals with a lower glycemic index and glycemic load and have higher alcohol consumption. One of these variants shows the hallmark of recent selection in Europe. Our data suggest that lower carbohydrate intake, consuming meals with a low glycemic index and glycemic load, and/or higher alcohol consumption, gave a survival advantage in Europeans since the agricultural revolution. This advantage could lie in overall health benefits, because lower carbohydrate intake, consuming meals with a low GI and GL, and/or higher alcohol consumption, are known to be associated with a lower risk of chronic diseases.

• Trynka G et al.
• Coeliac disease-associated risk variants in TNFAIP3 and REL implicate altered NF-kappaB signalling.
• Gut. 2009; 58: 1078-83
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• OBJECTIVE: Our previous coeliac disease genome-wide association study (GWAS) implicated risk variants in the human leucocyte antigen (HLA) region and eight novel risk regions. To identify more coeliac disease loci, we selected 458 single nucleotide polymorphisms (SNPs) that showed more modest association in the GWAS for genotyping and analysis in four independent cohorts. DESIGN: 458 SNPs were assayed in 1682 cases and 3258 controls from three populations (UK, Irish and Dutch). We combined the results with the original GWAS cohort (767 UK cases and 1422 controls); six SNPs showed association with p<1 x 10(-04) and were then genotyped in an independent Italian coeliac cohort (538 cases and 593 controls). RESULTS: We identified two novel coeliac disease risk regions: 6q23.3 (OLIG3-TNFAIP3) and 2p16.1 (REL), both of which reached genome-wide significance in the combined analysis of all 2987 cases and 5273 controls (rs2327832 p = 1.3 x 10(-08), and rs842647 p = 5.2 x 10(-07)). We investigated the expression of these genes in the RNA isolated from biopsies and from whole blood RNA. We did not observe any changes in gene expression, nor in the correlation of genotype with gene expression. CONCLUSIONS: Both TNFAIP3 (A20, at the protein level) and REL are key mediators in the nuclear factor kappa B (NF-kappaB) inflammatory signalling pathway. For the first time, a role for primary heritable variation in this important biological pathway predisposing to coeliac disease has been identified. Currently, the HLA risk factors and the 10 established non-HLA risk factors explain approximately 40% of the heritability of coeliac disease.

• Romanos J, Barisani D, Trynka G, Zhernakova A, Bardella MT, Wijmenga C
• Six new coeliac disease loci replicated in an Italian population confirm association with coeliac disease.
• J Med Genet. 2009; 46: 60-3
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• BACKGROUND AND AIMS: The first genome wide association study on coeliac disease (CD) and its follow-up have identified eight new loci that contribute significantly towards CD risk. Seven of these loci contain genes controlling adaptive immune responses, including IL2/IL21 (4q27), RGS1 (1q31), IL18RAP (2q11-2q12), CCR3 (3p21), IL12A (3q25-3q26), TAGAP (6q25) and SH2B3 (12q24). METHODS: We selected the nine most associated single nucleotide polymorphisms to tag the eight new loci in an Italian cohort comprising 538 CD patients and 593 healthy controls. RESULTS: Common variation in IL2/IL21, RGS1, IL12A/SCHIP and SH2B3 was associated with susceptibility to CD in our Italian cohort. The LPP and TAGAP regions also showed moderate association, whereas there was no association with CCR3 and IL18RAP. CONCLUSION: This is the first replication study of six of the eight new CD loci; it is also the first CD association study in a southern European cohort. Our results may imply there is a genuine population difference across Europe regarding the loci contributing to CD.

• Esparza-Gordillo J et al.
• A common variant on chromosome 11q13 is associated with atopic dermatitis.
• Nat Genet. 2009; 41: 596-601
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• We conducted a genome-wide association study in 939 individuals with atopic dermatitis and 975 controls as well as 270 complete nuclear families with two affected siblings. SNPs consistently associated with atopic dermatitis in both discovery sets were then investigated in two additional independent replication sets totalling 2,637 cases and 3,957 controls. Highly significant association was found with allele A of rs7927894 on chromosome 11q13.5, located 38 kb downstream of C11orf30 (P(combined) = 7.6 x 10(-10)). Approximately 13% of individuals of European origin are homozygous for rs7927894[A], and their risk of developing atopic dermatitis is 1.47 times that of noncarriers.

• Sareneva I et al.
• Linkage and association study of FcgammaR polymorphisms in celiac disease.
• Tissue Antigens. 2009; 73: 54-8
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• The Fcgamma receptor cluster on chromosome 1q23 contains a number of genes that may affect susceptibility to celiac disease, but previous studies have yielded contradictory results. We studied the FcgammaRIIa*A519G (rs1801274) and FcgammaRIIIa*A559C (rs396991) single nucleotide polymorphisms in celiac disease families from Hungary and Finland and in celiac disease case-control materials from Hungary and Italy. Neither the Hungarian nor the Italian case-control material or a meta-analysis of the combined case-control material showed significant single-marker or haplotype association. In addition, neither linkage nor family-based association tests showed evidence for association in the Finnish or Hungarian family material. This study thus does not support a previous publication showing FcgammaR association with celiac disease.

• Holm D, Fink DR, Gronlund J, Hansen S, Holmskov U
• Cloning and characterization of SCART1, a novel scavenger receptor cysteine-rich type I transmembrane molecule.
• Mol Immunol. 2009; 46: 1663-72
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• We have cloned and characterized a novel murine transmembrane molecule, mSCART1 belonging to the scavenger receptor cysteine-rich superfamily. The cDNA encodes a polypeptide chain of 989 amino acids, organized as a type I transmembrane protein that contains eight extracellular SRCR domains followed by a transmembrane region and a cytoplasmic domain. The cytoplasmic domain contains two putative src kinase consensus substrate sequences, three additional potential phosphorylation sites, and two potential internalization motifs. Two possible secreted forms that lack the transmembrane region arise by alternative splicing. The murine SCART1 gene maps to chromosome 7 band F5 and the analysis of the genomic organization showed that the gene spans 12.86 kb and contains 14 exons. Quantitative real-time PCR analyses on murine tissues showed high mSCART1 mRNA expression in the lymph node, the trachea, and the lung, and low expression was found in the thymus, the spleen, the skin, and in tissues throughout the gastrointestinal tract. Comparative studies of the domain organization as well as the cytoplasmic domain of mSCART1 with the other members of the SRCR superfamily show that mSCART1 is highly related to the WC1 family of the SRCR superfamily. Finally, a novel human scavenger receptor cysteine-rich molecule with high homology to mSCART1 was identified by searching in the human genomic databases using the mSCART1 cDNA sequence.

• Daha NA et al.
• Confirmation of STAT4, IL2/IL21, and CTLA4 polymorphisms in rheumatoid arthritis.
• Arthritis Rheum. 2009; 60: 1255-60
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• OBJECTIVE: Recent advances have led to novel identification of genetic polymorphisms that are associated with susceptibility to rheumatoid arthritis (RA). Currently, 5 loci (HLA, PTPN22, TRAF1/C5, TNFAIP3, and STAT4) have been consistently reported, whereas others have been observed less systematically. The aim of the present study was to independently replicate 3 recently described RA susceptibility loci, STAT4, IL2/IL21, and CTLA4, in a large Dutch case-control cohort, and to perform a meta-analysis of all published studies to date and investigate the relevance of the findings in clinically well-defined subgroups of RA patients with or without autoantibodies. METHODS: The STAT4, IL2/IL21, and CTLA4 gene polymorphisms (rs7574865, rs6822844, and rs3087243, respectively) were genotyped in 877 RA patients and 866 healthy individuals. A meta-analysis of all published studies of disease association with these polymorphisms was performed using the Mantel-Haenszel fixed-effects method. RESULTS: An association of STAT4, IL2/IL21, and CTLA4 with RA was detected in Dutch patients (odds ratio [OR] 1.19 [P=0.031], OR 0.84 [P=0.051], and OR 0.87 [P=0.041], respectively). Results from the meta-analysis confirmed an association of all 3 polymorphisms with RA in Caucasians (OR 1.24 [P=1.66x10(-11)], OR 0.78 [P=5.6x10(-5)], and OR 0.91 [P=1.8x10(-3)], respectively). The meta-analysis also revealed that STAT4 predisposed to disease development equally in patients with autoantibodies and those without autoantibodies, and that CTLA4 enhanced the development of anti-citrullinated protein antibody (ACPA)-positive RA as compared with ACPA-negative RA. CONCLUSION: Our results replicate and firmly establish the association of STAT4 and CTLA4 with RA and provide highly suggestive evidence for IL2/IL21 loci as a risk factor for RA. Given the strong statistical power of our meta-analysis to confirm a true-positive association, these findings provide considerable support for the involvement of CTLA4 in distinct subsets of RA patients.

• Li L et al.
• Molecular cloning and characterization of a novel transcript variant of Mtsarg1 gene.
• Mol Biol Rep. 2009; 36: 1023-32
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• Mtsarg1 (Mus musculus testis and spermatogenesis cell apoptosis-related gene 1) gene with 1103 bp in full length had been cloned previously (GenBank accession number: AF399971, 2002; re-designated as Spata3, Mus musculus spermatogenesis-associated 3, 2007). In the present study, we identified a novel transcript variant of Mtsarg1, named Mtsarg1-beta which is 887 bp in length (GenBank accession numbers: EU259321 and EF546784, 2007, designated as Spata3 variant 4) by reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing. Mtsarg1-beta which has high similarity with Mtsarg1 contains an entire open reading frame of 417 bp encoding a protein consisting of 138 amino acids. Mtsarg1-beta protein is a non-secretory protein with a theoretic molecular mass around 14.79 kD and an isoelectric point of 9.74, which shares the 100 N-terminal amino acids with Mtsarg1 followed by 38 amino acids differing from Mtsarg1. Multi-tissue RT-PCR results and Northern blot analysis for adult DBA/2 mice showed that Mtsarg1-beta and Mtsarg1 mRNAs were specifically expressed in testis at high level. RT-PCR results also showed that Mtsarg1-beta and Mtsarg1 mRNAs were not expressed in mouse GC-1 spermatogonia. In situ hybridization revealed that both Mtsarg1 and probably Mtsarg1-beta mRNAs were mainly expressed in mouse spermatocytes. Subcellular localization analysis suggested that Mtsarg1 protein was mainly localized in nucleus while Mtsarg1-beta protein was mainly localized in cytoplasm. All these results indicate that Mtsarg1 and Mtsarg1-beta may play an important role in mouse testicular function and in spermatocyte development.

• Gregersen PK, Olsson LM
• Recent advances in the genetics of autoimmune disease.
• Annu Rev Immunol. 2009; 27: 363-91
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• Extraordinary technical advances in the field of human genetics over the past few years have catalyzed an explosion of new information about the genetics of human autoimmunity. In particular, the ability to scan the entire genome for common polymorphisms that associate with disease has led to the identification of numerous new risk genes involved in autoimmune phenotypes. Several themes are emerging. Autoimmune disorders have a complex genetic basis; multiple genes contribute to disease risk, each with generally modest effects independently. In addition, it is now clear that common genes underlie multiple autoimmune disorders. There is also heterogeneity among subphenotypes within a disease and across major racial groups. The current crop of genetic associations are only the start of a complete catalog of genetic factors for autoimmunity, and it remains unclear to what extent common variation versus multiple rare variants contribute to disease susceptibility. The current review focuses on recent discoveries within functionally related groups of genes that provide clues to novel pathways of pathogenesis for human autoimmunity.

• Koskinen LL et al.
• Fine mapping of the CELIAC2 locus on chromosome 5q31-q33 in the Finnish and Hungarian populations.
• Tissue Antigens. 2009; 74: 408-16
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• Celiac disease is a chronic inflammation of the small intestine, arising in genetically predisposed individuals as a result of ingestion of dietary gluten. The only confirmed and functionally characterised genetic risk factors for celiac disease are the DQ2 or DQ8 heterodimers at the major histocompatibility complex (MHC) class II locus (CELIAC1). These genes are necessary but alone not sufficient for disease onset. Genome-wide linkage scans have suggested chromosome 5q31-q33 (CELIAC2) as an important risk locus for celiac disease. This region has also been associated to other inflammatory disorders, although as yet, no clear gene associations have been found. In the current study, 11 celiac disease candidate loci were screened for genetic linkage in the Hungarian population. As the CELIAC2 locus showed the strongest evidence for linkage, this locus was selected for follow-up. Seventeen candidate genes were selected from the CELIAC2 locus, and genotyped using 48 haplotype tagging single nucleotide polymorphisms (SNPs) in large Finnish and Hungarian family materials. A subset of these, 40 tagging SNPs in 15 genes, were genotyped in an independent set of Finnish and Hungarian cases and controls. We confirmed linkage of this region with celiac disease and report strong linkage in both the Finnish and Hungarian populations. The association analysis showed modest associations throughout the whole region. These association findings were not replicated in the case-control datasets. Our study strongly supports the role of the CELIAC2 locus in celiac disease, but it also highlights the need for a more powerful study design in the region, to locate the true disease risk variants.

• Forabosco P et al.
• Meta-analysis of genome-wide linkage studies across autoimmune diseases.
• Eur J Hum Genet. 2009; 17: 236-43
• Display abstract

• Autoimmune diseases are chronic disorders initiated by a loss of immunologic tolerance to self-antigens. They cluster within families, and patients may be diagnosed with more than one disease, suggesting pleiotropic genes are involved in the aetiology of different diseases. To identify potential loci, which confer susceptibility to autoimmunity independent of disease phenotype, we pooled results from genome-wide linkage studies, using the genome scan meta-analysis method (GSMA). The meta-analysis included 42 independent studies for 11 autoimmune diseases, using 7350 families with 18 291 affected individuals. In addition to the HLA region, which showed highly significant genome-wide evidence for linkage, we obtained suggestive evidence for linkage on chromosome 16, with peak evidence at 10.0-19.8 Mb. This region may harbour a pleiotropic gene (or genes) conferring risk for several diseases, although no such gene has been identified through association studies. We did not identify evidence for linkage at several genes known to confer increased risk to different autoimmune diseases (PTPN22, CTLA4), even in subgroups of diseases consistently found to be associated with these genes. The relative risks conferred by variants in these genes are modest (<1.5 in most cases), and even a large study like this meta-analysis lacks power to detect linkage. This study illustrates the concept that linkage and association studies have power to identify very different types of disease-predisposing variants.

• Fischer U, Radermacher J, Mayer J, Mehraein Y, Meese E
• Tumor hypoxia: Impact on gene amplification in glioblastoma.
• Int J Oncol. 2008; 33: 509-15
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• Gene amplification is frequently found in human glioblastoma but the mechanisms driving amplifications remain to be elucidated. Hypoxia as hallmark of glioblastoma is known to be involved in the induction of fragile sites that are central to gene amplification. We analyzed the potential of hypoxia (pO2 0%) and mini hypoxia (pO2 5%) to induce fragile sites within a homogeneously staining region (HSR) at 12q14-15 in a glioblastoma cell line (TX3868). Treatment of cells by hypoxia or by mini hypoxia induced double minutes (DMs) and caused breakage of the HSR structure at 12q14-15, suggesting a novel hypoxia inducible fragile site on 12q. Treatment with aphidicolin, a known fragile site inducer, indicates that the hypoxia inducible fragile site is a common fragile site. Reintegration of amplified sequences and occurrence of anaphase-bridge-like structures shows that mini hypoxia and hypoxia are able to initiate amplification processes in human glioblastoma cells. Hypoxia as known tumor microenvironment factor is crucial for the development of amplifications in glioblastoma. The identification and characterization of novel common fragile sites induced by hypoxia will improve the understanding of mechanisms underlying amplifications in glioblastoma.

• Sandhu MS et al.
• LDL-cholesterol concentrations: a genome-wide association study.
• Lancet. 2008; 371: 483-91
• Display abstract

• BACKGROUND: LDL cholesterol has a causal role in the development of cardiovascular disease. Improved understanding of the biological mechanisms that underlie the metabolism and regulation of LDL cholesterol might help to identify novel therapeutic targets. We therefore did a genome-wide association study of LDL-cholesterol concentrations. METHODS: We used genome-wide association data from up to 11,685 participants with measures of circulating LDL-cholesterol concentrations across five studies, including data for 293 461 autosomal single nucleotide polymorphisms (SNPs) with a minor allele frequency of 5% or more that passed our quality control criteria. We also used data from a second genome-wide array in up to 4337 participants from three of these five studies, with data for 290,140 SNPs. We did replication studies in two independent populations consisting of up to 4979 participants. Statistical approaches, including meta-analysis and linkage disequilibrium plots, were used to refine association signals; we analysed pooled data from all seven populations to determine the effect of each SNP on variations in circulating LDL-cholesterol concentrations. FINDINGS: In our initial scan, we found two SNPs (rs599839 [p=1.7x10(-15)] and rs4970834 [p=3.0x10(-11)]) that showed genome-wide statistical association with LDL cholesterol at chromosomal locus 1p13.3. The second genome screen found a third statistically associated SNP at the same locus (rs646776 [p=4.3x10(-9)]). Meta-analysis of data from all studies showed an association of SNPs rs599839 (combined p=1.2x10(-33)) and rs646776 (p=4.8x10(-20)) with LDL-cholesterol concentrations. SNPs rs599839 and rs646776 both explained around 1% of the variation in circulating LDL-cholesterol concentrations and were associated with about 15% of an SD change in LDL cholesterol per allele, assuming an SD of 1 mmol/L. INTERPRETATION: We found evidence for a novel locus for LDL cholesterol on chromosome 1p13.3. These results potentially provide insight into the biological mechanisms that underlie the regulation of LDL cholesterol and might help in the discovery of novel therapeutic targets for cardiovascular disease.

• Hahn P, Bose J, Edler S, Lengeling A
• Genomic structure and expression of Jmjd6 and evolutionary analysis in the context of related JmjC domain containing proteins.
• BMC Genomics. 2008; 9: 293-293
• Display abstract

• BACKGROUND: The jumonji C (JmjC) domain containing gene 6 (Jmjd6, previously known as phosphatidylserine receptor) has misleadingly been annotated to encode a transmembrane receptor for the engulfment of apoptotic cells. Given the importance of JmjC domain containing proteins in controlling a wide range of diverse biological functions, we undertook a comparative genomic analysis to gain further insights in Jmjd6 gene organisation, evolution, and protein function. RESULTS: We describe here a semiautomated computational pipeline to identify and annotate JmjC domain containing proteins. Using a sequence segment N-terminal of the Jmjd6 JmjC domain as query for a reciprocal BLAST search, we identified homologous sequences in 62 species across all major phyla. Retrieved Jmjd6 sequences were used to phylogenetically analyse corresponding loci and their genomic neighbourhood. This analysis let to the identification and characterisation of a bi-directional transcriptional unit compromising the Jmjd6 and 1110005A03Rik genes and to the recognition of a new, before overseen Jmjd6 exon in mammals. Using expression studies, two novel Jmjd6 splice variants were identified and validated in vivo. Analysis of the Jmjd6 neighbouring gene 1110005A03Rik revealed an incident deletion of this gene in two out of three earlier reported Jmjd6 knockout mice, which might affect previously described conflicting phenotypes. To determine potentially important residues for Jmjd6 function a structural model of the Jmjd6 protein was calculated based on sequence conservation. This approach identified a conserved double-stranded beta-helix (DSBH) fold and a HxDxnH facial triad as structural motifs. Moreover, our systematic annotation in nine species identified 313 DSBH fold-containing proteins that split into 25 highly conserved subgroups. CONCLUSION: We give further evidence that Jmjd6 most likely has a function as a nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenase as previously suggested. Further, we provide novel insights into the evolution of Jmjd6 and other related members of the superfamily of JmjC domain containing proteins. Finally, we discuss possibilities of the involvement of Jmjd6 and 1110005A03Rik in an antagonistic biochemical pathway.

• Kauffman C, Rangwala H, Karypis G
• Improving homology models for protein-ligand binding sites.
• Comput Syst Bioinformatics Conf. 2008; 7: 211-22
• Display abstract

• In order to improve the prediction of protein-ligand binding sites through homology modeling, we incorporate knowledge of the binding residues into the modeling framework. Residues are identified as binding or nonbinding based on their true labels as well as labels predicted from structure and sequence. The sequence predictions were made using a support vector machine framework which employs a sophisticated window-based kernel. Binding labels are used with a very sensitive sequence alignment method to align the target and template. Relevant parameters governing the alignment process are searched for optimal values. Based on our results, homology models of the binding site can be improved if a priori knowledge of the binding residues is available. For target-template pairs with low sequence identity and high structural diversity our sequence-based prediction method provided sufficient information to realize this improvement.

• Cooper JD et al.
• Meta-analysis of genome-wide association study data identifies additional type 1 diabetes risk loci.
• Nat Genet. 2008; 40: 1399-401
• Display abstract

• We carried out a meta-analysis of data from three genome-wide association (GWA) studies of type 1 diabetes (T1D), testing 305,090 SNPs in 3,561 T1D cases and 4,646 controls of European ancestry. We obtained further support for 4q27 (IL2-IL21, P = 1.9 x 10(-8)) and, after genotyping an additional 6,225 cases, 6,946 controls and 2,828 families, convincing evidence for four previously unknown and distinct risk loci in chromosome regions 6q15 (BACH2, P = 4.7 x 10(-12)), 10p15 (PRKCQ, P = 3.7 x 10(-9)), 15q24 (CTSH, P = 3.2 x 10(-15)) and 22q13 (C1QTNF6, P = 2.0 x 10(-8)).

• Ding YC, Weizman Z, Yerushalmi B, Elbedour K, Garner CP, Neuhausen SL
• An autosomal genome-wide screen for celiac disease in Bedouin families.
• Genes Immun. 2008; 9: 81-6
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• Celiac disease is a common, familial autoimmune disease caused by exposure to gliadin in wheat, and related prolamins in barley and rye. The prevalence of the disease is approximately 1:133. Celiac disease can cause significant morbidity. The only treatment is a gluten-free diet. A genome-wide search of 405 microsatellite markers was performed on samples from 18 Bedouin families with a minimum of two cases of celiac disease. Non-parametric and parametric (including both dominant and recessive models of inheritance) linkage analyses were performed. The most significant genome-wide linkage evidence was at chromosome 3p26 with an HLod of 3.21, under the dominant model. The only other HLod or NPL greater than 2 was at 4q35, with an HLod of 2.15 under a dominant model. The region at 3p26, previously reported in two linkage analyses, harbors interleukin receptor genes, plausible candidates for celiac disease.

• Franke L et al.
• Detection, imputation, and association analysis of small deletions and null alleles on oligonucleotide arrays.
• Am J Hum Genet. 2008; 82: 1316-33
• Display abstract

• Copy-number variation (CNV) is a major contributor to human genetic variation. Recently, CNV associations with human disease have been reported. Many genome-wide association (GWA) studies in complex diseases have been performed with sets of biallelic single-nucleotide polymorphisms (SNPs), but the available CNV methods are still limited. We present a new method (TriTyper) that can infer genotypes in case-control data sets for deletion CNVs, or SNPs with an extra, untyped allele at a high-resolution single SNP level. By accounting for linkage disequilibrium (LD), as well as intensity data, calling accuracy is improved. Analysis of 3102 unrelated individuals with European descent, genotyped with Illumina Infinium BeadChips, resulted in the identification of 1880 SNPs with a common untyped allele, and these SNPs are in strong LD with neighboring biallelic SNPs. Simulations indicate our method has superior power to detect associations compared to biallelic SNPs that are in LD with these SNPs, yet without increasing type I errors, as shown in a GWA analysis in celiac disease. Genotypes for 1204 triallelic SNPs could be fully imputed, with only biallelic-genotype calls, permitting association analysis of these SNPs in many published data sets. We estimate that 682 of the 1655 unique loci reflect deletions; this is on average 99 deletions per individual, four times greater than those detected by other methods. Whereas the identified loci are strongly enriched for known deletions, 61% have not been reported before. Genes overlapping with these loci more often have paralogs (p = 0.006) and biologically interact with fewer genes than expected (p = 0.004).

• Manolio TA, Brooks LD, Collins FS
• A HapMap harvest of insights into the genetics of common disease.
• J Clin Invest. 2008; 118: 1590-605
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• The International HapMap Project was designed to create a genome-wide database of patterns of human genetic variation, with the expectation that these patterns would be useful for genetic association studies of common diseases. This expectation has been amply fulfilled with just the initial output of genome-wide association studies, identifying nearly 100 loci for nearly 40 common diseases and traits. These associations provided new insights into pathophysiology, suggesting previously unsuspected etiologic pathways for common diseases that will be of use in identifying new therapeutic targets and developing targeted interventions based on genetically defined risk. In addition, HapMap-based discoveries have shed new light on the impact of evolutionary pressures on the human genome, suggesting multiple loci important for adapting to disease-causing pathogens and new environments. In this review we examine the origin, development, and current status of the HapMap; its prospects for continued evolution; and its current and potential future impact on biomedical science.

• Heinzen EL et al.
• Tissue-specific genetic control of splicing: implications for the study of complex traits.
• PLoS Biol. 2008; 6: 1-1
• Display abstract

• Numerous genome-wide screens for polymorphisms that influence gene expression have provided key insights into the genetic control of transcription. Despite this work, the relevance of specific polymorphisms to in vivo expression and splicing remains unclear. We carried out the first genome-wide screen, to our knowledge, for SNPs that associate with alternative splicing and gene expression in human primary cells, evaluating 93 autopsy-collected cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononucleated cell samples collected from living healthy donors. We identified 23 high confidence associations with total expression and 80 with alternative splicing as reflected by expression levels of specific exons. Fewer than 50% of the implicated SNPs however show effects in both tissue types, reflecting strong evidence for distinct genetic control of splicing and expression in the two tissue types. The data generated here also suggest the possibility that splicing effects may be responsible for up to 13 out of 84 reported genome-wide significant associations with human traits. These results emphasize the importance of establishing a database of polymorphisms affecting splicing and expression in primary tissue types and suggest that splicing effects may be of more phenotypic significance than overall gene expression changes.

• Adamovic S et al.
• Association study of IL2/IL21 and FcgRIIa: significant association with the IL2/IL21 region in Scandinavian coeliac disease families.
• Genes Immun. 2008; 9: 364-7
• Display abstract

• The first genome-wide association study performed in a UK coeliac disease (CD) case-control cohort revealed association with a linkage disequilibrium block containing the KIAA1109/Tenr/IL2/IL21 genes. Also recently, an association with a non-synonymous polymorphism in FcgammaRIIa (CD32a) was reported in CD with an unusually strong P-value. We aimed to replicate the reported associations with the single nucleotide polymorphisms rs13119723 A>G and rs6822844 G>T in the KIAA1109/Tenr/IL2/IL21 region and rs1801274 G>A in the FcgammaRIIa gene in a family sample consisting of 325 Swedish/Norwegian families using the robust transmission disequilibrium test. The family sample used in this study included 100 families with two or more children affected by CD and 225 families with one affected child. We could confirm significant association between the polymorphisms rs13119723 A>G and rs6822844 G>T located in the KIAA1109/Tenr/IL2/IL21 region and CD (P-value 0.001 and 0.002, respectively). However, we found no association with the FcgammaRIIa rs1801274 G>A polymorphism (P-value=0.3). In conclusion, our results support the KIAA1109/Tenr/IL2/IL21 region as a true CD susceptibility region.

• Monsuur AJ et al.
• Effective detection of human leukocyte antigen risk alleles in celiac disease using tag single nucleotide polymorphisms.
• PLoS One. 2008; 3: 2270-2270
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• BACKGROUND: The HLA genes, located in the MHC region on chromosome 6p21.3, play an important role in many autoimmune disorders, such as celiac disease (CD), type 1 diabetes (T1D), rheumatoid arthritis, multiple sclerosis, psoriasis and others. Known HLA variants that confer risk to CD, for example, include DQA1*05/DQB1*02 (DQ2.5) and DQA1*03/DQB1*0302 (DQ8). To diagnose the majority of CD patients and to study disease susceptibility and progression, typing these strongly associated HLA risk factors is of utmost importance. However, current genotyping methods for HLA risk factors involve many reactions, and are complicated and expensive. We sought a simple experimental approach using tagging SNPs that predict the CD-associated HLA risk factors. METHODOLOGY: Our tagging approach exploits linkage disequilibrium between single nucleotide polymorphism (SNPs) and the CD-associated HLA risk factors DQ2.5 and DQ8 that indicate direct risk, and DQA1*0201/DQB1*0202 (DQ2.2) and DQA1*0505/DQB1*0301 (DQ7) that attribute to the risk of DQ2.5 to CD. To evaluate the predictive power of this approach, we performed an empirical comparison of the predicted DQ types, based on these six tag SNPs, with those executed with current validated laboratory typing methods of the HLA-DQA1 and -DQB1 genes in three large cohorts. The results were validated in three European celiac populations. CONCLUSION: Using this method, only six SNPs were needed to predict the risk types carried by >95% of CD patients. We determined that for this tagging approach the sensitivity was >0.991, specificity >0.996 and the predictive value >0.948. Our results show that this tag SNP method is very accurate and provides an excellent basis for population screening for CD. This method is broadly applicable in European populations.

• Burglin TR
• Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif.
• BMC Genomics. 2008; 9: 127-127
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• BACKGROUND: The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini. RESULTS: We performed extensive genome searches such as the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria. CONCLUSION: In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are found in many protists indicates that this gene family must have arisen in very early eukaryotic evolution, and gave rise eventually to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins are three families of Hint domain proteins that evolved in parallel in eukaryotes.

• Comabella M et al.
• Identification of a novel risk locus for multiple sclerosis at 13q31.3 by a pooled genome-wide scan of 500,000 single nucleotide polymorphisms.
• PLoS One. 2008; 3: 3490-3490
• Display abstract

• Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with an important genetic component and strongest association driven by the HLA genes. We performed a pooling-based genome-wide association study of 500,000 SNPs in order to find new loci associated with the disease. After applying several criteria, 320 SNPs were selected from the microarrays and individually genotyped in a first and independent Spanish Caucasian replication cohort. The 8 most significant SNPs validated in this cohort were also genotyped in a second US Caucasian replication cohort for confirmation. The most significant association was obtained for SNP rs3129934, which neighbors the HLA-DRB/DQA loci and validates our pooling-based strategy. The second strongest association signal was found for SNP rs1327328, which resides in an unannotated region of chromosome 13 but is in linkage disequilibrium with nearby functional elements that may play important roles in disease susceptibility. This region of chromosome 13 has not been previously identified in MS linkage genome screens and represents a novel risk locus for the disease.

• Salinas CA et al.
• Multiple independent genetic variants in the 8q24 region are associated with prostate cancer risk.
• Cancer Epidemiol Biomarkers Prev. 2008; 17: 1203-13
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• Recently, the 8q24 region has been identified as a prostate cancer susceptibility locus in a genome-wide scan of prostate cancer families in Iceland and an admixture scan of African Americans. Further investigations of variants at 8q24 have shown the existence of additional single nucleotide polymorphisms (SNPs) that enhance prostate cancer risk, suggesting the possibility of multiple regions harboring variants for the disease. In the present population-based study of Caucasians (1,308 cases and 1,266 controls) and African Americans (149 cases and 85 controls), we tested the association between prostate cancer and 23 SNPs in the 8q24 region. Fourteen SNPs in Caucasians and 5 SNPs in African Americans were significantly associated with risk of prostate cancer after adjusting for multiple comparisons; of these, 5 SNPs in Caucasians and 3 in African Americans were independently associated with risk. The strongest association was for rs6983561 (carriers of any C allele) with an odds ratio of 1.6 (95% confidence interval, 1.1-2.1) in Caucasians; variants in rs979200, rs1016343, rs7837328, and rs10090154 were also independently associated with risk. In African Americans, the strongest association was for rs7000448 (carriers of any T allele) with an odds ratio of 3.4 (95% confidence interval, 1.3-8.7). In addition, two SNPs that extend the boundaries of the 8q24 region were significantly associated with risk: rs979200 at the centromeric boundary and rs3891248, located in the first intron of the c-MYC gene (IVS1-355), which identifies a new telomeric boundary.

• Liu XQ et al.
• IL5RA and TNFRSF6B gene variants are associated with sporadic IgA nephropathy.
• J Am Soc Nephrol. 2008; 19: 1025-33
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• Familial clustering and genome-wide linkage scans strongly support a genetic susceptibility to familial IgA nephropathy (IgAN), but genetic factors that predispose to sporadic IgAN are unknown. A high-throughput single nucleotide polymorphism (SNP) association study was conducted using a customized Illumina BeadChip in 732 white patients with biopsy-proven IgAN and 503 control subjects from Canada, France, and Finland. Approximately 93% of 1536 SNPs on the array were tag SNPs from Phase I+II of the HapMap with a minor allele frequency > or =5%, designed to capture the common variants of genes within the critical interval of IGAN1 on chromosome 6q22 and 69 biologic candidate genes for IgAN. SNPs of suggestive or significant association were identified by using logistic regression to adjust for age, gender, study site, and population stratification. Despite using a dense marker set that covered an average interval of 6.5 kb between SNPs, there was no strong and consistent association signal within the IGAN1 critical interval. Among the biologic candidate genes examined, two significant association signals were found at IL5RA and TNFRSF6B, the latter being particularly interesting because this gene encodes a decoy receptor for a TNF family ligand that causes IgAN in mice when overexpressed. Pending replication, these data suggest that variants of IL5RA and TNFRSF6B may predispose to sporadic IgAN.

• Liu GY, Ge CR, Zhang X, Gao SZ
• Isolation, sequence identification and tissue expression distribution of three novel porcine genes--RAB14, S35A3 and ITM2A.
• Mol Biol Rep. 2008; 35: 201-6
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• The complete coding sequences of three porcine genes--RAB14, S35A3 and ITM2A were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals. The nucleotide sequence analysis of these three genes revealed that porcine RAB14 gene encodes a protein of 215 amino acids that contains the conserved putative Ras-related protein Rab-14 domain and has high homology with the Ras-related protein Rab-14 (RAB14) of four species--human and mouse (99%) and rat (100%), dictyostelium discoideum (71%). The porcine S35A3 gene encodes a protein of 325 amino acids that contains the conserved putative nucleotide-sugar transporter domain and has high homology with the UDP-N-acetylglucosamine transporter (S35A3) of five species--cattle (98%), dog (97%), human (96%), mouse (95%) and rat (94%). The porcine ITM2A gene encodes a protein of 254 amino acids that contains the conserved putative BRICHOS domain and has high homology with the integral membrane protein 2A (ITM2A) of two species--human (89%), and mouse (88%). The tissue expression analysis indicated that the swine RAB14 gene was over-expressed in fat, lung, spleen, and kidney, moderately in large intestine, weakly in small intestine, and hardly expressed in muscle and liver. The swine S35A3 gene was moderately expressed in large intestine, fat, and spleen, weakly in liver and lung, and almost not expressed in muscle, small intestine, and liver. The swine ITM2A gene was over-expressed in fat and spleen, moderately in lung, weakly in muscle, and hardly expressed in liver, small intestine, large intestine, and kidney. Our experiment established the primary foundation for further research on these three swine genes.

• Hunt KA, Franke L, Deloukas P, Wijmenga C, van Heel DA
• No evidence in a large UK collection for celiac disease risk variants reported by a Spanish study.
• Gastroenterology. 2008; 134: 1629-30

• Bracken S, Byrne G, Kelly J, Jackson J, Feighery C
• Altered gene expression in highly purified enterocytes from patients with active coeliac disease.
• BMC Genomics. 2008; 9: 377-377
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• BACKGROUND: Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. RESULTS: Enterocyte suspensions of high purity (98-99%) were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p < 0.05). Analysis of these altered genes revealed a number of biological processes that are potentially modified in active coeliac disease. These processes include events likely to contribute to coeliac pathology, such as altered cell proliferation, differentiation, survival, structure and transport. CONCLUSION: This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease.

• Nebert DW, Zhang G, Vesell ES
• From human genetics and genomics to pharmacogenetics and pharmacogenomics: past lessons, future directions.
• Drug Metab Rev. 2008; 40: 187-224
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• A brief history of human genetics and genomics is provided, comparing recent progress in those fields with that in pharmacogenetics and pharmacogenomics, which are subsets of genetics and genomics, respectively. Sequencing of the entire human genome, the mapping of common haplotypes of single-nucleotide polymorphisms (SNPs), and cost-effective genotyping technologies leading to genome-wide association (GWA) studies - have combined convincingly in the past several years to demonstrate the requirements needed to separate true associations from the plethora of false positives. While research in human genetics has moved from monogenic to oligogenic to complex diseases, its pharmacogenetics branch has followed, usually a few years behind. The continuous discoveries, even today, of new surprises about our genome cause us to question reviews declaring that "personalized medicine is almost here" or that "individualized drug therapy will soon be a reality." As summarized herein, numerous reasons exist to show that an "unequivocal genotype" or even an "unequivocal phenotype" is virtually impossible to achieve in current limited-size studies of human populations. This problem (of insufficiently stringent criteria) leads to a decrease in statistical power and, consequently, equivocal interpretation of most genotype-phenotype association studies. It remains unclear whether personalized medicine or individualized drug therapy will ever be achievable by means of DNA testing alone.

• Smyth DJ et al.
• Shared and distinct genetic variants in type 1 diabetes and celiac disease.
• N Engl J Med. 2008; 359: 2767-77
• Display abstract

• BACKGROUND: Two inflammatory disorders, type 1 diabetes and celiac disease, cosegregate in populations, suggesting a common genetic origin. Since both diseases are associated with the HLA class II genes on chromosome 6p21, we tested whether non-HLA loci are shared. METHODS: We evaluated the association between type 1 diabetes and eight loci related to the risk of celiac disease by genotyping and statistical analyses of DNA samples from 8064 patients with type 1 diabetes, 9339 control subjects, and 2828 families providing 3064 parent-child trios (consisting of an affected child and both biologic parents). We also investigated 18 loci associated with type 1 diabetes in 2560 patients with celiac disease and 9339 control subjects. RESULTS: Three celiac disease loci--RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, and TAGAP on chromosome 6q25--were associated with type 1 diabetes (P<1.00x10(-4)). The 32-bp insertion-deletion variant on chromosome 3p21 was newly identified as a type 1 diabetes locus (P=1.81x10(-8)) and was also associated with celiac disease, along with PTPN2 on chromosome 18p11 and CTLA4 on chromosome 2q33, bringing the total number of loci with evidence of a shared association to seven, including SH2B3 on chromosome 12q24. The effects of the IL18RAP and TAGAP alleles confer protection in type 1 diabetes and susceptibility in celiac disease. Loci with distinct effects in the two diseases included INS on chromosome 11p15, IL2RA on chromosome 10p15, and PTPN22 on chromosome 1p13 in type 1 diabetes and IL12A on 3q25 and LPP on 3q28 in celiac disease. CONCLUSIONS: A genetic susceptibility to both type 1 diabetes and celiac disease shares common alleles. These data suggest that common biologic mechanisms, such as autoimmunity-related tissue damage and intolerance to dietary antigens, may be etiologic features of both diseases.

• Ouyang W, Kolls JK, Zheng Y
• The biological functions of T helper 17 cell effector cytokines in inflammation.
• Immunity. 2008; 28: 454-67
• Display abstract

• T helper 17 (Th17) cells belong to a recently identified T helper subset, in addition to the traditional Th1 and Th2 subsets. These cells are characterized as preferential producers of interleukin-17A (IL-17A), IL-17F, IL-21, and IL-22. Th17 cells and their effector cytokines mediate host defensive mechanisms to various infections, especially extracellular bacteria infections, and are involved in the pathogenesis of many autoimmune diseases. The receptors for IL-17 and IL-22 are broadly expressed on various epithelial tissues. The effector cytokines of Th17 cells, therefore, mediate the crucial crosstalk between immune system and tissues, and play indispensable roles in tissue immunity.

• Bernsel A, Viklund H, Elofsson A
• Remote homology detection of integral membrane proteins using conserved sequence features.
• Proteins. 2008; 71: 1387-99
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• Compared with globular proteins, transmembrane proteins are surrounded by a more intricate environment and, consequently, amino acid composition varies between the different compartments. Existing algorithms for homology detection are generally developed with globular proteins in mind and may not be optimal to detect distant homology between transmembrane proteins. Here, we introduce a new profile-profile based alignment method for remote homology detection of transmembrane proteins in a hidden Markov model framework that takes advantage of the sequence constraints placed by the hydrophobic interior of the membrane. We expect that, for distant membrane protein homologs, even if the sequences have diverged too far to be recognized, the hydrophobicity pattern and the transmembrane topology are better conserved. By using this information in parallel with sequence information, we show that both sensitivity and specificity can be substantially improved for remote homology detection in two independent test sets. In addition, we show that alignment quality can be improved for the most distant homologs in a public dataset of membrane protein structures. Applying the method to the Pfam domain database, we are able to suggest new putative evolutionary relationships for a few relatively uncharacterized protein domain families, of which several are confirmed by other methods. The method is called Searcher for Homology Relationships of Integral Membrane Proteins (SHRIMP) and is available for download at http://www.sbc.su.se/shrimp/.

• Castellanos-Rubio A et al.
• Combined functional and positional gene information for the identification of susceptibility variants in celiac disease.
• Gastroenterology. 2008; 134: 738-46
• Display abstract

• BACKGROUND & AIMS: Celiac disease is a complex, immune-mediated disorder of the intestinal mucosa with a strong genetic component. HLA-DQ2 is the major determinant of risk, but other minor genes, still to be identified, also are involved. METHODS: We designed a strategy that combines gene expression profiling of intestinal biopsy specimens, linkage region information, and different bioinformatics tools for the selection of potentially regulatory single-nucleotide polymorphisms (SNPs) involved in the disease. We selected 361 SNPs from 71 genes that fulfilled stringent functional (changes in expression level) and positional criteria (located in regions that have been linked to the disease, other than HLA). These polymorphisms were genotyped in 262 celiac patients and 214 controls. RESULTS: We detected strong evidence of association with several SNPs (the most significant were rs6747096, P = 2.38 x 10(-5); rs7040561, P = 6.55 x 10(-5); and rs458046, P = 1.35 x 10(-4)) that pinpoint novel candidate determinants of predisposition to the disease in previously identified linkage regions (eg, SERPINE2 in 2q33, and PBX3 or PPP6C in 9q34). CONCLUSIONS: Our study shows that the combination of function and position is a valid strategy for the genetic dissection of complex traits.

• Rahim NG, Harismendy O, Topol EJ, Frazer KA
• Genetic determinants of phenotypic diversity in humans.
• Genome Biol. 2008; 9: 215-215
• Display abstract

• New technologies for rapidly assaying DNA sequences have revealed that the degree and nature of human genetic variation is far more complex then previously realized. These same technologies have also resulted in the identification of common genetic variants associated with more than 30 human diseases and traits.

• Chang M et al.
• A large-scale rheumatoid arthritis genetic study identifies association at chromosome 9q33.2.
• PLoS Genet. 2008; 4: 1000107-1000107
• Display abstract

• Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting both joints and extra-articular tissues. Although some genetic risk factors for RA are well-established, most notably HLA-DRB1 and PTPN22, these markers do not fully account for the observed heritability. To identify additional susceptibility loci, we carried out a multi-tiered, case-control association study, genotyping 25,966 putative functional SNPs in 475 white North American RA patients and 475 matched controls. Significant markers were genotyped in two additional, independent, white case-control sample sets (661 cases/1322 controls from North America and 596 cases/705 controls from The Netherlands) identifying a SNP, rs1953126, on chromosome 9q33.2 that was significantly associated with RA (OR(common) = 1.28, trend P(comb) = 1.45E-06). Through a comprehensive fine-scale-mapping SNP-selection procedure, 137 additional SNPs in a 668 kb region from MEGF9 to STOM on 9q33.2 were chosen for follow-up genotyping in a staged-approach. Significant single marker results (P(comb)<0.01) spanned a large 525 kb region from FBXW2 to GSN. However, a variety of analyses identified SNPs in a 70 kb region extending from the third intron of PHF19 across TRAF1 into the TRAF1-C5 intergenic region, but excluding the C5 coding region, as the most interesting (trend P(comb): 1.45E-06 --> 5.41E-09). The observed association patterns for these SNPs had heightened statistical significance and a higher degree of consistency across sample sets. In addition, the allele frequencies for these SNPs displayed reduced variability between control groups when compared to other SNPs. Lastly, in combination with the other two known genetic risk factors, HLA-DRB1 and PTPN22, the variants reported here generate more than a 45-fold RA-risk differential.

• Kingsmore SF, Lindquist IE, Mudge J, Gessler DD, Beavis WD
• Genome-wide association studies: progress and potential for drug discovery and development.
• Nat Rev Drug Discov. 2008; 7: 221-30
• Display abstract

• Although genetic studies have been critically important for the identification of therapeutic targets in Mendelian disorders, genetic approaches aiming to identify targets for common, complex diseases have traditionally had much more limited success. However, during the past year, a novel genetic approach - genome-wide association (GWA) - has demonstrated its potential to identify common genetic variants associated with complex diseases such as diabetes, inflammatory bowel disease and cancer. Here, we highlight some of these recent successes, and discuss the potential for GWA studies to identify novel therapeutic targets and genetic biomarkers that will be useful for drug discovery, patient selection and stratification in common diseases.

• Chen R et al.
• FitSNPs: highly differentially expressed genes are more likely to have variants associated with disease.
• Genome Biol. 2008; 9: 170-170
• Display abstract

• BACKGROUND: Candidate single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWASs) were often selected for validation based on their functional annotation, which was inadequate and biased. We propose to use the more than 200,000 microarray studies in the Gene Expression Omnibus to systematically prioritize candidate SNPs from GWASs. RESULTS: We analyzed all human microarray studies from the Gene Expression Omnibus, and calculated the observed frequency of differential expression, which we called differential expression ratio, for every human gene. Analysis conducted in a comprehensive list of curated disease genes revealed a positive association between differential expression ratio values and the likelihood of harboring disease-associated variants. By considering highly differentially expressed genes, we were able to rediscover disease genes with 79% specificity and 37% sensitivity. We successfully distinguished true disease genes from false positives in multiple GWASs for multiple diseases. We then derived a list of functionally interpolating SNPs (fitSNPs) to analyze the top seven loci of Wellcome Trust Case Control Consortium type 1 diabetes mellitus GWASs, rediscovered all type 1 diabetes mellitus genes, and predicted a novel gene (KIAA1109) for an unexplained locus 4q27. We suggest that fitSNPs would work equally well for both Mendelian and complex diseases (being more effective for cancer) and proposed candidate genes to sequence for their association with 597 syndromes with unknown molecular basis. CONCLUSIONS: Our study demonstrates that highly differentially expressed genes are more likely to harbor disease-associated DNA variants. FitSNPs can serve as an effective tool to systematically prioritize candidate SNPs from GWASs.

• Matsuoka Y, Kobayashi T, Kihara K, Nagahama Y
• Molecular cloning of Plk1 and Nek2 and their expression in mature gonads of the teleost fish Nile tilapia (Oreochromis niloticus).
• Mol Reprod Dev. 2008; 75: 989-1001
• Display abstract

• Polo-like kinase 1 (Plk1) and NIMA-related kinase 2 (Nek2) are serine/threonine kinases that are involved in the G2/M phase transition of the cell cycle in vertebrates. Although they also play critical roles in the regulation of spermatogenesis and oogenesis, their characterization in meiosis has not been elucidated fully, particularly in teleost fish. To investigate the evolutionary significance of serine/threonine kinases in the reproductive system of fish, we cloned the cDNAs of Plk1 and Nek2 from a Nile tilapia (Oreochromis niloticus) testicular cDNA library. Tilapia Plk1 encodes a protein of 582 amino acids that shares 75% homology with human Plk1, while tilapia Nek2 encodes a putative protein of 446 amino acids that shares 70% homology with human Nek2. Analyses of tissue distribution by RT-PCR and Southern blotting revealed that Plk1 and Nek2 are strongly expressed in the ovary and testis. Northern blot analysis revealed two Nek2 transcripts in the ovary and testis with different expression patterns, which indicates the presence of two structural variants for tilapia Nek2. Moreover, the localization of Plk1 and Nek2 mRNAs in tilapia gonads was determined by in situ hybridization analysis. In the ovary, Plk1 and Nek2 were expressed predominantly in oocytes. In the testis, on the other hand, Plk1 was expressed in primary spermatocytes, while Nek2 was generally expressed in primary and secondary spermatocytes. These results suggest that Plk1 and Nek2 are key factors in the progression of meiosis in fish.

• Rainbow DB et al.
• Commonality in the genetic control of Type 1 diabetes in humans and NOD mice: variants of genes in the IL-2 pathway are associated with autoimmune diabetes in both species.
• Biochem Soc Trans. 2008; 36: 312-5
• Display abstract

• Variants within the IL-2 (interleukin 2) and CD25 genes are associated with T1DM (Type 1 diabetes mellitus) in mice and humans respectively. Both gene products are essential for optimal immune tolerance and a partial failure to tolerize is linked to the autoimmune responses to insulin and other beta-cell proteins that precede T1DM onset. Gene variants that contribute to common disease susceptibility often alter gene expression only modestly. Small expression changes can be technically challenging to measure robustly, especially since biological variation usually contributes negatively to this goal. The present review focuses on allele-specific expression assays that can be used to quantify genotype-determined expression differences such as those observed for IL-2, where the susceptibility allele is transcribed 2-fold less than the resistance allele.

• Tusnady GE, Kalmar L, Hegyi H, Tompa P, Simon I
• TOPDOM: database of domains and motifs with conservative location in transmembrane proteins.
• Bioinformatics. 2008; 24: 1469-70
• Display abstract

• The TOPDOM database is a collection of domains and sequence motifs located consistently on the same side of the membrane in alpha-helical transmembrane proteins. The database was created by scanning well-annotated transmembrane protein sequences in the UniProt database by specific domain or motif detecting algorithms. The identified domains or motifs were added to the database if they were uniformly annotated on the same side of the membrane of the various proteins in the UniProt database. The information about the location of the collected domains and motifs can be incorporated into constrained topology prediction algorithms, like HMMTOP, increasing the prediction accuracy. AVAILABILITY: The TOPDOM database and the constrained HMMTOP prediction server are available on the page http://topdom.enzim.hu CONTACT: tusi@enzim.hu; lkalmar@enzim.hu.

• Rubio-Tapia A, Murray JA
• Celiac disease beyond the gut.
• Clin Gastroenterol Hepatol. 2008; 6: 722-3

• Xavier RJ, Rioux JD
• Genome-wide association studies: a new window into immune-mediated diseases.
• Nat Rev Immunol. 2008; 8: 631-43
• Display abstract

• Given the recent explosion of genetic discoveries, 2007 is becoming known to human geneticists as the year of genome-wide association studies. In fact, more genetic risk factors for common diseases were identified in this one year than had been collectively reported before 2007. In particular, 2007 witnessed the discovery of many genes that influence susceptibility to individual immune-mediated diseases, as well as other genes that are associated with susceptibility to more than one disease. Although much work remains to be done, in this Review we discuss what effect these studies are having on our understanding of disease pathogenesis and their potential impact on future immunology studies.

• Hofmann S et al.
• Genome-wide association study identifies ANXA11 as a new susceptibility locus for sarcoidosis.
• Nat Genet. 2008; 40: 1103-6
• Display abstract

• Sarcoidosis is a complex chronic inflammatory disorder with predominant manifestation in the lung. In the first genome-wide association study (> 440,000 SNPs) of this disease, comprising 499 German individuals with sarcoidosis and 490 controls, we detected a series of genetic associations. The strongest association signal maps to the ANXA11 (annexin A11) gene on chromosome 10q22.3. Validation in an independent sample (1,649 cases, 1,832 controls) confirmed the association (SNP rs2789679: P = 3.0 x 10(-13), rs7091565: P = 1.0 x 10(-5), allele-based test). Extensive fine mapping located the association signal to a region between exon 5 and exon 14 of ANXA11. A common nonsynonymous SNP (rs1049550, C > T, [corrected] R230C) was found to be strongly associated with sarcoidosis. The GWAS lead SNP and additional risk variants in the region (rs1953600, rs2573346, rs2784773) were in strong linkage disequilibrium with rs1049550. Annexin A11 has complex and essential functions in several biological pathways, including apoptosis and proliferation.

• Rafiq S et al.
• Gene variants influencing measures of inflammation or predisposing to autoimmune and inflammatory diseases are not associated with the risk of type 2 diabetes.
• Diabetologia. 2008; 51: 2205-13
• Display abstract

• AIMS/HYPOTHESIS: There are strong associations between measures of inflammation and type 2 diabetes, but the causal directions of these associations are not known. We tested the hypothesis that common gene variants known to alter circulating levels of inflammatory proteins, or known to alter autoimmune-related disease risk, influence type 2 diabetes risk. METHODS: We selected 46 variants: (1) eight variants known to alter circulating levels of inflammatory proteins, including those in the IL18, IL1RN, IL6R, MIF, PAI1 (also known as SERPINE1) and CRP genes; and (2) 38 variants known to predispose to autoimmune diseases, including type 1 diabetes. We tested the associations of these variants with type 2 diabetes using a meta-analysis of 4,107 cases and 5,187 controls from the Wellcome Trust Case Control Consortium, the Diabetes Genetics Initiative, and the Finland-United States Investigation of NIDDM studies. We followed up associated variants (p < 0.01) in a further set of 3,125 cases and 3,596 controls from the UK. RESULTS: We found no evidence that inflammatory or autoimmune disease variants are associated with type 2 diabetes (at p

• Dubois PC, van Heel DA
• Translational mini-review series on the immunogenetics of gut disease: immunogenetics of coeliac disease.
• Clin Exp Immunol. 2008; 153: 162-73
• Display abstract

• Recent advances in immunological and genetic research in coeliac disease provide new and complementary insights into the immune response driving this chronic intestinal inflammatory disorder. Both approaches confirm the central importance of T cell-mediated immune responses to disease pathogenesis and have further begun to highlight other relevant components of the mucosal immune system, including innate immunity and the control of lymphocyte trafficking to the mucosa. In the last year, the first genome wide association study in celiac disease led to the identification of multiple new risk variants. These risk regions implicate genes involved in the immune system. Overlap with autoimmune diseases is striking with several of these regions being shown to confer susceptibility to other chronic immune-mediated diseases, particularly type 1 diabetes.

• Koning F
• A tertiary twist to the transglutaminase tale.
• PLoS Biol. 2007; 5: 337-337

• Cheng J
• DOMAC: an accurate, hybrid protein domain prediction server.
• Nucleic Acids Res. 2007; 35: 3546-3546
• Display abstract

• Protein domain prediction is important for protein structure prediction, structure determination, function annotation, mutagenesis analysis and protein engineering. Here we describe an accurate protein domain prediction server (DOMAC) combining both template-based and ab initio methods. The preliminary version of the server was ranked among the top domain prediction servers in the seventh edition of Critical Assessment of Techniques for Protein Structure Prediction (CASP7), 2006. DOMAC server and datasets are available at: http://www.bioinfotool.org/domac.html.

• Estivill X, Armengol L
• Copy number variants and common disorders: filling the gaps and exploring complexity in genome-wide association studies.
• PLoS Genet. 2007; 3: 1787-99
• Display abstract

• Genome-wide association scans (GWASs) using single nucleotide polymorphisms (SNPs) have been completed successfully for several common disorders and have detected over 30 new associations. Considering the large sample sizes and genome-wide SNP coverage of the scans, one might have expected many of the common variants underpinning the genetic component of various disorders to have been identified by now. However, these studies have not evaluated the contribution of other forms of genetic variation, such as structural variation, mainly in the form of copy number variants (CNVs). Known CNVs account for over 15% of the assembled human genome sequence. Since CNVs are not easily tagged by SNPs, might have a wide range of copy number variability, and often fall in genomic regions not well covered by whole-genome arrays or not genotyped by the HapMap project, current GWASs have largely missed the contribution of CNVs to complex disorders. In fact, some CNVs have already been reported to show association with several complex disorders using candidate gene/region approaches, underpinning the importance of regions not investigated in current GWASs. This reveals the need for new generation arrays (some already in the market) and the use of tailored approaches to explore the full dimension of genome variability beyond the single nucleotide scale.

• Amundsen SS et al.
• A comprehensive screen for SNP associations on chromosome region 5q31-33 in Swedish/Norwegian celiac disease families.
• Eur J Hum Genet. 2007; 15: 980-7
• Display abstract

• Celiac disease (CD) is a gluten-induced enteropathy, which results from the interplay between environmental and genetic factors. There is a strong human leukocyte antigen (HLA) association with the disease, and HLA-DQ alleles represent a major genetic risk factor. In addition to HLA-DQ, non-HLA genes appear to be crucial for CD development. Chromosomal region 5q31-33 has demonstrated linkage with CD in several genome-wide studies, including in our Swedish/Norwegian cohort. In a European meta-analysis 5q31-33 was the only region that reached a genome-wide level of significance except for the HLA region. To identify the genetic variant(s) responsible for this linkage signal, we performed a comprehensive single nucleotide polymorphism (SNP) association screen in 97 Swedish/Norwegian multiplex families who demonstrate linkage to the region. We selected tag SNPs from a 16 Mb region representing the 95% confidence interval of the linkage peak. A total of 1,404 SNPs were used for the association analysis. We identified several regions with SNPs demonstrating moderate single- or multipoint associations. However, the isolated association signals appeared insufficient to account for the linkage signal seen in our cohort. Collective effects of multiple risk genes within the region, incomplete genetic coverage or effects related to copy number variation are possible explanations for our findings.

• Standley DM, Toh H, Nakamura H
• ASH structure alignment package: sensitivity and selectivity in domain classification.
• BMC Bioinformatics. 2007; 8: 116-116
• Display abstract

• BACKGROUND: Structure alignment methods offer the possibility of measuring distant evolutionary relationships between proteins that are not visible by sequence-based analysis. However, the question of how structural differences and similarities ought to be quantified in this regard remains open. In this study we construct a training set of sequence-unique CATH and SCOP domains, from which we develop a scoring function that can reliably identify domains with the same CATH topology and SCOP fold classification. The score is implemented in the ASH structure alignment package, for which the source code and a web service are freely available from the PDBj website http://www.pdbj.org/ASH/. RESULTS: The new ASH score shows increased selectivity and sensitivity compared with values reported for several popular programs using the same test set of 4,298,905 structure pairs, yielding an area of .96 under the receiver operating characteristic (ROC) curve. In addition, weak sequence homologies between similar domains are revealed that could not be detected by BLAST sequence alignment. Also, a subset of domain pairs is identified that exhibit high similarity, even though their CATH and SCOP classification differs. Finally, we show that the ranking of alignment programs based solely on geometric measures depends on the choice of the quality measure. CONCLUSION: ASH shows high selectivity and sensitivity with regard to domain classification, an important step in defining distantly related protein sequence families. Moreover, the CPU cost per alignment is competitive with the fastest programs, making ASH a practical option for large-scale structure classification studies.

• Christensen K, Murray JC
• What genome-wide association studies can do for medicine.
• N Engl J Med. 2007; 356: 1094-7

• Callebaut I, Mornon JP, Monget P
• Isolated ZP-N domains constitute the N-terminal extensions of Zona Pellucida proteins.
• Bioinformatics. 2007; 23: 1871-4
• Display abstract

• Zona Pellucida (ZP) domains have been found in a wide variety of extracellular proteins, in which they play essential role for polymerization. They are shared by the ZP proteins, which constitute the extracellular coat of animal eggs. Except from ZP3, constituting the primary sperm receptor, the ZP proteins possess, in addition to their C-terminal ZP domains, N-terminal extensions, which are thought to play an important role in the species-specific gamete recognition. Here, we show that these extensions are made of single or multiple copies of a small globular domain, which can be significantly related to the N-terminal region of ZP domains (ZP-N domains). This finding brings new insights into the molecular evolution of ZP proteins, which may have evolved around a common ZP-N architecture, and more generally into the noticeable sequence diversity of ZP-N domains, which can be found as isolated subunits or tightly associated with ZP-C domains to form complete, canonical ZP domains.

• Yeager M et al.
• Genome-wide association study of prostate cancer identifies a second risk locus at 8q24.
• Nat Genet. 2007; 39: 645-9
• Display abstract

• Recently, common variants on human chromosome 8q24 were found to be associated with prostate cancer risk. While conducting a genome-wide association study in the Cancer Genetic Markers of Susceptibility project with 550,000 SNPs in a nested case-control study (1,172 cases and 1,157 controls of European origin), we identified a new association at 8q24 with an independent effect on prostate cancer susceptibility. The most significant signal is 70 kb centromeric to the previously reported SNP, rs1447295, but shows little evidence of linkage disequilibrium with it. A combined analysis with four additional studies (total: 4,296 cases and 4,299 controls) confirms association with prostate cancer for rs6983267 in the centromeric locus (P = 9.42 x 10(-13); heterozygote odds ratio (OR): 1.26, 95% confidence interval (c.i.): 1.13-1.41; homozygote OR: 1.58, 95% c.i.: 1.40-1.78). Each SNP remained significant in a joint analysis after adjusting for the other (rs1447295 P = 1.41 x 10(-11); rs6983267 P = 6.62 x 10(-10)). These observations, combined with compelling evidence for a recombination hotspot between the two markers, indicate the presence of at least two independent loci within 8q24 that contribute to prostate cancer in men of European ancestry. We estimate that the population attributable risk of the new locus, marked by rs6983267, is higher than the locus marked by rs1447295 (21% versus 9%).

• Maurer-Stroh S, Koranda M, Benetka W, Schneider G, Sirota FL, Eisenhaber F
• Towards complete sets of farnesylated and geranylgeranylated proteins.
• PLoS Comput Biol. 2007; 3: 66-66
• Display abstract

• Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS) has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase) and can be queried for verification status, type of modifying enzymes (anchor type), and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs) used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI), and Rab28 (short isoform) as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for the selective farnesylation of targets with an evolutionary conserved modification site.

• Latiano A et al.
• Analysis of candidate genes on chromosomes 5q and 19p in celiac disease.
• J Pediatr Gastroenterol Nutr. 2007; 45: 180-6
• Display abstract

• BACKGROUND AND AIM: Celiac disease (CD) is a multifactorial disease with involvement of both environmental and genetic susceptibility factors. The HLA-DQ loci account for <40% of CD heritability, but linkage studies have delineated other loci at the 5q31-33 (CELIAC2), and 19p13 regions (CELIAC4), similarly as in inflammatory bowel diseases. However, data in association studies are contradictory. To evaluate whether single nucleotide polymorphisms (SNPs) tagging the MYO9B susceptibility haplotype and the IBD5 locus (5q31-33) are involved in CD predisposition, we performed case-control and family-based analyses. Additionally, any possible correlation with the HLA-DQ status was investigated. Finally, our data were pooled with the results of other studies by a meta-analysis. PATIENTS AND METHODS: In all, 337 unrelated patients with CD, 424 parents (212 sets), and 452 healthy individuals were genotyped for the IGR2198a_1, rs12521868, rs1050152, and rs2631367 SNPs (IBD5 locus) and the rs962917, rs2305764, and rs1545620 SNPs of the MYO9B gene by the restriction enzyme method and the TaqMan system ABI PRISM 7700, respectively. RESULTS: In comparison with healthy control individuals, the allele, genotype, and haplotype frequencies of all investigated SNPs were not different in the CD patients, nor was any correlation observed with the HLA-DQ status or clinical presentation. The transmission disequilibrium test did not show a transmission distortion. Five other studies were available for meta-analysis on MYO9B variants; by pooling of data, no significant association was demonstrated by the random effect model. A significant heterogeneity (P < 0.002) among the studies was present, mainly explained by a single study in the Dutch population. CONCLUSIONS: Our results and those of the meta-analysis (>2000 CD patients and 4000 control individuals) question the role of MYO9B at the CELIAC4 locus as a disease-causing gene. Moreover, none of the investigated SNPs explain the linkage at the CELIAC2 locus.

• Asano K et al.
• Molecular scanning of interleukin-21 gene and genetic susceptibility to type 1 diabetes.
• Hum Immunol. 2007; 68: 384-91
• Display abstract

• A recent study in the nonobese diabetic (NOD) mouse demonstrated the involvement of interleukin (IL)-21 in the pathogenesis of type 1 diabetes. A strong susceptibility locus, Idd3, has also been mapped to the interval containing the murine gene for IL-21 (Il21), making Il21 and the human orthologue IL21 a functional and positional candidate gene for type 1 diabetes. To investigate the contribution of the human genes for IL-21 and its receptor (IL-21R) to susceptibility to type 1 diabetes, we re-sequenced IL21 to identify novel sequence variants, searched for informative variants of IL21R, and studied the association of these variants with the disease. Two polymorphisms, a single nucleotide polymorphism (SNP) and a mononucleotide repeat polymorphism, were identified for IL21, and an allele of the mononucleotide repeat polymorphism was positively associated with the disease. Two novel microsatellite polymorphisms of IL21R were identified, one of which was associated with the disease. Scoring of individuals according to the status of these alleles showed a significant trend for high scores for susceptibility in diabetes patients, suggesting the contribution of IL21 and IL21R to disease susceptibility in an additive manner. These data suggest a contribution of IL21 and IL21R to genetic susceptibility to type 1 diabetes and possible involvement of IL-21 and its receptor system in the disease pathogenesis.

• Wang HT, Chang JW, Guo Z, Li BG
• In silico-initiated cloning and molecular characterization of cortexin 3, a novel human gene specifically expressed in the kidney and brain, and well conserved in vertebrates.
• Int J Mol Med. 2007; 20: 501-10
• Display abstract

• We report on the in silico-initiated cloning and molecular characterization of CTXN3 (cortexin 3), a new human gene that was specifically expressed in the kidney and brain due to tissue-specific alternative exon 1 usage, on chromosome 5q23.2 using digital gene expression displayer (DGED) and a novel in silico cloning approach based on both expressed sequence tags (ESTs) and genomic sequence. The gene CTXN3 included 3 exons and spanned an approximate 9.6-kb region of human chromosome 5q23. Two alternative transcript variants (GenBank accession nos. AB219764 and AB219832) were 1660 and 1458 bp long, respectively, encoding for an 81-amino acid protein with a predicted molecular weight of 8933.4 Da. The predicted human CTXN3 protein had 43% identity with function-unknown protein cortexin, which showed brain-specific expression. Further analysis of the encoded protein using PSORT II, TMpred, and PSIPRED programs demonstrated a putative single membrane-spanning domain in the middle of the CTXN3 amino acid sequence, indicating that it might be an integral membrane protein which may mediate extracellular or intracellular signaling of the kidney or brain. Analysis of the predicted CTXN3 orthologs from different species showed that these proteins are highly conserved in vertebrates. In conclusion, a combination of bioinformatics and molecular approaches is useful in the identification of genes expressed in specific tissues. Selective expression of CTXN3 in the kidney and brain, the amino acid identity to cortexin, and its high conservation among different species indicate that CTXN3 may be involved in a process specifically restricted to kidney and brain tissue function.

• Zhernakova A et al.
• Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a general risk locus for autoimmune diseases.
• Am J Hum Genet. 2007; 81: 1284-8
• Display abstract

• Recently, association of celiac disease with common single-nucleotide polymorphism (SNP) variants in an extensive linkage-disequilibrium block of 480 kb containing the KIAA1109, Tenr, IL2, and IL21 genes has been demonstrated in three independent populations (rs6822844P combined=1.3 x 10(-14)). The KIAA1109/Tenr/IL2/IL21 block corresponds to the Idd3 locus in the nonobese diabetic mouse model of type 1 diabetes (T1D). This block was recently found to be associated with T1D in a genomewide association study, although this finding lacks unequivocal confirmation. We therefore aimed to investigate whether the KIAA1109/Tenr/IL2/IL21 region is involved in susceptibility to multiple autoimmune diseases. We tested SNP rs6822844 for association with disease in 350 T1D-affected and 1,047 rheumatoid arthritis (RA)-affected Dutch patients and in 929 controls. We replicated the association with T1D (P=.0006; OR 0.64 [95% CI 0.50-0.83]), and revealed a similar novel association with RA (P=.0002; OR 0.72 [95% CI 0.61-0.86]). Our results replicate and extend the association found in the KIAA1109/Tenr/IL2/IL21 gene region with autoimmune diseases, implying that this locus is a general risk factor for multiple autoimmune diseases.

• Scuteri A et al.
• Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits.
• PLoS Genet. 2007; 3: 115-115
• Display abstract

• The obesity epidemic is responsible for a substantial economic burden in developed countries and is a major risk factor for type 2 diabetes and cardiovascular disease. The disease is the result not only of several environmental risk factors, but also of genetic predisposition. To take advantage of recent advances in gene-mapping technology, we executed a genome-wide association scan to identify genetic variants associated with obesity-related quantitative traits in the genetically isolated population of Sardinia. Initial analysis suggested that several SNPs in the FTO and PFKP genes were associated with increased BMI, hip circumference, and weight. Within the FTO gene, rs9930506 showed the strongest association with BMI (p = 8.6 x10(-7)), hip circumference (p = 3.4 x 10(-8)), and weight (p = 9.1 x 10(-7)). In Sardinia, homozygotes for the rare "G" allele of this SNP (minor allele frequency = 0.46) were 1.3 BMI units heavier than homozygotes for the common "A" allele. Within the PFKP gene, rs6602024 showed very strong association with BMI (p = 4.9 x 10(-6)). Homozygotes for the rare "A" allele of this SNP (minor allele frequency = 0.12) were 1.8 BMI units heavier than homozygotes for the common "G" allele. To replicate our findings, we genotyped these two SNPs in the GenNet study. In European Americans (N = 1,496) and in Hispanic Americans (N = 839), we replicated significant association between rs9930506 in the FTO gene and BMI (p-value for meta-analysis of European American and Hispanic American follow-up samples, p = 0.001), weight (p = 0.001), and hip circumference (p = 0.0005). We did not replicate association between rs6602024 and obesity-related traits in the GenNet sample, although we found that in European Americans, Hispanic Americans, and African Americans, homozygotes for the rare "A" allele were, on average, 1.0-3.0 BMI units heavier than homozygotes for the more common "G" allele. In summary, we have completed a whole genome-association scan for three obesity-related quantitative traits and report that common genetic variants in the FTO gene are associated with substantial changes in BMI, hip circumference, and body weight. These changes could have a significant impact on the risk of obesity-related morbidity in the general population.

• McCauley JL et al.
• SNPs in Multi-species Conserved Sequences (MCS) as useful markers in association studies: a practical approach.
• BMC Genomics. 2007; 8: 266-266
• Display abstract

• BACKGROUND: Although genes play a key role in many complex diseases, the specific genes involved in most complex diseases remain largely unidentified. Their discovery will hinge on the identification of key sequence variants that are conclusively associated with disease. While much attention has been focused on variants in protein-coding DNA, variants in noncoding regions may also play many important roles in complex disease by altering gene regulation. Since the vast majority of noncoding genomic sequence is of unknown function, this increases the challenge of identifying "functional" variants that cause disease. However, evolutionary conservation can be used as a guide to indicate regions of noncoding or coding DNA that are likely to have biological function, and thus may be more likely to harbor SNP variants with functional consequences. To help bias marker selection in favor of such variants, we devised a process that prioritizes annotated SNPs for genotyping studies based on their location within Multi-species Conserved Sequences (MCSs) and used this process to select SNPs in a region of linkage to a complex disease. This allowed us to evaluate the utility of the chosen SNPs for further association studies. Previously, a region of chromosome 1q43 was linked to Multiple Sclerosis (MS) in a genome-wide screen. We chose annotated SNPs in the region based on location within MCSs (termed MCS-SNPs). We then obtained genotypes for 478 MCS-SNPs in 989 individuals from MS families. RESULTS: Analysis of our MCS-SNP genotypes from the 1q43 region and comparison to HapMap data confirmed that annotated SNPs in MCS regions are frequently polymorphic and show subtle signatures of selective pressure, consistent with previous reports of genome-wide variation in conserved regions. We also present an online tool that allows MCS data to be directly exported to the UCSC genome browser so that MCS-SNPs can be easily identified within genomic regions of interest. CONCLUSION: Our results showed that MCS can easily be used to prioritize markers for follow-up and candidate gene association studies. We believe that this novel approach demonstrates a paradigm for expediting the search for genes contributing to complex diseases.

• Tan XJ et al.
• Molecular cloning and preliminary function study of a novel human gene, TSARG7, related to spermatogenesis.
• Yi Chuan Xue Bao. 2006; 33: 294-303
• Display abstract

• A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the mTSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2,463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56,295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.

• Gewehr JE, Zimmer R
• SSEP-Domain: protein domain prediction by alignment of secondary structure elements and profiles.
• Bioinformatics. 2006; 22: 181-7
• Display abstract

• MOTIVATION: The prediction of protein domains is a crucial task for functional classification, homology-based structure prediction and structural genomics. In this paper, we present the SSEP-Domain protein domain prediction approach, which is based on the application of secondary structure element alignment (SSEA) and profile-profile alignment (PPA) in combination with InterPro pattern searches. SSEA allows rapid screening for potential domain regions while PPA provides us with the necessary specificity for selecting significant hits. The combination with InterPro patterns allows finding domain regions without solved structural templates if sequence family definitions exist. RESULTS: A preliminary version of SSEP-Domain was ranked among the top-performing domain prediction servers in the CASP 6 and CAFASP 4 experiments. Evaluation of the final version shows further improvement over these results together with a significant speed-up. AVAILABILITY: The server is available at http://www.bio.ifi.lmu.de/SSEP/

• Benitez-Paez A
• Sequence analysis of the Receptor Activity-Modifying Proteins family, new putative peptides and structural conformation inference.
• In Silico Biol. 2006; 6: 467-83
• Display abstract

• The Receptor Activity-Modifying Proteins (RAMP) is a family constituted by a single N-terminal extracellular domain and a transmembrane region ending in a short cytoplasmic region. Due to their specific role in modulating the specificity of ligand binding in many class II G-Protein Coupled Receptors, these proteins are awaiting further characterization and elucidation of their structure. This was the aim of this study. We were able to find 13 new RAMP sequences including new protein sequences and predicted peptides from Expressed Sequence Tags and genomic DNA, all of them annotated in databases such as GeneBank, EMBL, Swissprot and ENSEMBL. The predicted peptides came from an array of different organisms including Teleostei and Elasmobranchii species, of which the latter was the most ancient RAMP sequence found. It was also possible to efficiently predict the 1D structure of the extracellular RAMP domain and its 3D conformation was inferred through a combination of bioinformatic approaches such as threading. The 1D structure of the extracellular RAMP domain was predicted as three alpha-helix domain. The most highly conserved residues in the RAMP family were found to be involved in critical functions. Bioinformatic data mining and multiple sequence alignment analysis were crucial for improving the characterization of RAMP proteins and prediction of their 1D and 3D configurations.

• Redon R et al.
• Global variation in copy number in the human genome.
• Nature. 2006; 444: 444-54
• Display abstract

• Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

• van Heel DA, West J
• Recent advances in coeliac disease.
• Gut. 2006; 55: 1037-46

• Cao YW, Jiang CL, Jiang T
• Molecular cloning and preliminary analysis of a fragile site associated gene.
• Biomed Environ Sci. 2006; 19: 392-8
• Display abstract

• OBJECTIVE: To analyze the molecular coining of a fragile site-associated gene. METHODS: Genomic Chinese hamster ovary (CHO) DNA library was constructed using high molecular weight CHO DNA partially digested with MboI restriction enzyme from cultured CHO cells. Screening of genomic DNA library followed the established procedures. Genomic CHO in the positive clones was sequenced. Appropriate primers were designed for the reverse transcriptase-polymerase chain reactions (RT-PCR). The RT-PCR products were cloned into a pCRII TOPO vector and confirmed by DNA sequencing. Antibodies were prepared using synthetic peptides as antigens by immunizing the rabbits. Immunohistochemical analyses were performed to evaluate the expression of the novel gene in different tissues. RESULTS: To investigate the molecular mechanism underlying the initial events of mdr1a amplification, we cloned 1q31 fragile site DNA. Strikingly, we found that this fragile site contained a novel gene which was designated as a fragile site-associated (FSA) gene. FSA encoded an unusually large mRNA of approximately16 kb. Full-length human FSA cDNA was cloned. FSA mRNA was expressed in many cultured cells and tissue types. Immunohistochemical analyses also revealed an expression pattern of the encoded proteins in postmitotic, well-differentiated epithelial compartments of many organs, including colon, mammary glands, ovary, prostate, and bladder. CONCLUSION: FSA plays an important role in regulating mammalian epithelial cell growth and differentiation.

• Kuo MT et al.
• Association of fragile site-associated (FSA) gene expression with epithelial differentiation and tumor development.
• Biochem Biophys Res Commun. 2006; 340: 887-93
• Display abstract

• A novel gene designated as fragile site-associated (FSA) gene was recently identified by positional cloning from the CHO 1q31 fragile site which plays an important role in regulating amplification of multidrug resistance (mdr1) gene in multidrug-resistant cells. FSA produces a message of approximately 16 kb which encodes an open-reading frame of 5005 amino acids. FSA shares sequence similarity with that in Caenorhabditis elegans lpd-3, a lipid storage gene. Using immunohistochemical staining and RNA in situ hybridization we report here that expression of FSA is associated with developmental programs of spermatogenesis and mammary gland in mice. Real-time RT-PCR results also support the upregulation of FSA expression in mammary gland development. Expression of FSA in many tissues including colon, skin, ovary, prostate, and bladder is mainly in the postmitotic, well-differentiated compartments. Moreover, levels of FSA expression are downregulated in tumors of these tissue origins. These results suggest that FSA also plays important roles in regulating mammalian epithelial growth and differentiation and tumor development.

• Luz H, Vingron M
• Family specific rates of protein evolution.
• Bioinformatics. 2006; 22: 1166-71
• Display abstract

• MOTIVATION: Amino acid changing mutations in proteins are contstrained by purifying selection and accumulate at different rates. We estimate evolutionary rates on multiple alignments of eukaryotic protein families in a maximum likelihood framework and spot sets of slow and fast evolving proteins. RESULTS: We find that the evolution of indispensable proteins is constrained by selection and that protein secretion is coupled to an increased evolutionary rate.

• Ma Q et al.
• Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells.
• Mol Reprod Dev. 2006; 73: 1075-83
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• Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.

• Zhang D, Martyniuk CJ, Trudeau VL
• SANTA domain: a novel conserved protein module in Eukaryota with potential involvement in chromatin regulation.
• Bioinformatics. 2006; 22: 2459-62
• Display abstract

• Since packaging of DNA in the chromatin structure restricts the accessibility for regulatory factors, chromatin remodeling is required to facilitate nuclear processes such as gene transcription, replication, and genome recombination. Many conserved non-enzymatic protein domains have been identified that contribute to the activities of multiprotein remodeling complexes. Here we identified a novel conserved protein domain in Eukaryota whose putative function may be in regulating chromatin remodeling. Since this domain is associated with a known SANT domain in several vertebrate proteins, we named it the SANTA (SANT Associated) domain. Sequence analysis showed that the SANTA domain is approximately a 90 amino acid module and likely composed of four central beta-sheets and three flanking alpha-helices. Many hydrophobic residues exhibited high conservation along the domain, implying a possible function in protein-protein interactions. The SANTA domain was identified in mammals, chicken, frog, fish, sea squirt, sea urchin, worms and plants. Furthermore, a phylogenetic tree of SANTA domains showed that one plant-specific duplication event happened in the Viridiplantae lineage.

• Lin K, Zhu L, Zhang DY
• An initial strategy for comparing proteins at the domain architecture level.
• Bioinformatics. 2006; 22: 2081-6
• Display abstract

• MOTIVATION: Ideally, only proteins that exhibit highly similar domain architectures should be compared with one another as homologues or be classified into a single family. By combining three different indices, the Jaccard index, the Goodman-Kruskal gamma function and the domain duplicate index, into a single similarity measure, we propose a method for comparing proteins based on their domain architectures. RESULTS: Evaluation of the method using the eukaryotic orthologous groups of proteins (KOGs) database indicated that it allows the automatic and efficient comparison of multiple-domain proteins, which are usually refractory to classic approaches based on sequence similarity measures. As a case study, the PDZ and LRR_1 domains are used to demonstrate how proteins containing promiscuous domains can be clearly compared using our method. For the convenience of users, a web server was set up where three different query interfaces were implemented to compare different domain architectures or proteins with domain(s), and to identify the relationships among domain architectures within a given KOG from the Clusters of Orthologous Groups of Proteins database. CONCLUSION: The approach we propose is suitable for estimating the similarity of domain architectures of proteins, especially those of multidomain proteins. AVAILABILITY: http://cmb.bnu.edu.cn/pdart/.

• Guo J, Cheng H, Zhao S, Yu L
• GG: a domain involved in phage LTF apparatus and implicated in human MEB and non-syndromic hearing loss diseases.
• FEBS Lett. 2006; 580: 581-4
• Display abstract

• Here, we report the identification of a novel domain--GG (domain in KIAA1199, FAM3, POMGnT1 and Tmem2 proteins, with two well-conserved glycine residues), present in eukaryotic FAM3 superfamily (FAM3A, FAM3B, FAM3C and FAM3D), POMGnT1 (protein O-linked mannose beta-1,2-N-acetylglucosaminyltransferase), TEM2 proteins as well as phage gp35 proteins. GG domain has been revealed to be implicated in muscle-eye-brain disease and non-syndromic hearing loss. The presence of GG domain in Bacteriophage gp35 hinge connector of long tail fiber might reflect the horizontal gene transfer from organisms. And we proposed that GG domain might function as important structural element in phage LTF.

• He QY, Liu XH, Li Q, Studholme DJ, Li XW, Liang SP
• G8: a novel domain associated with polycystic kidney disease and non-syndromic hearing loss.
• Bioinformatics. 2006; 22: 2189-91
• Display abstract

• We report a novel protein domain-G8-which contains five repeated beta-strand pairs and is present in some disease-related proteins such as PKHD1, KIAA1199, TMEM2 as well as other uncharacterized proteins. Most G8-containing proteins are predicted to be membrane-integral or secreted. The G8 domain may be involved in extracellular ligand binding and catalysis. It has been reported that mis-sense mutations in the two G8 domains of human PKHD1 protein resulted in a less stable protein and are associated with autosomal-recessive polycystic kidney disease, indicating the importance of the domain structure. G8 is also present in the N-terminus of some non-syndromic hearing loss disease-related proteins such as KIAA1109 and TMEM2. Discovery of G8 domain will be important for the research of the structure/function of related proteins and beneficial for the development of novel therapeutics. CONTACT: liangsp@hunnu.edu.cn

• Lai WC, Peach ML, Lybrand TP, Hazelbauer GL
• Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity.
• Protein Sci. 2006; 15: 94-101
• Display abstract

• Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg(E) from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg(E) domain was created using the homodimeric, four-helix bundle structure of the Tar(S) domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg(E) periplasmic domain is very similar to the Tar(S) domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.

• Lai X, Guo J, Zhang X, Wang H
• Identification of a novel domain--DIM, which defines a new family composed mainly of bacterial membrane proteins.
• FEMS Microbiol Lett. 2005; 246: 87-90
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• We report here the identification of a novel domain - DIM (N-terminal domain in bacterial membrane proteins and other proteins) present exclusively in bacterial species including mycobacteria, revealed by PSI-BLAST iterative searches. DIM comprises about 53 amino acids in length with conserved Leu, Ile and Gly residues. Secondary structure prediction indicated that this domain contains two alpha-helices. DIM occurs at the N-terminus of proteins, and was found particularly but not exclusively in proteins with a transmembrane domain, and also in proteins with a FHA domain or RPT repeats. DIM-containing proteins have been reported to be involved in pathogenicity, signal transduction or small solute transport.

• Hiesberger T, Shao X, Gourley E, Reimann A, Pontoglio M, Igarashi P
• Role of the hepatocyte nuclear factor-1beta (HNF-1beta) C-terminal domain in Pkhd1 (ARPKD) gene transcription and renal cystogenesis.
• J Biol Chem. 2005; 280: 10578-86
• Display abstract

• Hepatocyte nuclear factor-1beta (HNF-1beta) is a homeodomain-containing transcription factor that regulates tissue-specific gene expression in the kidney and other epithelial organs. Mutations of HNF-1beta produce congenital cystic abnormalities of the kidney, and previous studies showed that HNF-1beta regulates the expression of the autosomal recessive polycystic kidney disease (ARPKD) gene, Pkhd1. Here we show that the C-terminal region of HNF-1beta contains an activation domain that is functional when fused to a heterologous DNA-binding domain. An HNF-1beta deletion mutant lacking the C-terminal domain interacts with wild-type HNF-1beta, binds DNA, and functions as a dominant-negative inhibitor of a chromosomally integrated Pkhd1 promoter. The activation of the Pkhd1 promoter by wild-type HNF-1beta is stimulated by sodium butyrate or coactivators CREB (cAMP-response element)-binding protein (CBP) and P/CAF. The interaction with CBP and P/CAF requires the C-terminal domain. Expression of an HNF-1beta C-terminal deletion mutant in transgenic mice produces renal cysts, increased cell proliferation, and dilatation of the ureter similar to mice with kidney-specific inactivation of HNF-1beta. Pkhd1 expression is inhibited in cystic collecting ducts but not in non-cystic proximal tubules, despite transgene expression in this nephron segment. We conclude that the C-terminal domain of HNF-1beta is required for the activation of the Pkhd1 promoter. Deletion mutants lacking the C-terminal domain function as dominant-negative mutants, possibly by preventing the recruitment of histone acetylases to the promoter. Cyst formation correlates with inhibition of Pkhd1 expression, which argues that mutations of HNF-1beta produce kidney cysts by down-regulating the ARPKD gene, Pkhd1. Expression of HNF-1alpha in proximal tubules may protect against cystogenesis.

• Tekin M, Akcayoz D, Incesulu A
• A novel missense mutation in a C2 domain of OTOF results in autosomal recessive auditory neuropathy.
• Am J Med Genet A. 2005; 138: 6-10
• Display abstract

• Screening of 12 Turkish families with apparently autosomal recessive nonsyndromic sensorineural deafness without GJB2 and mtDNA m.1555A > G mutations for 11 previously mapped recessive deafness loci showed a family in which hearing loss cosegregated with the DFNB9 (OTOF) locus. Three affected children were later found to carry a novel homozygous c.3032T > C (p.Leu1011Pro) mutation in the OTOF gene. Both parents were heterozygous for the mutation. p.Leu1011Pro alters a conserved leucine residue in the C2D domain of otoferlin. Pure tone audiometry of the family showed severe to profound sensorineural hearing loss (with U-shape audiograms) in children, and normal hearing in the parents. Otoacoustic emissions and auditory brainstem response (ABR) suggested the presence of auditory neuropathy in affected individuals.

• Bateman A, Holden MT, Yeats C
• The G5 domain: a potential N-acetylglucosamine recognition domain involved in biofilm formation.
• Bioinformatics. 2005; 21: 1301-3
• Display abstract

• SUMMARY: Biofilms are complex microbial communities found at surfaces that are often associated with extracellular polysaccharides. Biofilm formation is a complex process that is being understood at the molecular level only recently. We have identified a novel domain that we call the G5 domain (named after its conserved glycine residues), which is found in a variety of enzymes such as Streptococcal IgA peptidases and various glycosyl hydrolases in bacteria. The G5 domain is found in the Accumulation Associated Protein (AAP), which is an important component in biofilm formation in Staphylococcus aureus. A common feature of the proteins containing G5 domains is N-acetylglucosamine binding, and we attribute this function to the G5 domain. CONTACT: agb@sanger.ac.uk.

• Gough J
• Convergent evolution of domain architectures (is rare).
• Bioinformatics. 2005; 21: 1464-71
• Display abstract

• MOTIVATION: In this paper, we shall examine the evolution of domain architectures across 62 genomes of known phylogeny including all kingdoms of life. We look in particular at the possibility of convergent evolution, with a view to determining the extent to which the architectures observed in the genomes are due to functional necessity or evolutionary descent. We used domains of known structure, because from this and other information we know their evolutionary relationships. We use a range of methods including phylogenetic grouping, sequence similarity/alignment, mutation rates and comparative genomics to approach this difficult problem from several angles. RESULTS: Although we do not claim an exhaustive analysis, we conclude that between 0.4 and 4% of sequences are involved in convergent evolution of domain architectures, and expect the actual number to be close to the lower bound. We also made two incidental observations, albeit on a small sample: the events leading to convergent evolution appear to be random with no functional or structural preferences, and changes in the number of tandem repeat domains occur more readily than changes which alter the domain composition. CONCLUSION: The principal conclusion is that the observed domain architectures of the sequences in the genomes are driven by evolutionary descent rather than functional necessity. CONTACT: gough@supfam.org.

• Andre S, Siebert HC, Nishiguchi M, Tazaki K, Gabius HJ
• Evidence for lectin activity of a plant receptor-like protein kinase by application of neoglycoproteins and bioinformatic algorithms.
• Biochim Biophys Acta. 2005; 1725: 222-32
• Display abstract

• Detection of genes for putative receptor-like protein kinases, which contain an extracellular domain related to leguminous lectins, in plant genomes inspired the hypothesis that this part acts as sensor. Initial support for this concept came from proof for protein kinase activity. The next step, focusing on the protein of lombardy poplar (Populus nigra var. italica), is scrutiny for lectin activity. Consequently, we first pinpointed sets of high-scoring sequence pairs by extensive databank search. The calculations resulted in P-values in the range from 10(-14) to 10(-18) exclusively for leguminous lectins, the Pterocarpus angolensis agglutinin being front runner with P=3 x 10(-18) and thus most suitable template for modeling. The superimposition of the two folds gave notable similarity in the region responsible for binding carbohydrate and Ca(2+)/Mn(2+)-ions. Binding activity toward carbohydrates was detected by assaying a panel of (neo)glycoproteins as polyvalent probes, especially for alpha-l-rhamnose and glycans of asialofetuin. It was strictly dependent on Ca(2+)-ions, enhanced by Mn(2+)-ions and reached a K(D)-value of 34.3 nM for the neoglycoprotein with rhamnose as ligand. These results give further research direction to define physiological ligands, plant/bacterial rhamnose-containing saccharides and rhamnose-mimetic glycans or peptides being potential candidates.

• Lim G et al.
• An integrated mBAND and submegabase resolution tiling set (SMRT) CGH array analysis of focal amplification, microdeletions, and ladder structures consistent with breakage-fusion-bridge cycle events in osteosarcoma.
• Genes Chromosomes Cancer. 2005; 42: 392-403
• Display abstract

• Osteosarcoma (OS) is characterized by chromosomal instability and high-copy-number gene amplification. The breakage-fusion-bridge (BFB) cycle is a well-established mechanism of genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high-resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB-dicentric chromosomes, inverted duplications, and intra- and interchromosomal amplifications-were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG-63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21-p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.

• Losekoot M, Haarloo C, Ruivenkamp C, White SJ, Breuning MH, Peters DJ
• Analysis of missense variants in the PKHD1-gene in patients with autosomal recessive polycystic kidney disease (ARPKD).
• Hum Genet. 2005; 118: 185-206
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of polycystic kidney disease characterized by enlarged kidneys and congenital hepatic fibrosis. Given the poor prognosis for the majority of children with the severe perinatal ARPKD phenotype, there is a regular request for prenatal testing. ARPKD is caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which consists of 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest transcript, comprising 67 exons, encodes the protein fibrocystin/polyductin. We have set up mutation analysis by direct sequencing of these 67 exons. In 39 mainly Dutch families we identified: 11 nonsense mutations, 15 deletions/insertions, 5 splice site mutations, and 39 missense mutations. To classify missense variants we combined evolutionary conservation, using the human, chimpanzee, dog, mouse, chicken and frog Pkhd1 sequences, with the Grantham score for chemical differences. Thirty-three missense mutations were considered pathogenic and seven were classified as rare, probably pathogenic variants. In addition to sequence analysis, multiplex ligation-dependent probe amplification (MLPA) was used to identify multiple exon deletions. However, no large deletions in the PKHD1 gene were identified. In 31 index patients two mutations were found, in 6 patients one mutation was found, leading to a mutation detection rate of 87%. The analysis of amino acid conservation as well as applying the Grantham score for chemical differences allowed us to determine the pathogeneity for nearly all new missense mutations and thus proved to be useful tools to classify missense variants.

• Wohlke A, Drogemuller C, Kuiper H, Leeb T, Distl O
• Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2.
• Gene. 2005; 348: 73-81
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• The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

• Shen H, Chou KC
• Using optimized evidence-theoretic K-nearest neighbor classifier and pseudo-amino acid composition to predict membrane protein types.
• Biochem Biophys Res Commun. 2005; 334: 288-92
• Display abstract

• Knowledge of membrane protein type often provides crucial hints toward determining the function of an uncharacterized membrane protein. With the avalanche of new protein sequences emerging during the post-genomic era, it is highly desirable to develop an automated method that can serve as a high throughput tool in identifying the types of newly found membrane proteins according to their primary sequences, so as to timely make the relevant annotations on them for the reference usage in both basic research and drug discovery. Based on the concept of pseudo-amino acid composition [K.C. Chou, Proteins: Struct. Funct. Genet. 43 (2001) 246-255; Erratum: Proteins: Struct. Funct. Genet. 44 (2001) 60] that has made it possible to incorporate a considerable amount of sequence-order effects by representing a protein sample in terms of a set of discrete numbers, a novel predictor, the so-called "optimized evidence-theoretic K-nearest neighbor" or "OET-KNN" classifier, was proposed. It was demonstrated via the self-consistency test, jackknife test, and independent dataset test that the new predictor, compared with many previous ones, yielded higher success rates in most cases. The new predictor can also be used to improve the prediction quality for, among many other protein attributes, structural class, subcellular localization, enzyme family class, and G-protein coupled receptor type. The OET-KNN classifier will be available as a web-server at http://www.pami.sjtu.edu.cn/kcchou.

• Qi G, Lee R, Hayward S
• A comprehensive and non-redundant database of protein domain movements.
• Bioinformatics. 2005; 21: 2832-8
• Display abstract

• MOTIVATION: The current DynDom database of protein domain motions is a user-created database that suffers from selectivity and redundancy. The aim of the analysis presented here was to overcome both these limitations and to produce both a comprehensive and a non-redundant description of domain movements from structures stored in the current protein data bank. RESULTS: A multi-step procedure is applied that starts with grouping proteins in the structural databank into families based on sequence similarity. Multiple sequence alignment, conformational clustering and a dimensional clustering method based on the Gram-Schmidt algorithm are applied to members of each family to remove dynamic redundancy in their domain movements. Representative domain movements are described in terms of domains, hinge axes and hinge-bending residues using the DynDom program. The results show that within an average family of 11.5 members, there are on average only 1.31 different domain movements indicating a high redundancy in the movements these structures represent. This verifies earlier findings that domain movements are usually highly controlled. Despite the removal of this considerable redundancy, the process has resulted in double the number of domain movements stored in the user-created database. The data are organized in a relational database with a web-interface. AVAILABILITY: The database can be browsed and searched at http://www.cmp.uea.ac.uk/dyndom CONTACT: sjh@cmp.uea.ac.uk.

• Jimenez A et al.
• Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis.
• J Biol Chem. 2004; 279: 34971-82
• Display abstract

• Mammalian germ cells are endowed with a complete set of thioredoxins (Trx), a class of redox proteins located in specific structures of the spermatid and sperm tail. We report here the characterization, under normal and pathological conditions, of a novel thioredoxin with a germ line-restricted expression pattern, named spermatocyte/spermatid-specific thioredoxin-3 (SPTRX-3). The human SPTRX-3 gene maps at 9q32, only 50 kb downstream from the TRX-1 gene from which it probably originated as genomic duplication. Therefore, human SPTRX-3 protein comprises a unique thioredoxin domain displaying high homology with the ubiquitously expressed TRX-1. Among the tissues investigated, Sptrx-3 mRNA is found exclusively in the male germ cells at pachytene spermatocyte and round spermatid stages. Light and electron microscopy show SPTRX-3 protein to be predominately located in the Golgi apparatus of pachytene spermatocytes and round and elongated spermatids, with a transient localization in the developing acrosome of round spermatids. In addition, increased levels of SPTRX-3, possibly caused by overexpression, are observed in morphologically abnormal human spermatozoa from infertile men. In addition, SPTRX-3 is identified as a novel postobstruction autoantigen. In this report, we propose that SPTRX-3 can be used as a specific marker for diverse sperm and testis pathologies. SPTRX-3 is the first thioredoxin specific to the Golgi apparatus, and its function within this organelle might be related to the post-translational modification of proteins required for germ cell-specific functions, such as acrosomal biogenesis.

• Bergmann C et al.
• PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD).
• Hum Mutat. 2004; 23: 453-63
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD) is an important cause of childhood renal- and liver-related morbidity and mortality. The clinical spectrum is widely variable. About 30 to 50% of affected individuals die in the neonatal period, while others survive into adulthood. ARPKD is caused by mutations in the PKHD1 (polycystic kidney and hepatic disease 1) gene on chromosome 6p12, which is among the largest human genes, with a minimum of 86 exons assembled into a variety of alternatively spliced transcripts. The longest continuous open reading frame is predicted to yield a 4,074-aa (447-kDa) multidomain integral membrane protein (fibrocystin/polyductin) of unknown function. This update compiles all known PKHD1 mutations and polymorphisms/sequence variants. Mutations were found to be scattered throughout the gene without evidence of clustering at specific sites. Most PKHD1 mutations are unique to single families ("private mutations") hampering genotype-phenotype correlations. Correlations have been drawn for the type of mutation rather than for the site of individual mutations. All patients carrying two truncating mutations displayed a severe phenotype with perinatal or neonatal demise, while patients surviving the neonatal period bear at least one missense mutation. However, some missense changes are obviously as devastating as truncating mutations. The present article intends 1) to provide an overview of PKHD1 mutations and polymorphisms/sequence variants identified so far, 2) to discuss potential genotype-phenotype correlations, and 3) to review them in the context of their clinical implications. A constantly updated list of mutations is available online (www.humgen.rwth-aachen.de) and investigators are invited to submit their novel data to this PKHD1 mutation database.

• Gebauer D, Li J, Jogl G, Shen Y, Myszka DG, Tong L
• Crystal structure of the PH-BEACH domains of human LRBA/BGL.
• Biochemistry. 2004; 43: 14873-80
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• The beige and Chediak-Higashi syndrome (BEACH) domain defines a large family of eukaryotic proteins that have diverse cellular functions in vesicle trafficking, membrane dynamics, and receptor signaling. The domain is the only module that is highly conserved among all of these proteins, but the exact functions of this domain and the molecular basis for its actions are currently unknown. Our previous studies showed that the BEACH domain is preceded by a novel, weakly conserved pleckstrin homology (PH) domain. We report here the crystal structure at 2.4 A resolution of the PH-BEACH domain of human LRBA/BGL. The PH domain has the same backbone fold as canonical PH domains, despite sharing no sequence homology with them. However, our binding assays demonstrate that the PH domain in the BEACH proteins cannot bind phospholipids. The BEACH domain contains a core of several partially extended peptide segments that is flanked by helices on both sides. The structure suggests intimate association between the PH and the BEACH domains, and surface plasmon resonance studies confirm that the two domains of the protein FAN have high affinity for each other, with a K(d) of 120 nM.

• Yang H, Wu C, Zhao S, Guo J
• Identification and characterization of D8C, a novel domain present in liver-specific LZP, uromodulin and glycoprotein 2, mutated in familial juvenile hyperuricaemic nephropathy.
• FEBS Lett. 2004; 578: 236-8
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• Present work reported a novel domain--D8C (domain with conserved eight cysteines in liver-specific ZP domain-containing protein, glycoprotein 2 (GP-2) and uromodulin (UMOD)), present in liver-specific LZP, UMOD, GP-2 and some uncharacterized proteins, most of which are membrane proteins, extracellular proteins or nuclear membrane proteins. D8C contains eight well-conserved cysteine residues, which were predicted to form four pairs of disulfide bridges. D8C is composed mainly of beta-strands. Mutation in the D8C at Cys217 in human UMOD is associated with familial juvenile hyperuricaemic nephropathy, which might be due to the disruption of the disulfide bridge. Identification of D8C would further the understandings of related proteins.

• Anantharaman V, Aravind L
• Novel conserved domains in proteins with predicted roles in eukaryotic cell-cycle regulation, decapping and RNA stability.
• BMC Genomics. 2004; 5: 45-45
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• BACKGROUND: The emergence of eukaryotes was characterized by the expansion and diversification of several ancient RNA-binding domains and the apparent de novo innovation of new RNA-binding domains. The identification of these RNA-binding domains may throw light on the emergence of eukaryote-specific systems of RNA metabolism. RESULTS: Using sensitive sequence profile searches, homology-based fold recognition and sequence-structure superpositions, we identified novel, divergent versions of the Sm domain in the Scd6p family of proteins. This family of Sm-related domains shares certain features of conventional Sm domains, which are required for binding RNA, in addition to possessing some unique conserved features. We also show that these proteins contain a second previously uncharacterized C-terminal domain, termed the FDF domain (after a conserved sequence motif in this domain). The FDF domain is also found in the fungal Dcp3p-like and the animal FLJ22128-like proteins, where it fused to a C-terminal domain of the YjeF-N domain family. In addition to the FDF domains, the FLJ22128-like proteins contain yet another divergent version of the Sm domain at their extreme N-terminus. We show that the YjeF-N domains represent a novel version of the Rossmann fold that has acquired a set of catalytic residues and structural features that distinguish them from the conventional dehydrogenases. CONCLUSIONS: Several lines of contextual information suggest that the Scd6p family and the Dcp3p-like proteins are conserved components of the eukaryotic RNA metabolism system. We propose that the novel domains reported here, namely the divergent versions of the Sm domain and the FDF domain may mediate specific RNA-protein and protein-protein interactions in cytoplasmic ribonucleoprotein complexes. More specifically, the protein complexes containing Sm-like domains of the Scd6p family are predicted to regulate the stability of mRNA encoding proteins involved in cell cycle progression and vesicular assembly. The Dcp3p and FLJ22128 proteins may localize to the cytoplasmic processing bodies and possibly catalyze a specific processing step in the decapping pathway. The explosive diversification of Sm domains appears to have played a role in the emergence of several uniquely eukaryotic ribonucleoprotein complexes, including those involved in decapping and mRNA stability.

• Zhu Q, Deng Y, Vanka P, Brown SJ, Muthukrishnan S, Kramer KJ
• Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome.
• Bioinformatics. 2004; 20: 161-9
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• MOTIVATION: Multiple chitinases as well as lectins closely related to them have been characterized previously from many insect species and the corresponding genes/cDNAs have been cloned. However, the identification of the entire assortment of genes for chitinase family proteins and their differences in biochemical properties have not been carried out in any individual insect species. The completion of the entire DNA sequence of Drosophila melanogaster (fruit fly) genome and identification of open reading frames presents an opportunity to study the structures and functions of chitinase-like proteins, and also to identify new members of this family in Drosophila. We are, therefore, interested in studying the functional genomics of chitinase-like gene families in insects. METHODS: We searched the Drosophila protein sequences database using fully characterized insect chitinase sequences and BLASTP software, identified all the putative chitinase-like proteins encoded in Drosophila genome, and predicted their structures using domain analysis tools. A phylogenetic analysis of the chitinase-like proteins from Drosophila and several other insect species was carried out. The structures of these chitinases were modeled using homology modeling software. RESULTS: Our analysis revealed the presence of 18 chitinase-like proteins in the Drosophila protein database. Among these are seven novel chitinase-like proteins that contain four signature amino acid sequences of chitinases belonging to family 18 glycosylhydrolases, including both acidic and hydrophobic amino acid residues critical for enzyme activity. All the proteins contain at least one catalytic domain with one having four catalytic domains. Phylogenetic analysis of chitinase-like proteins from Drosophila and other insects revealed an evolutionary relationship among all these proteins, which indicated gene duplication and domain shuffling to generate the observed diversity in the encoded proteins. Homology modeling showed that all the Drosophila chitinase-like proteins contain one or more catalytic domains with a (alpha/beta)8 barrel-like structure. Our results suggest that insects utilize multiple family 18 chitinolytic enzymes and also non-enzymatic chitinase-like proteins for degrading/remodeling/binding to chitin in different insect anatomical extracellular structures, such as the cuticle, peritrophic membrane, trachea and mouth parts during insect development, and possibly for other roles including chitin synthesis. AVAILABILITY: Perl program and supplementary material are available at http://www.ksu.edu/bioinformatics/supplementary.htm

• Tominaga K, Johmura Y, Nishizuka M, Imagawa M
• Fad24, a mammalian homolog of Noc3p, is a positive regulator in adipocyte differentiation.
• J Cell Sci. 2004; 117: 6217-26
• Display abstract

• Adipocyte differentiation is controlled by complex actions involving gene expression and signal transduction. From metaphase to anaphase, peroxisome proliferator-activated receptor gamma, the CCAAT/enhancer-binding protein family and sterol regulatory element-binding protein-1 are known to function as master regulators. However, the mechanism underlying the earliest step, which triggers the initiation of differentiation, remains unknown. In previous reports, we have isolated a number of genes, whose expression increases in the early stage of differentiation in the mouse 3T3-L1 preadipocyte cell line. Here we report the cloning of the full-length cDNA and characterization of an unknown gene isolated previously and named fad24 (factor for adipocyte differentiation 24). Fad24 encodes a protein consisting of 807 amino acids. The deduced amino acid sequence was shown to have a basic leucine zipper motif and a NOC domain. Expression of fad24 was rapidly induced after stimulation with inducers. Furthermore, overexpression of fad24 in NIH-3T3 cells promoted adipogenesis in the presence of a ligand for peroxisome proliferator-activated receptor gamma. FAD24 localizes in the nucleus, especially within nuclear speckles. As the nuclear speckle functions as a nascent transcription and pre-mRNA splicing machinery, there is a possibility that FAD24 functions as one of the components for transcription and/or pre-mRNA splicing and positively regulates adipocyte differentiation.

• Wang Z, Peters B, Klussmann S, Bender H, Herb A, Krieglstein K
• Gene structure and evolution of Tieg3, a new member of the Tieg family of proteins.
• Gene. 2004; 325: 25-34
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• TGF beta-inducible immediate early gene, Tieg, belongs to the superfamily of Sp1-like transcription factors containing three C(2)H(2)-zinc finger DNA binding motifs close to the C-terminus. So far, Tieg1 and Tieg2 have been identified in human and mouse. We identified Tieg3, a new member of the Tieg protein family by screening a mouse cDNA library. Tieg3 has almost all the known features of the Tieg protein family: it shares a highly conserved C(2)H(2) zinc finger DNA binding domain and is 96% identical to Tieg2 and 86% to Tieg1, respectively. In addition, the three repression domains at the N-terminus, R1, R2 and R3 are conserved in all the Tiegs. Similar to Tieg1 and Tieg2, Tieg3 mRNA is up-regulated in response to TGF beta 1 treatment and can perform the Sp1 sites mediated repression of transcription. A 4 kilobase (kb) long transcript of mouse Tieg3 can be detected using Northern-blot analysis. The gene of mouse Tieg3 contains four exons. Due to the amino acid sequence similarity, mouse Tieg2 is regarded as an orthologue of human Tieg2. However, the mouse Tieg3 gene is localized in a conserved segment on mouse chromosome 12 corresponding to human Tieg2 on chromosome 2 with the same gene order. An interesting explanation for this apparent contradiction might be a homologous recombination leading to loci exchange between the mouse Tieg3 and Tieg2.

• Zhang MZ et al.
• PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells.
• Proc Natl Acad Sci U S A. 2004; 101: 2311-6
• Display abstract

• Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.

• Tan JM, Tock EP, Chow VT
• The novel human MOST-1 (C8orf17) gene exhibits tissue specific expression, maps to chromosome 8q24.2, and is overexpressed/amplified in high grade cancers of the breast and prostate.
• Mol Pathol. 2003; 56: 109-15
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• AIMS: To elucidate genes that participate in the process of oncogenesis, primers based on the E6 genes of genital human papillomaviruses (HPVs) were used to amplify potential expressed sequence tags (ESTs) from the MOLT-4 T lymphoblastic leukaemia cell line. METHODS: Using the polymerase chain reaction (PCR) with human papillomavirus E6 gene primers, an EST from the MOLT-4 T lymphoblastic leukaemia cell line was amplified. Via rapid amplification of cDNA ends (RACE) and cycle sequencing from MOLT-4 and fetal lung cDNA libraries, overlapping cDNAs of 2786 bp and 2054 bp of the corresponding novel human intronless gene designated MOST-1 (for MOLT-4 sequence tag-1) were characterised and assigned the symbol C8orf17 by the HUGO Nomenclature Committee. RESULTS: Both cDNAs contained a potential open reading frame (ORF) of 297 bp incorporating a methionine codon with an ideal Kozak consensus sequence for translation initiation, and encoding a putative hydrophilic polypeptide of 99 amino acids. Although reverse transcription PCR (RT-PCR) demonstrated MOST-1 expression in all 19 cancer and two normal cell lines tested, differential expression was seen in only nine of 16 normal tissues tested (heart, kidney, liver, pancreas, small intestine, ovary, testis, prostate, and thymus). A 388 bp fragment was amplified from the NS-1 mouse myeloma cell line, the sequence of which was identical to that within the MOST-1 ORF. The MOST-1 gene was mapped by fluorescent in situ hybridisation to chromosome 8q24.2, a region amplified in many breast cancers and prostate cancers, which is also the candidate site of potential oncogene(s) other than c-myc located at 8q24.1. Analysis of paired biopsies of invasive ductal breast cancer and adjacent normal tissue by semiquantitative and real time RT-PCR revealed average tumour to normal ratios of MOST-1 expression that were two times greater in grade 3 cancers than in grade 1 and 2 cancers. Quantitative real time PCR of archival prostatic biopsies displayed MOST-1 DNA values that were 9.9, 7.5, 4.2, and 1.4 times higher in high grade carcinomas, intermediate grade carcinomas, low grade carcinomas, and benign hyperplasias, respectively, than in normal samples. CONCLUSIONS: These data suggest a role for MOST-1 in cellular differentiation, proliferation, and carcinogenesis.

• Bergmann C et al.
• Spectrum of mutations in the gene for autosomal recessive polycystic kidney disease (ARPKD/PKHD1).
• J Am Soc Nephrol. 2003; 14: 76-89
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• Autosomal recessive polycystic kidney disease (ARPKD/PKHD1) is an important cause of renal-related and liver-related morbidity and mortality in childhood. Recently mutations in the PKHD1 gene on chromosome 6p21.1-p12 have been identified as the molecular cause of ARPKD. The longest continuous open reading frame (ORF) is encoded by a 67-exon transcript and predicted to yield a 4074-amino acid protein ("polyductin") of thus far unknown function. By now, a total of 29 different PKHD1 mutations have been described. This study reports mutation screening in 90 ARPKD patients and identifies mutations in 110 alleles making up a detection rate of 61%. Thirty-four of the detected mutations have not been reported previously. Two underlying mutations in 40 patients and one mutation in 30 cases are disclosed, and no mutation was detected on the remaining chromosomes. Mutations were found to be scattered throughout the gene without evidence of clustering at specific sites. About 45% of the changes were predicted to truncate the protein. All missense mutations were nonconservative, with the affected amino acid residues found to be conserved in the murine polyductin orthologue. One recurrent missense mutation (T36M) likely represents a mutational hotspot and occurs in a variety of populations. Two founder mutations (R496X and V3471G) make up about 60% of PKHD1 mutations in the Finnish population. Preliminary genotype-phenotype correlations could be established for the type of mutation rather than for the site of the individual mutation. All patients carrying two truncating mutations displayed a severe phenotype with perinatal or neonatal demise. PKHD1 mutation analysis is a powerful tool to establish the molecular cause of ARPKD in a given family. Direct identification of mutations allows an unequivocal diagnosis and accurate genetic counseling even in families displaying diagnostic challenges.

• Domingues FS, Lengauer T
• Protein function from sequence and structure data.
• Appl Bioinformatics. 2003; 2: 3-12
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• With the large amount of genomics and proteomics data that we are confronted with, computational support for the elucidation of protein function becomes more and more pressing. Many different kinds of biological data harbour signals of protein function, but these signals are often concealed. Computational methods that use protein sequence and structure data can be used for discovering these signals. They provide information that can substantially speed up experimental function elucidation. In this review we concentrate on such methods.

• Hogan MC, Griffin MD, Rossetti S, Torres VE, Ward CJ, Harris PC
• PKHDL1, a homolog of the autosomal recessive polycystic kidney disease gene, encodes a receptor with inducible T lymphocyte expression.
• Hum Mol Genet. 2003; 12: 685-98
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• Autosomal-recessive polycystic kidney disease (ARPKD) is caused by mutation to a large gene, PKHD1, encoding a putative receptor protein, fibrocystin. We have identified, through analysis of human genomic sequence, a PKHD1 homolog, PKHDL1, in chromosome region 8q23. The PKHDL1 transcript of 13081 bp was amplified as 16 fragments and sequenced; the sequence of the murine ortholog, Pkhdl1 (chromosome region 15B3) was also determined. PKHDL1 contains 78 exons, covers a genomic region of approximately 168 kb and encodes a large protein, fibrocystin-L. Screening PKHDL1 in ARPKD patients with no PKHD1 mutations revealed several sequence variants but no clear mutations, making it unlikely that it is ARPKD-associated. Human fibrocystin-L is predicted to be a large receptor protein (4243 aa; 466 kDa) with a signal peptide, single transmembrane domain and short cytoplasmic tail. Fibrocystin-L is homologous to fibrocystin throughout most of the extracellular region with overall identity of 25.0% and similarity of 41.5%. Fibrocystin-L has extracellular domains similar to fibrocystin with 14 copies of the TIG domain and two regions of significant homology to the protein TMEM2. Genomic sequence analysis identified no other full-length fibrocystin homologs in humans, mice or other sequenced organisms. The Fugu fish has a fibrocystin-L ortholog but no fibrocystin, suggesting that the newly identified protein may be the ancestral form. PKHDL1 and Pkhdl1 are widely expressed at a low level in most tissues but only detected in blood-derived cell-lines. Low level expression was detected in many primary immune cell subtypes but up-regulated specifically in T lymphocytes, following activation signals, suggesting a role in cellular immunity.

• Ludes-Meyers JH, Bednarek AK, Popescu NC, Bedford M, Aldaz CM
• WWOX, the common chromosomal fragile site, FRA16D, cancer gene.
• Cytogenet Genome Res. 2003; 100: 101-10
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• Gross chromosomal rearrangements and aneuploidy are among the most common somatic genomic abnormalities that occur during cancer initiation and progression, in particular in human solid tumor carcinogenesis. The loss of large chromosomal regions as consequence of gross rearrangements (e.g. deletions, monosomies, unbalanced translocations and mitotic recombination) have been traditionally associated with the existence of tumor suppressor genes within the areas affected by the loss of genetic material. The long arm of chromosome 16 was identified as being frequently associated with structural abnormalities in multiple neoplasias, that led us to focus attention on the detailed genetic dissection of this region resulting in the cloning of the putative tumor suppressor gene, WWOX (WW domain containing Oxidoreductase). Interestingly, the WWOX gene resides in the very same region as that of the common chromosomal fragile site 16D (FRA16D). The WWOX gene encodes a protein that contains two WW domains, involved in protein-protein interactions, and a short chain dehydrogenase (SDR) domain, possibly involved in sex-steroid metabolism. We have identified the WWOX WW domain ligand as the PPXY motif confirming the biochemical activity of this domain. WWOX normally resides in the Golgi and we will demonstrate that Golgi localization requires an intact SDR. Inactivation of the WWOX gene during tumorigenesis can occur by homozygous deletions and possibly mutation, however, aberrantly spliced forms of WWOX mRNA have been observed even when one allele is still intact. The aberrantly spliced mRNAs have deletions of the exons that encode the SDR and these WWOX protein isoforms display abnormal intracellular localization to the nucleus possibly functioning as dominant negative inhibitors of full length WWOX. Thus, generation of aberrant transcripts of WWOX may represent a novel mechanism to functionally inactivate WWOX without genomic alteration of the remaining allele. In this article we will review the cloning and identification of WWOX as the target of FRA16D. In addition, we will discuss the possible biochemical functions of WWOX and present evidence that ectopic WWOX expression inhibits tumor growth.

• Suyama M, Ohara O
• DomCut: prediction of inter-domain linker regions in amino acid sequences.
• Bioinformatics. 2003; 19: 673-4
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• DomCut is a program to predict inter-domain linker regions solely by amino acid sequence information. The prediction is made by using linker index deduced from a data set of domain/linker segments. The linker preference profile, which is the averaged linker index along a sequence, can be visualized in the graphical interface.

• Ishizuka M et al.
• Molecular cloning and characteristics of a novel zinc finger protein and its splice variant whose transcripts are expressed during spermatogenesis.
• Biochem Biophys Res Commun. 2003; 301: 1079-85
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• Testicular zinc finger protein (TZF) has a zinc finger motif of the Cys2-His2 type and its transcript is expressed predominantly in mouse spermatogenic cells. Using the fragment of TZF as a probe, we isolated the alternative splice variant form (TZF-L) from mouse testis cDNA library. Analysis of the open reading frame of each cDNA indicated that TZF and TZF-L were polypeptides of 942 and 2025 amino acid residues, respectively, and the N-terminal 902 amino acids of TZF-L were identical to those of TZF. The C-terminal region of TZF-L had more a zinc finger motif of the Cys2-His2 type and poly-Glu and poly-Pro regions. The mouse TZF/TZF-L gene spanned >20 kb and consisted of 11 exons. RT-PCR analysis of the expression level of mRNAs for mouse TZF and TZF-L showed that both transcripts are highly expressed in testis and moderately in kidney and ovary. Elevated expression of both transcripts during testicular development in mice was restricted to spermatocytes at the pachytene stage of meiotic prophase. Fusion proteins with GFP also demonstrated the nuclear localization of TZF and TZF-L. These experiments suggest that TZF and TZF-L may act to control the gene activity during spermatogenesis.

• Furu L et al.
• Milder presentation of recessive polycystic kidney disease requires presence of amino acid substitution mutations.
• J Am Soc Nephrol. 2003; 14: 2004-14
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD; MIM 263200) is a hereditary and severe form of polycystic disease affecting the kidneys and biliary tract with an estimated incidence of 1 in 20,000 live births. The clinical spectrum is widely variable: up to 50% of affected neonates die shortly after birth, whereas others survive to adulthood. Mutations at a single locus, polycystic kidney and hepatic disease 1 (PKHD1), are responsible for all typical forms of ARPKD. Mutation detection was performed in PKHD1 by DHPLC in 85 affected, unrelated individuals. Seventy-four amplicons were amplified and analyzed from the PKHD1 genomic locus. Sequence variants were considered pathogenic when they were not observed in 160 control individuals (320 chromosomes). For purposes of genotype-phenotype comparisons, families were stratified by clinical presentation into two groups: the severe perinatal group, in which at least one affected child presented with perinatal disease and neonatal demise, and the less severe, nonperinatal group, in which none of the affected children died in the neonatal period. Forty-one mutations were found in 55 affected disease chromosomes; 32 of these mutations have not been reported previously. Mutations were distributed throughout the portions of gene encoding the predicted extracellular portion of the protein product. The most commonly encountered mutation, T36M, was found in 8 of 55 disease chromosomes. Amino acid substitutions were found to be more commonly associated with a nonlethal presentation, whereas chain terminating mutations were more commonly associated with neonatal demise (chi(2) = 11.54, P = 0.003). All patients who survive the neonatal period have at least one amino acid substitution mutation, suggesting that such substitutions produce milder disease through production of partially functional protein products. The nature of the germline mutations in ARPKD plays a significant role in determining clinical outcome.

• Karell K et al.
• HLA types in celiac disease patients not carrying the DQA1*05-DQB1*02 (DQ2) heterodimer: results from the European Genetics Cluster on Celiac Disease.
• Hum Immunol. 2003; 64: 469-77
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• Genetic susceptibility to celiac disease is strongly associated with HLA-DQA1*05-DQB1*02 (DQ2) and HLA-DQA1*03-DQB1*0302 (DQ8). Study of the HLA associations in patients not carrying these heterodimers has been limited by the rarity of such patients. This European collaboration has provided a unique opportunity to study a large series of such patients. From 1008 European coeliacs, 61 were identified who neither carry the DQ2 nor DQ8 heterodimers. Fifty seven of these encoded half of the DQ2 heterodimer. The remaining 4 patients had a variety of clinical presentations. Three of them carried the DQA1*01-DQB*05 haplotype as did 20/61 of those carrying neither DQ2 nor DQ8. This may implicate a role of the DQA1*01-DQB*05 haplotype. None of these four patients carried the DQB1*06 allele that has previously been reported in this sub-group of patients. Of the 16 DQ2 heterodimer negative patients without DRB1*04 or DRB1*07 haplotypes, it was inferred that none encoded the previously implicated DRB4 gene as none had a DRB1*09 haplotype. These results underline the primary importance of HLA-DQ alleles in susceptibility to celiac disease, and the extreme rarity of celiac patients carrying neither the DQ2 or DQ8 heterodimers nor one half of the DQ2 heterodimer alone.

• Li A, Tian X, Sung SW, Somlo S
• Identification of two novel polycystic kidney disease-1-like genes in human and mouse genomes.
• Genomics. 2003; 81: 596-608
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• Mutations to the prototypical members of the two general classes of polycystins, polycystin-1 encoded by PKD1 and polycystin-2 encoded by PKD2, underlie autosomal-dominant polycystic kidney disease. Here we report the identification of a pair of genes homologous to PKD1 from both the human and mouse genomes. PKD1L2 and PKD1L3 are located on human chromosome 16q22-q23 and mouse chromosome 8 and are alternatively spliced. The human and mouse forms of PKD1L2 are highly conserved, with each one consisting of 43 exons and approximately 2,460 codons. PKD1L3 shows regional sequence divergence, with the mouse form having two additional exons and a much larger exon 5. The predicted protein products of PKD1L2 and PKD1L3 contain the combination of GPS and PLAT/LH2 domains that uniquely define them as polycystin-1 family members. They are predicted to have 11 membrane-spanning regions with a large extracellular domain consistent with the proposed receptor function of this protein family. PKD1L2 and PKD1L3 contain strong ion channel signature motifs that suggest their possible function as components of cation channel pores. Polycystin-1-related proteins may not only regulate channels, but may actually be part of the pore-forming unit.

• Van Belzen MJ et al.
• A major non-HLA locus in celiac disease maps to chromosome 19.
• Gastroenterology. 2003; 125: 1032-41
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• BACKGROUND AND AIMS: The pathogenesis of celiac disease is still unknown despite its well-known association with human leukocyte antigen (HLA)-DQ2 and DQ8. It is clear that non-HLA genes contribute to celiac disease development as well, but none of the previous genome-wide screens in celiac disease have resulted in identification of these genes. METHODS: We, therefore, performed a 2-stage, genome-wide screen in 101 affected sibpairs from 82 Dutch families who met strict diagnostic criteria. The small intestinal biopsy samples, on which the original celiac disease diagnoses had been based, showed a Marsh III lesion in all patients on reevaluation by 1 pathologist. For association analysis of markers in regions linked to celiac disease, 216 independent MIII patients and 216 age- and sex-matched controls were available. RESULTS: As expected, highly significant linkage to the HLA-region was detected (multipoint maximum lod score [MMLS] = 8.14). More importantly, significant linkage was also present at 19p13.1 (MMLS = 4.31), with the peak at marker D19S899. Moreover, this marker was also significantly associated with celiac disease in the case-control study (corrected P = 0.016). Furthermore, we identified suggestive linkage to 6q21-22, which is approximately 70 cM downstream from the HLA region (MMLS = 3.10). CONCLUSIONS: Significant linkage of celiac disease to chromosome region 19p13.1 was detected in our genome-wide screen. These results were confirmed by the association of D19S899 to celiac disease in an independent case-control cohort. Furthermore, we identified a possible second celiac disease locus on chromosome region 6q21-22.

• Li A et al.
• Mutations in PRKCSH cause isolated autosomal dominant polycystic liver disease.
• Am J Hum Genet. 2003; 72: 691-703
• Display abstract

• Autosomal dominant polycystic liver disease (ADPLD) is a distinct clinical and genetic entity that can occur independently from autosomal dominant polycystic kidney disease (ADPKD). We previously studied two large kindreds and reported localization of a gene for ADPLD to an approximately 8-Mb region, flanked by markers D19S586/D19S583 and D19S593/D19S579, on chromosome 19p13.2-13.1. Expansion of these kindreds and identification of an additional family allowed us to define flanking markers CA267 and CA048 in an approximately 3-Mb region containing >70 candidate genes. We used a combination of denaturing high-performance liquid chromatography (DHPLC) heteroduplex analysis and direct sequencing to screen a panel of 15 unrelated affected individuals for mutations in genes from this interval. We found sequence variations in a known gene, PRKCSH, that were not observed in control individuals, that segregated with the disease haplotype, and that were predicted to be chain-terminating mutations. In contrast to PKD1, PKD2, and PKHD1, PRKCSH encodes a previously described human protein termed "protein kinase C substrate 80K-H" or "noncatalytic beta-subunit of glucosidase II." This protein is highly conserved, is expressed in all tissues tested, and contains a leader sequence, an LDLa domain, two EF-hand domains, and a conserved C-terminal HDEL sequence. Its function may be dependent on calcium binding, and its putative actions include the regulation of N-glycosylation of proteins and signal transduction via fibroblast growth-factor receptor. In light of the focal nature of liver cysts in ADPLD, the apparent loss-of-function mutations in PRKCSH, and the two-hit mechanism operational in dominant polycystic kidney disease, ADPLD may also occur by a two-hit mechanism.

• Anantharaman V, Aravind L
• Application of comparative genomics in the identification and analysis of novel families of membrane-associated receptors in bacteria.
• BMC Genomics. 2003; 4: 34-34
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• BACKGROUND: A great diversity of multi-pass membrane receptors, typically with 7 transmembrane (TM) helices, is observed in the eukaryote crown group. So far, they are relatively rare in the prokaryotes, and are restricted to the well-characterized sensory rhodopsins of various phototropic prokaryotes. RESULTS: Utilizing the currently available wealth of prokaryotic genomic sequences, we set up a computational screen to identify putative 7 (TM) and other multi-pass membrane receptors in prokaryotes. As a result of this procedure we were able to recover two widespread families of 7 TM receptors in bacteria that are distantly related to the eukaryotic 7 TM receptors and prokaryotic rhodopsins. Using sequence profile analysis, we were able to establish that the first members of these receptor families contain one of two distinct N-terminal extracellular globular domains, which are predicted to bind ligands such as carbohydrates. In their intracellular portions they contain fusions to a variety of signaling domains, which suggest that they are likely to transduce signals via cyclic AMP, cyclic diguanylate, histidine phosphorylation, dephosphorylation, and through direct interactions with DNA. The second family of bacterial 7 TM receptors possesses an alpha-helical extracellular domain, and is predicted to transduce a signal via an intracellular HD hydrolase domain. Based on comparative analysis of gene neighborhoods, this receptor is predicted to function as a regulator of the diacylglycerol-kinase-dependent glycerolipid pathway. Additionally, our procedure also recovered other types of putative prokaryotic multi-pass membrane associated receptor domains. Of these, we characterized two widespread, evolutionarily mobile multi-TM domains that are fused to a variety of C-terminal intracellular signaling domains. One of these typified by the Gram-positive LytS protein is predicted to be a potential sensor of murein derivatives, whereas the other one typified by the Escherichia coli UhpB protein is predicted to function as sensor of conformational changes occurring in associated membrane proteins CONCLUSIONS: We present evidence for considerable variety in the types of uncharacterized surface receptors in bacteria, and reconstruct the evolutionary processes that model their diversity. The identification of novel receptor families in prokaryotes is likely to aid in the experimental analysis of signal transduction and environmental responses of several bacteria, including pathogens such as Leptospira, Treponema, Corynebacterium, Coxiella, Bacillus anthracis and Cytophaga.

• Edwards YJ, Cottage A
• Bioinformatics methods to predict protein structure and function. A practical approach.
• Mol Biotechnol. 2003; 23: 139-66
• Display abstract

• Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.

• Xiong H et al.
• A novel gene encoding a TIG multiple domain protein is a positional candidate for autosomal recessive polycystic kidney disease.
• Genomics. 2002; 80: 96-104
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.

• Chong JM, Uren A, Rubin JS, Speicher DW
• Disulfide bond assignments of secreted Frizzled-related protein-1 provide insights about Frizzled homology and netrin modules.
• J Biol Chem. 2002; 277: 5134-44
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• Secreted Frizzled-related protein-1 (sFRP-1), a soluble protein that binds to Wnts and modulates Wnt signaling, contains an N-terminal domain homologous to the putative Wnt-binding site of Frizzled (Fz domain) and a C-terminal heparin-binding domain with weak homology to netrin. Both domains are cysteine-rich, having 10 and 6 cysteines in the Fz and heparin-binding domains, respectively. In this study, the disulfide linkages of recombinant sFRP-1 were determined. Numbering sFRP-1 cysteines sequentially from the N terminus, the five disulfide linkages in the Fz domain are 1-5, 2-4, 3-8, 6-10, and 7-9, consistent with the disulfide pattern determined for homologous domains of several other proteins. The disulfide linkages of the heparin-binding domain are 11-14, 12-15, and 13-16. This latter set of assignments provides experimental verification of one of the disulfide patterns proposed for netrin (NTR) modules and thereby supports the prediction that the C-terminal heparin-binding domain of sFRP-1 is an NTR-type domain. Interestingly, two subsets of sFRPs appear to have alternate disulfide linkage patterns compared with sFRP-1, one of which involves the loss of a disulfide due to deletion of a single cysteine from the NTR module, whereas the remaining cysteine may pair with a new cysteine introduced in the Fz domain of the protein. Analysis of glycosylation sites showed that sFRP-1 contains a relatively large carbohydrate moiety on Asn(172) (approximately 2.8 kDa), whereas Asn(262), the second potential N-linked glycosylation site, is not modified. No O-linked carbohydrate groups were detected. There was evidence of heterogeneous proteolytic processing at both the N and C termini of the recombinant protein. The predominant N terminus was Ser(31), although minor amounts of the protein with Asp(41) and Phe(50) as the N termini were observed. The major C-terminal processing event was removal of the terminal amino acid (Lys(313)) with only a trace amount of unprocessed protein detected.

• Ward CJ et al.
• The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.
• Nat Genet. 2002; 30: 259-69
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD) is characterized by dilation of collecting ducts and by biliary dysgenesis and is an important cause of renal- and liver-related morbidity and mortality. Genetic analysis of a rat with recessive polycystic kidney disease revealed an orthologous relationship between the rat locus and the ARPKD region in humans; a candidate gene was identified. A mutation was characterized in the rat and screening the 66 coding exons of the human ortholog (PKHD1) in 14 probands with ARPKD revealed 6 truncating and 12 missense mutations; 8 of the affected individuals were compound heterozygotes. The PKHD1 transcript, approximately 16 kb long, is expressed in adult and fetal kidney, liver and pancreas and is predicted to encode a large novel protein, fibrocystin, with multiple copies of a domain shared with plexins and transcription factors. Fibrocystin may be a receptor protein that acts in collecting-duct and biliary differentiation.

• Gabriel SB et al.
• The structure of haplotype blocks in the human genome.
• Science. 2002; 296: 2225-9
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• Haplotype-based methods offer a powerful approach to disease gene mapping, based on the association between causal mutations and the ancestral haplotypes on which they arose. As part of The SNP Consortium Allele Frequency Projects, we characterized haplotype patterns across 51 autosomal regions (spanning 13 megabases of the human genome) in samples from Africa, Europe, and Asia. We show that the human genome can be parsed objectively into haplotype blocks: sizable regions over which there is little evidence for historical recombination and within which only a few common haplotypes are observed. The boundaries of blocks and specific haplotypes they contain are highly correlated across populations. We demonstrate that such haplotype frameworks provide substantial statistical power in association studies of common genetic variation across each region. Our results provide a foundation for the construction of a haplotype map of the human genome, facilitating comprehensive genetic association studies of human disease.

• Veal CD et al.
• Family-based analysis using a dense single-nucleotide polymorphism-based map defines genetic variation at PSORS1, the major psoriasis-susceptibility locus.
• Am J Hum Genet. 2002; 71: 554-64
• Display abstract

• Psoriasis is a common skin disorder of multifactorial origin. Genomewide scans for disease susceptibility have repeatedly demonstrated the existence of a major locus, PSORS1 (psoriasis susceptibility 1), contained within the major histocompatibility complex (MHC), on chromosome 6p21. Subsequent refinement studies have highlighted linkage disequilibrium (LD) with psoriasis, along a 150-kb segment that includes at least three candidate genes (encoding human leukocyte antigen-C [HLA-C], alpha-helix-coiled-coil-rod homologue, and corneodesmosin), each of which has been shown to harbor disease-associated alleles. However, the boundaries of the minimal PSORS1 region remain poorly defined. Moreover, interpretations of allelic association with psoriasis are compounded by limited insight of LD conservation within MHC class I interval. To address these issues, we have pursued a high-resolution genetic characterization of the PSORS1 locus. We resequenced genomic segments along a 220-kb region at chromosome 6p21 and identified a total of 119 high-frequency SNPs. Using 59 SNPs (18 coding and 41 noncoding SNPs) whose position was representative of the overall marker distribution, we genotyped a data set of 171 independently ascertained parent-affected offspring trios. Family-based association analysis of this cohort highlighted two SNPs (n.7 and n.9) respectively lying 7 and 4 kb proximal to HLA-C. These markers generated highly significant evidence of disease association (P<10-9), several orders of magnitude greater than the observed significance displayed by any other SNP that has previously been associated with disease susceptibility. This observation was replicated in a Gujarati Indian case/control data set. Haplotype-based analysis detected overtransmission of a cluster of chromosomes, which probably originated by ancestral mutation of a common disease-bearing haplotype. The only markers exclusive to the overtransmitted chromosomes are SNPs n.7 and n.9, which define a 10-kb PSORS1 core risk haplotype. These data demonstrate the power of SNP haplotype-based association analyses and provide high-resolution dissection of genetic variation across the PSORS1 interval, the major susceptibility locus for psoriasis.

• Hellman A et al.
• A role for common fragile site induction in amplification of human oncogenes.
• Cancer Cell. 2002; 1: 89-97
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• Oncogene amplification is an important process in human tumorigenesis, but its underlying mechanism is currently unknown. Cytogenetic analysis indicates that amplification of drug-selected genes in rodent cells is driven by recurrent breaks within chromosomal common fragile sites (CFSs), via the breakage-fusion-bridge (BFB) mechanism. Here we show that BFB cycles drive the intrachromosomal amplification of the MET oncogene in a human gastric carcinoma. Our molecular evidence includes a "ladder-like" structure and inverted repeat organization of the MET amplicons. Furthermore, we show that the breakpoints, setting the centromeric amplicon boundaries, are within the CFS FRA7G region. Upon replication stress, this region showed perturbed chromatin organization, predisposing it to breakage. Thus, in vivo induction of CFSs can play an important role in human oncogenesis.

• Honda H, Nakamura H, Otsuki M
• The elongated PAP II/Reg III mRNA is upregulated in rat pancreas during acute experimental pancreatitis.
• Pancreas. 2002; 25: 192-7
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• INTRODUCTION: The pathogenesis and the mechanism of the development of severe acute pancreatitis are not clearly understood. AIMS: We performed differential display analysis to find genes that show transcriptional changes in the pancreas during the development of severe acute pancreatitis in the rat. METHODOLOGY: Twenty candidate pancreatitis-associated complementary DNA (cDNA) fragments were isolated. cDNA sequencing and subsequent database analysis revealed that one fragment (C18-2) among the 20 cDNA fragments showed no significant sequence similarity to previously reported genes, suggesting that it represented a novel gene. The rapid and high expression of C18-2 during the acute phase of pancreatitis suggested that the gene was involved in the development of acute pancreatitis. Therefore, we used rapid amplification of cDNA ends and identified the full-length cDNA. RESULTS: Analysis of the open reading frame of the cDNA indicated that the deduced protein from the messenger RNA (mRNA) was a polypeptide of 174 amino acids, unexpectedly similar to that of a known gene, rat pancreatitis-associated protein II/regenerating gene III (PAP II/Reg III). However, the length of the identified mRNA (1,467 base pairs) was longer than that of rat PAP II mRNA (885 base pairs), because the elongated mRNA was generated through the different polyadenylation site in the same gene. The elongated mRNA after acute pancreatitis was strongly induced in the restricted early phase, in comparison with the original mRNA. CONCLUSION: It is considered that the elongated mRNA affects the function of PAP II/Reg III protein because the elongated mRNA with long 3; untranslated regions is known to be involved in the translation efficiency. The identified mRNA may play an important role in the progression of pancreatitis.

• Marchler-Bauer A, Panchenko AR, Ariel N, Bryant SH
• Comparison of sequence and structure alignments for protein domains.
• Proteins. 2002; 48: 439-46
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• Profile search methods based on protein domain alignments have proven to be useful tools in comparative sequence analysis. Domain alignments used by currently available search methods have been computed by sequence comparison. With the growth of the protein structure database, however, alignments of many domain pairs have also been computed by structure comparison. Here, we examine the extent to which information from these two sources agrees. We measure agreement with respect to identification of homologous regions in each protein, that is, with respect to the location of domain boundaries. We also measure agreement with respect to identification of homologous residue sites by comparing alignments and assessing the accuracy of the molecular models they predict. We find that domain alignments in publicly available collections based on sequence and structure comparison are largely consistent. However, the homologous regions identified by sequence comparison are often shorter than those identified by 3D structure comparison. In addition, when overall sequence similarity is low alignments from sequence comparison produce less accurate molecular models, suggesting that they less accurately identify homologous sites. These observations suggest that structure comparison results might be used to improve the overall accuracy of domain alignment collections and the performance of profile search methods based on them.

• Anantharaman V, Aravind L
• The PRC-barrel: a widespread, conserved domain shared by photosynthetic reaction center subunits and proteins of RNA metabolism.
• Genome Biol. 2002; 3: 61-61
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• BACKGROUND: The H subunit of the purple bacterial photosynthetic reaction center (PRC-H) is important for the assembly of the photosynthetic reaction center and appears to regulate electron transfer during the reduction of the secondary quinone. It contains a distinct cytoplasmic beta-barrel domain whose fold has no close structural relationship to any other well known beta-barrel domain. RESULTS: We show that the PRC-H beta-barrel domain is the prototype of a novel superfamily of protein domains, the PRC-barrels, approximately 80 residues long, which is widely represented in bacteria, archaea and plants. This domain is also present at the carboxyl terminus of the pan-bacterial protein RimM, which is involved in ribosomal maturation and processing of 16S rRNA. A family of small proteins conserved in all known euryarchaea are composed entirely of a single stand-alone copy of the domain. Versions of this domain from photosynthetic proteobacteria contain a conserved acidic residue that is thought to regulate the reduction of quinones in the light-induced electron-transfer reaction. Closely related forms containing this acidic residue are also found in several non-photosynthetic bacteria, as well as in cyanobacteria, which have reaction centers with a different organization. We also show that the domain contains several determinants that could mediate specific protein-protein interactions. CONCLUSIONS: The PRC-barrel is a widespread, ancient domain that appears to have been recruited to a variety of biological systems, ranging from RNA processing to photosynthesis. Identification of this versatile domain in numerous proteins could aid investigation of unexplored aspects of their biology.

• Corbin S, Neilly ME, Espinosa R 3rd, Davis EM, McKeithan TW, Le Beau MM
• Identification of unstable sequences within the common fragile site at 3p14.2: implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3p14.2 in tumors.
• Cancer Res. 2002; 62: 3477-84
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• The FRA3B, at 3p14.2, lies within the fragile histidine triad (FHIT) gene and is the most highly expressed of the common fragile sites observed when DNA replication is perturbed by aphidicolin. Common fragile sites are highly unstable regions of the genome. Large intragenic deletions within FHIT, localized within the FRA3B sequences, have been identified in a variety of tumor cells. To characterize the FRA3B deletions in tumor cells and identify FRA3B sequences that are required for fragile site induction, we used microcell-mediated chromosome transfer to isolate hybrid cell clones that retain chromosome 3 homologues with various deletions within FRA3B. Detailed molecular mapping of the FHIT/FRA3B locus in the resultant hybrid cells revealed a complex pattern of instability within FRA3B. Each tumor cell line contained multiple chromosome 3 homologues with variable deletion patterns, often with discontinuous deletions, suggesting that the process of breakage and repair within FRA3B is an ongoing one. By comparing the approximate location of the breakpoints in the hybrid clones, we identified 11 recurring breakpoint/repair regions within the FRA3B. A comparison of the frequency of breaks/gaps within FRA3B in the hybrid clones with various deletions of FRA3B sequences revealed that the loss of FRA3B sequences does not reduce the overall rate of breakage and instability within the remaining FRA3B sequences. The majority of breaks occurred in the proximal portion of the FRA3B, in a 300-kb interval between exon 4 and the proximal 50 kb of intron 5. Our observations suggest that there is no single sequence within the FRA3B that influences breakage or recombination within this region; however, we cannot rule out the presence of multiple "hot spots" within the FHIT/FRA3B locus. Together, the results suggest that factors other than the DNA sequence per se are responsible for the formation of DNA breaks/gaps.

• Scheel H, Tomiuk S, Hofmann K
• A common protein interaction domain links two recently identified epilepsy genes.
• Hum Mol Genet. 2002; 11: 1757-62
• Display abstract

• Until recently, all genes found to be mutated in hereditary idiopathic epilepsies encoded subunits of ion channels, leading to the view of this class of diseases as channelopathies. Two apparent exceptions to this rule are the MASS1 gene, which is mutated in the Frings mouse model of audiogenic epilepsy, and the LGI1 gene, which is mutated in autosomal dominant partial epilepsy with auditory features (ADPEAF). Careful sequence analysis of the two protein products encoded by those genes shows a common feature: both sequences harbour a novel homology domain consisting of a 7-fold repeated 44-residue motif. The architecture and structural features of this new domain make it a likely member of the growing class of protein interaction domains with a seven-bladed beta-propeller fold. In the MASS1 gene product, which has recently been shown to be a fragment of the very large G-protein-coupled receptor VLGR1, this EAR domain (for epilepsy-associated repeat) is part of the ligand-binding ectodomain. LGI1, as well as a number of newly identified LGI1 relatives, is predicted to be a secreted protein, and consists of an N-terminal leucine-rich repeat region and a C-terminal EAR region. The known portion of the human genome encodes six EAR proteins, some of which map to chromosome regions associated with seizure disorders. The EAR domain is likely to play an important role in the pathogenesis of epilepsy, either by binding to an unknown anti-epileptic ligand, or more likely by interfering with axon guidance or synaptogenesis.

• Sollid LM
• Coeliac disease: dissecting a complex inflammatory disorder.
• Nat Rev Immunol. 2002; 2: 647-55
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• The disease mechanisms of complex inflammatory disorders are difficult to define because of extensive interactions between genetic and environmental factors. Coeliac disease is a typical complex inflammatory disorder, but this disease is unusual in that crucial genetic and environmental factors have been identified. This knowledge has allowed functional studies of the predisposing HLA molecules, the identification of antigenic epitopes and detailed studies of disease-relevant T cells in coeliac disease. This dissection of the pathogenic mechanisms of coeliac disease has uncovered principles that are relevant to other chronic inflammatory diseases.

• Onuchic LF et al.
• PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.
• Am J Hum Genet. 2002; 70: 1305-17
• Display abstract

• Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of polycystic kidney disease that presents primarily in infancy and childhood and that is characterized by enlarged kidneys and congenital hepatic fibrosis. We have identified PKHD1, the gene mutated in ARPKD. PKHD1 extends over > or =469 kb, is primarily expressed in human fetal and adult kidney, and includes a minimum of 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest continuous open reading frame encodes a 4,074-amino-acid protein, polyductin, that is predicted to have a single transmembrane (TM)-spanning domain near its carboxyl terminus, immunoglobulin-like plexin-transcription-factor domains, and parallel beta-helix 1 repeats in its amino terminus. Several transcripts encode truncated products that lack the TM and that may be secreted if translated. The PKHD1-gene products are members of a novel class of proteins that share structural features with hepatocyte growth-factor receptor and plexins and that belong to a superfamily of proteins involved in regulation of cell proliferation and of cellular adhesion and repulsion.

• Harris PC
• Molecular basis of polycystic kidney disease: PKD1, PKD2 and PKHD1.
• Curr Opin Nephrol Hypertens. 2002; 11: 309-14
• Display abstract

• Recent developments have helped elucidate the function of the autosomal dominant polycystic kidney disease proteins, polycystin-1 and polycystin-2, and have revealed the primary defect in autosomal recessive polycystic kidney disease, by positional cloning of the gene, PKHD1. Several studies demonstrating that polycystin-2 can act as a calcium-ion-permeable cation channel, and that polycystin-1 may be involved in regulating/localizing this channel, have provided compelling evidence of the function of these proteins. A role in regulating intracellular calcium levels seems likely, with the many cellular abnormalities associated with cystogenesis due to a disruption of calcium homeostasis. Improved mutation analysis in autosomal dominant polycystic kidney disease has led to the finding of genotype/phenotype correlations which could be related to possible cleavage of polycystin-1. A major recent breakthrough has revealed the primary defect in autosomal recessive polycystic kidney disease. Genetic analysis showed that the PCK rat model is orthologous to autosomal recessive polycystic kidney disease, and allowed the human gene, PKHD1, to be precisely localized and identified. PKHD1 is a large gene, encoding a protein, fibrocystin, of 4074 amino acids, which is predicted to have a large extracellular region, a single transmembrane domain and a short cytoplasmic tail. Fibrocystin may act as a receptor with critical roles in collecting-duct and biliary development.

• Tanaka H, Tapscott SJ, Trask BJ, Yao MC
• Short inverted repeats initiate gene amplification through the formation of a large DNA palindrome in mammalian cells.
• Proc Natl Acad Sci U S A. 2002; 99: 8772-7
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• Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.

• Ulyanova T, Shah DD, Thomas ML
• Molecular cloning of MIS, a myeloid inhibitory siglec, that binds protein-tyrosine phosphatases SHP-1 and SHP-2.
• J Biol Chem. 2001; 276: 14451-8
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• We describe the molecular cloning and characterization of a novel myeloid inhibitory siglec, MIS, that belongs to the family of sialic acid-binding immunoglobulin-like lectins. A full-length MIS cDNA was obtained from murine bone marrow cells. MIS is predicted to contain an extracellular region comprising three immunoglobulin-like domains (V-set amino-terminal domain followed by two C-set domains), a transmembrane domain and a cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. The closest relative of MIS in the siglec family is human siglec 8. Extracellular regions of these two siglecs share 47% identity at the amino acid level. Southern blot analysis suggests the presence of one MIS gene. MIS is expressed in the spleen, liver, heart, kidney, lung and testis tissues. Several isoforms of MIS protein exist due to the alternative splicing. In a human promonocyte cell line, MIS was able to bind Src homology 2-containing protein-tyrosine phosphatases, SHP-1 and SHP-2. This binding was mediated by the membrane-proximal ITIM of MIS. Moreover, MIS exerted an inhibitory effect on FcgammaRI receptor-induced calcium mobilization. These data suggest that MIS can play an inhibitory role through its ITIM sequences.

• Yamano Y et al.
• A novel spermatogenesis-related factor-1 gene expressed in maturing rat testis.
• Biochem Biophys Res Commun. 2001; 289: 888-93
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• A rat gene with testis-specific expression coinciding with spermatogenesis was cloned by differential display. This spermatogenesis-related factor-1 (SRF-1) gene was not expressed in other organs. Testicular expression was detected from 5 weeks of age and increased up to 15 weeks; this level of expression was maintained for 63 weeks. The 750-bp cloned gene was coded for an open reading frame of 202 amino acids. According to in situ hybridization at 7 weeks, this gene was expressed mainly in spermatocyte. The gene product may function as a molecular motor in meiosis, as the deduced amino acid sequence showed partial homology with kinesin-related proteins. The action of this gene and its product with respect to division of reproductive cells requires further investigation.

• LaCount DJ, El-Sayed NM, Kaul S, Wanless D, Turner CM, Donelson JE
• Analysis of a donor gene region for a variant surface glycoprotein and its expression site in African trypanosomes.
• Nucleic Acids Res. 2001; 29: 2012-9
• Display abstract

• African trypanosomes evade the immune response of their mammalian hosts by sequentially expressing genes for different variant surface glycoproteins (VSGs) from telomere-linked VSG expression sites. In the Trypanosoma brucei clone whose genome is being sequenced (GUTat 10.1), we show that the expressed VSG (VSG 10.1) is duplicated from a silent donor VSG located at another telomere-linked site. We have determined two 130 kb sequences representing the VSG 10.1 donor and expression sites. The telomere-linked donor VSG 10.1 resembles metacyclic VSG expression sites, and is preceded by a cluster of 35 or more tandem housekeeping genes, all of which are transcribed away from the telomere. The 45 kb telomere-linked VSG 10.1 expression site contains a promoter followed by seven expression site-associated genes (ESAGs), three pseudo ESAGs, two pseudo VSGs and VSG 10.1. The 80 kb preceding the expression site has few, if any, functional ORFs, but contains 50 bp repeats, INGI retrotransposon-like elements, and novel 4-12 kb repeats found near other telomeres. This analysis provides the first look over a 130 kb range of a telomere-linked donor VSG and its corresponding telomere-linked VSG expression site and forms the basis for studies on antigenic variation in the context of a completely sequenced genome.

• Stover C, Gradl G, Jentsch I, Speicher MR, Wieser R, Schwaeble W
• cDNA cloning, chromosome assignment, and genomic structure of a human gene encoding a novel member of the RBM family.
• Cytogenet Cell Genet. 2001; 92: 225-30
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• We have cloned and characterised a novel human gene mapping to chromosome 20q11.2. A partial transcript was initially isolated from a human cDNA library transcribed from RNA of the colon carcinoma cell line T-84. In order to determine the full coding sequence of this novel mRNA, we isolated seven cDNA clones from a human cDNA library transcribed from RNA of the acute monocytic leukemia cell line THP1 by colony hybridization. On Northern blot analysis of four human cell lines, the cDNAs isolated hybridize with an abundantly expressed mRNA species of 3.5 kb. A full-length cDNA transcript of this novel mRNA has an open reading frame of 2,796 bp encoding a protein with a calculated molecular weight of 97 kDa. Two repetitive structural consensus motifs are contained within the deduced protein sequence, namely five distinct RNA binding motifs and two proline rich regions. The derived protein sequence also contains putative transmembrane domains. These structural motifs identify this novel protein as a member of an expanding protein family containing RNA binding motifs (RBM). As seen from recently completed sequence of the genomic area encoding this novel mRNA by the Sanger Centre Human Genome Project, the coding region of this gene, RBM12, is intronless.

• Gonzalez LC Jr, Weis WI, Scheller RH
• A novel snare N-terminal domain revealed by the crystal structure of Sec22b.
• J Biol Chem. 2001; 276: 24203-11
• Display abstract

• Intra-cellular membrane fusion is facilitated by the association of SNAREs from opposite membranes into stable alpha-helical bundles. Many SNAREs, in addition to their alpha-helical regions, contain N-terminal domains that likely have essential regulatory functions. To better understand this regulation, we have determined the 2.4-A crystal structure of the 130-amino acid N-terminal domain of mouse Sec22b (mSec22b), a SNARE involved in endoplasmic reticulum/Golgi membrane trafficking. The domain consists of a mixed alpha-helical/beta-sheet fold that resembles a circular permutation of the actin/poly-proline binding protein, profilin, and the GAF/PAS family of regulatory modules. The structure is distinct from the previously characterized N-terminal domain of syntaxin 1A, and, unlike syntaxin 1A, the N-terminal domain of mSec22b has no effect on the rate of SNARE assembly in vitro. An analysis of surface conserved residues reveals a potential protein interaction site. Key residues in this site are distinct in two mammalian Sec22 variants that lack SNARE domains. Finally, sequence analysis indicates that a similar domain is likely present in the endosomal/lysosomal SNARE VAMP7.

• Mougel C, Zhulin IB
• CHASE: an extracellular sensing domain common to transmembrane receptors from prokaryotes, lower eukaryotes and plants.
• Trends Biochem Sci. 2001; 26: 582-4
• Display abstract

• A novel extracellular ligand-binding domain, termed CHASE, is described in sensory adenylyl and diguanylate cyclases, and histidine kinases, in several bacterial species, Dictyostelium and plants. The CHASE domain is predicted to sense stimuli that are specific for the developmental program of an organism.

• Fang JM, Arlt MF, Burgess AC, Dagenais SL, Beer DG, Glover TW
• Translocation breakpoints in FHIT and FRA3B in both homologs of chromosome 3 in an esophageal adenocarcinoma.
• Genes Chromosomes Cancer. 2001; 30: 292-8
• Display abstract

• Common fragile sites have been proposed to play a mechanistic role in chromosome translocations and other rearrangements in cancer cells in vivo based on their behavior in vitro and their co-localization with cancer translocation breakpoints. This hypothesis has been the subject of controversy, because associations have been made at the chromosomal level and because of the large number of both fragile sites and cancer chromosome breakpoints. Tests of this hypothesis at the molecular level are now possible with the cloning of common fragile site loci and the use of fragile site clones in the analysis of rearranged chromosomes. FRA3B, the most frequently seen common fragile site, lies within the large FHIT gene. It is now well established that this region is the site of frequent, large intragenic deletions and aberrant transcripts in a number of tumors and tumor cell lines. In contrast, only one tumor-associated translocation involving the FHIT gene has been reported. We have found translocations in both homologs of chromosome 3 in an early-passage esophageal adenocarcinoma cell line. This cell line showed no normal FHIT transcripts by reverse transcription polymerase chain reaction. Subsequent chromosome analysis showed translocations of the short arms of both homologs of chromosome 3: t(3;16) and t(3;4). The breakpoints of both translocations were shown by fluorescence in situ hybridization and polymerase chain reaction to be in the FHIT gene, at or near the center of the fragile site region. Using rapid amplification of cDNA ends with FHIT primers, a noncoding chimeric transcript resulting from t(3;16) was identified. These data provide direct support for the hypothesis that FRA3B, and likely other common fragile sites, may be "hot spots" for translocations in certain cancers, as they are for deletions, and that such translocations have the potential to form abnormal chimeric transcripts. In addition, the results suggest selection for loss of a functional FHIT gene by the translocation events.

• Verpy E et al.
• Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.
• Nat Genet. 2001; 29: 345-9
• Display abstract

• Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein-stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

• Masmoudi S et al.
• Novel missense mutations of TMPRSS3 in two consanguineous Tunisian families with non-syndromic autosomal recessive deafness.
• Hum Mutat. 2001; 18: 101-8
• Display abstract

• Recently the TMPRSS3 gene, which encodes a transmembrane serine protease, was found to be responsible for two non-syndromic recessive deafness loci located on human chromosome 21q22.3, DFNB8 and DFNB10. We found evidence for linkage to the DFNB8/10 locus in two unrelated consanguineous Tunisian families segregating congenital autosomal recessive sensorineural deafness. The audiometric tests showed a loss of hearing greater than 70 dB, in all affected individuals of both families. Mutation screening of TMPRSS3 revealed two novel missense mutations, W251C and P404L, altering highly conserved amino acids of the serine protease domain. Both mutations were not found in 200 control Tunisian chromosomes. The detection of naturally-occurring TMPRSS3 missense mutations in deafness families identifies functionally important amino acids. Comparative protein modeling of the TMPRSS3 protease domain predicted that W251C might lead to a structural rearrangement affecting the active site H257 and that P404L might alter the geometry of the active site loop and therefore affect the serine protease activity.

• Liepinsh E et al.
• NMR structure of the LCCL domain and implications for DFNA9 deafness disorder.
• EMBO J. 2001; 20: 5347-53
• Display abstract

• The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch-5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch-5b2, where dominant mutations leading to DFNA9 deafness disorder have been identified. The fold is novel. Four of the five known DFNA9 mutations are shown to involve at least partially solvent-exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent-accessible residues might be attributed to interference with the folding pathway of this disulfide-containing domain.

• Chomiki N, Voss JC, Warden CH
• Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.
• Eur J Biochem. 2001; 268: 903-13
• Display abstract

• Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.

• Valve EM, Ruohola JK, Tasanen MJ, Glover JF, Darbre PD, Harkonen PL
• Expression of the androgen-dependent MMTV-specific orf gene in Shionogi 115 mouse mammary tumor cells.
• J Steroid Biochem Mol Biol. 2001; 78: 389-400
• Display abstract

• The Shionogi 115 (S115) mouse mammary tumor cells express the MMTV-specific 1.7 kb mRNA (orf) at a high level in the presence of androgens. In lymphoid cells the orf-gene encodes a superantigen which has an important role in establishing self-tolerance but in mammary and breast cancer cells the function of the orf gene is unclear. In the present work we studied the expression of the S115 mammary tumor cell orf sequence and its role in the androgen regulated growth of S115 cells. The cloning and sequencing of the cDNA specific for the 1.7 kb mRNA from the S115 mouse mammary tumor cells revealed a 990 bp DNA sequence with a 99.8% homology to the Mtv-17 proviral strain. There was a difference of only one amino acid (isoleu-tyr) in the coding region. A peptide was synthesized according to the hypervariable C-terminal part of the predicted protein and used to raise a rabbit antiserum. The anti-S115-orf antiserum immunoprecipitated an approximately 45 kDa protein from the metabolically labeled S115 cell lysates. In order to analyze the putative functions of the protein, the orf-sequence was linked to MoMLV-LTR and to the human ss-actin promoter in the mammalian expression vectors pLTRpoly and pHssAPr-1-neo, respectively, and transfected into NIH3T3 and S115 cells. NIH3T3 transfectants expressing orf mRNA did not show a transformed phenotype in vitro. The S115 orf transfectants proliferated somewhat more slowly than the vector transfected control cells in cell culture, both in the presence or absence of androgen, but there was no obvious change in the phenotype of S115 cells or in expression of the fibroblast growth factor 8 (FGF-8). This factor is activated by Mtv-6 integration and mediates androgen effects in these cells. Unexpectedly, however, the formation of tumors by S115 orf cells in nude mice was considerably prolonged and tumor growth retarded when compared with vector transfected control or parent S115 cells. The results suggest that MMTV-orf can be functional in breast cancer cells but the mechanism of the growth repressive effect in mammary tumor remains to be analyzed.

• Muscarella P et al.
• Identification and sequencing of the Syrian Golden hamster (Mesocricetus auratus) p16(INK4a) and p15(INK4b) cDNAs and their homozygous gene deletion in cheek pouch and pancreatic tumor cells.
• Gene. 2001; 278: 235-43
• Display abstract

• Previous studies have shown that the p16(INK4a) tumor suppressor gene is inactivated in up to 98% of human pancreatic cancer specimens and 83% of oral squamous cell carcinomas. Inactivation of the related p15(INK4b) gene has also been identified in a number of tumors and cell lines, however, its role as an independent tumor suppressor remains to be elucidated. Chemically-induced tumors in the Syrian Golden hamster (Mesocricetus auratus) have been shown to be excellent representative models for the comparative development and progression of a number of human malignancies. The purpose of this study was to determine the importance of the p16(INK4a) and p15(INK4b) genes in two experimental hamster models for human pancreatic and oral carcinogenesis. First, hamster p16(INK4a) and p15(INK4b) cDNAs were cloned and sequenced. The hamster p16(INK4a) cDNA open reading frame (ORF) shares 78%, 80%, and 81% identity with the human, mouse, and rat p16(INK4a) sequences, respectively. Similarly, the hamster p15(INK4b) cDNA ORF shares 82% and 89% sequence identity with human and mouse p15(INK4b), respectively. Second, a deletion analysis of hamster p16(INK4a) and p15(INK4b) genes was performed for several tumorigenic and non-tumorigenic hamster cell lines and revealed that both p16(INK4a) and p15(INK4b) were homozygously deleted in a cheek pouch carcinoma cell line (HCPC) and two pancreatic adenocarcinoma cell lines (KL5B, H2T), but not in tissue matched, non-tumorigenic cheek pouch (POT2) or pancreatic (KL5N) cell lines. These data strongly suggest that homozygous deletion of the p16(INK4a) and p15(INK4b) genes plays a prominent role in hamster pancreatic and oral tumorigenesis, as has been well established in correlative studies in comparable human tumors. Furthermore, this study supports the comparative importance of the hamster pancreatic and cheek pouch models of carcinogenesis in subsequent mechanistic-, therapeutic-, and preventive-based studies aimed at providing important translational data applicable to pancreatic adenocarcinoma and oral squamous cell carcinoma in humans.

• Scarman AL et al.
• Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis.
• Eur J Biochem. 2001; 268: 1250-8
• Display abstract

• The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans approximately 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at approximately day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.

• Tan YC, Chow VT
• Novel human HALR (MLL3) gene encodes a protein homologous to ALR and to ALL-1 involved in leukemia, and maps to chromosome 7q36 associated with leukemia and developmental defects.
• Cancer Detect Prev. 2001; 25: 454-69
• Display abstract

• We have identified and characterized the approximately 12-kb cDNA of a novel human gene (designated HALR for "homologous to ALR" and given the symbol MLL3 by the HUGO Gene Nomenclature Committee) for which open reading frame (ORF) encodes a predicted large hydrophilic nuclear protein comprising 4,025 amino acids with a calculated molecular mass of approximately 443 kD. Within the amino acid sequence of HALR were identified a SUVAR3-9, enhancer of zeste, trithorax (SET) domain, three plant homeodomain (PHD)-type zinc fingers, a high motility group (HMG)-1 box, a leucine-zipper-like pattern, two potential transactivating domains, several nuclear localization signals, and multiple nuclear receptor interaction signature motifs. Especially within the SET domain, PHD fingers and several other regions, the HALR protein exhibits significant similarity to ALR (acute lymphoblastic leukemia [ALL]-1 related), ALL-1/myeloid/lymphoid or mixed-lineage leukemia (ALL-1/MLL), and trithorax, evolutionarily conserved proteins that influence differentiation and development. Northern blot analysis demonstrated transcripts of approximately 11-12 kb, while reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that HALR is expressed in a wide range of human tissues and cancer cell lines. The HALR gene contains 46 exons, is estimated to span >101 kb, and is located on chromosome region 7q36. Terminal 7q deletions are common chromosomal aberrations encountered in hematological neoplasia and in holoprosencephaly 3, a midline embryonic defect involving forebrain development. We have also isolated the partial cDNA of the murine homologue of HALR, which displays high homology to its human counterpart. Taking into consideration its notable protein motifs, ubiquitous expression, evolutionary conservation and chromosomal position, HALR is likely to play a housekeeping role in transcriptional regulation, and may be involved in leukemogenesis and developmental disorders.

• Yasunaga S et al.
• OTOF encodes multiple long and short isoforms: genetic evidence that the long ones underlie recessive deafness DFNB9.
• Am J Hum Genet. 2000; 67: 591-600
• Display abstract

• We have recently reported that OTOF underlies an autosomal recessive form of prelingual sensorineural deafness, DFNB9. The isolated 5-kb cDNA predicted a 1,230 amino acid (aa) C-terminus membrane-anchored cytosolic protein with three C2 domains. This protein belongs to a family of mammalian proteins sharing homology with the Caenorhabditis elegans fer-1. The two other known members of this family, dysferlin and myoferlin, both have six predicted C2 domains. By northern blot analysis, a 7-kb otoferlin mRNA could be detected in the human brain. We isolated the corresponding cDNA, which is expected to encode a 1,977-aa-long form of otoferlin with six C2 domains. A 7-kb cDNA derived from the murine orthologous gene, Otof, was also identified in the inner ear and the brain. The determination of the exon-intron structure of the human and murine genes showed that they are composed of 48 coding exons and extend approximately 90 kb and approximately 80 kb, respectively. Alternatively spliced transcripts could be detected that predict several long isoforms (six C2 domains) in humans and mice and short isoforms (three C2 domains) only in humans. Primers were designed to explore the first 19 OTOF exons, henceforth permitting exploration of the complete coding sequence of the gene in DFNB9 patients. In a southwestern Indian family affected by DFNB9, a mutation in the acceptor splice site of intron 8 was detected, which demonstrates that the long otoferlin isoforms are required for inner ear function.

• Hubbard C, Singleton D, Rauch M, Jayasinghe S, Cafiso D, Castle D
• The secretory carrier membrane protein family: structure and membrane topology.
• Mol Biol Cell. 2000; 11: 2933-47
• Display abstract

• Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

• Ried K et al.
• Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells.
• Hum Mol Genet. 2000; 9: 1651-63
• Display abstract

• Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cyto-genetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells.

• Misra S, Beach BM, Hurley JH
• Structure of the VHS domain of human Tom1 (target of myb 1): insights into interactions with proteins and membranes.
• Biochemistry. 2000; 39: 11282-90
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• VHS domains are found at the N-termini of select proteins involved in intracellular membrane trafficking. We have determined the crystal structure of the VHS domain of the human Tom1 (target of myb 1) protein to 1.5 A resolution. The domain consists of eight helices arranged in a superhelix. The surface of the domain has two main features: (1) a basic patch on one side due to several conserved positively charged residues on helix 3 and (2) a negatively charged ridge on the opposite side, formed by residues on helix 2. We compare our structure to the recently obtained structure of tandem VHS-FYVE domains from Hrs [Mao, Y., Nickitenko, A., Duan, X., Lloyd, T. E., Wu, M. N., Bellen, H., and Quiocho, F. A. (2000) Cell 100, 447-456]. Key features of the interaction surface between the FYVE and VHS domains of Hrs, involving helices 2 and 4 of the VHS domain, are conserved in the VHS domain of Tom1, even though Tom1 does not have a FYVE domain. We also compare the structures of the VHS domains of Tom1 and Hrs to the recently obtained structure of the ENTH domain of epsin-1 [Hyman, J., Chen, H., Di Fiore, P. P., De Camilli, P., and Brunger, A. T. (2000) J. Cell Biol. 149, 537-546]. Comparison of the two VHS domains and the ENTH domain reveals a conserved surface, composed of helices 2 and 4, that is utilized for protein-protein interactions. In addition, VHS domain-containing proteins are often localized to membranes. We suggest that the conserved positively charged surface of helix 3 in VHS and ENTH domains plays a role in membrane binding.

• Gnanasekaran TV, Peri S, Arockiasamy A, Krishnaswamy S
• Profiles from structure based sequence alignment of porins can identify beta stranded integral membrane proteins.
• Bioinformatics. 2000; 16: 839-42

• Shuster MI et al.
• A consistent pattern of RIN1 rearrangements in oral squamous cell carcinoma cell lines supports a breakage-fusion-bridge cycle model for 11q13 amplification.
• Genes Chromosomes Cancer. 2000; 28: 153-63
• Display abstract

• Gene amplification is a common feature of tumors. Overexpression of some amplified genes plays a role in tumor progression. Gene amplification can occur either extrachromosomally as double-minute chromosomes (dmin) or intrachromosomally in the form of homogeneously staining regions (hsrs). Approximately one-half of our oral squamous cell carcinomas (OSCCs) are characterized by amplification of band 11q13, usually as an hsr located entopically (occurring or situated at the normal chromosomal site, as opposed to ectopically). Using chromosomal fluorescence in situ hybridization (FISH), we confirmed the amplification of the cyclin D1 (CCND1/PRAD1) and fibroblast growth factor types 3 and 4 (FGF3/INT2 and FGF4/HSTF1) genes within the 11q13 amplicon in our series of primary OSCCs and derived cell lines. The human RIN1 gene was isolated as an RAS interaction/interference protein in a genetic selection in yeast and has been described as a putative effector of both the RAS and ABL oncogenes. We mapped RIN1 to 11q13.2. FISH analysis of 10 11q13-amplified OSCC cell lines revealed high-level RIN1 amplification in two cell lines. Three additional cell lines have what appear to be duplications and/or low-level amplification of RIN1, visible in both interphase and metaphase cells. The hybridization pattern of RIN1 on the metaphase chromosomes is particularly revealing; RIN1 signals flank the 11q13 hsr, possibly as a result of an inverted duplication. The gene amplification model of Coquelle et al. (1997) predicted that gene amplification occurs by breakage-fusion-bridge (BFB) cycles involving fragile sites. Our data suggest that the pattern of gene amplification at 11q13 in OSCC cell lines is consistent with a BFB model. RIN1 appears to be a valuable probe for investigating the process of gene amplification in general and, specifically, 11q13 amplification in oral cancer.

• Cao X, Rogers SW, Butler J, Beevers L, Rogers JC
• Structural requirements for ligand binding by a probable plant vacuolar sorting receptor.
• Plant Cell. 2000; 12: 493-506
• Display abstract

• How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.

• Treves S, Feriotto G, Moccagatta L, Gambari R, Zorzato F
• Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane.
• J Biol Chem. 2000; 275: 39555-68
• Display abstract

• Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).

• Veldhuisen B, Spruit L, Dauwerse HG, Breuning MH, Peters DJ
• Genes homologous to the autosomal dominant polycystic kidney disease genes (PKD1 and PKD2).
• Eur J Hum Genet. 1999; 7: 860-72
• Display abstract

• Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common inherited disease leading to progressive renal failure, can be caused by a mutation in either the PKD1 or PKD2 gene. Both genes encode for putative transmembrane proteins, polycystin-1 and polycystin-2, which show significant homology to each other and are believed to interact at their carboxy termini. To identify genes that code for related proteins we searched for homologous sequences in several databases and identified one partial cDNA and two genomic sequences with significant homology to both polycystin-1 and - 2. Further analysis revealed one novel gene, PKD2L2, located on chromosome band 5q31, and two recently described genes, PKD2L and PKDREJ, located on chromosome bands 10q31 and 22q13.3, respectively. PKD2L2 and PKD2L, which encode proteins of 613 and 805 amino acids, are approximately 65% similar to polycystin-2. The third gene, PKDREJ, encodes a putative 2253 amino acid protein and shows about 35% similarity to both polycystin-1 and polycystin-2. For all the genes expression was found in testis. Additional expression of PKD2L was observed in retina, brain, liver and spleen by RT-PCR. Analyses of five ADPKD families without clear linkage to either the PKD1 or PKD2 locus showed no linkage to any of the novel loci, excluding these genes as the cause of ADPKD in these families. Although these genes may not be involved in renal cystic diseases, their striking homology to PKD2 and PKD1 implies similar roles and may contribute to elucidating the function of both polycystin-1 and polycystin-2.

• Tsiokas L, Arnould T, Zhu C, Kim E, Walz G, Sukhatme VP
• Specific association of the gene product of PKD2 with the TRPC1 channel.
• Proc Natl Acad Sci U S A. 1999; 96: 3934-9
• Display abstract

• The function(s) of the genes (PKD1 and PKD2) responsible for the majority of cases of autosomal dominant polycystic kidney disease is unknown. While PKD1 encodes a large integral membrane protein containing several structural motifs found in known proteins involved in cell-cell or cell-matrix interactions, PKD2 has homology to PKD1 and the major subunit of the voltage-activated Ca2+ channels. We now describe sequence homology between PKD2 and various members of the mammalian transient receptor potential channel (TRPC) proteins, thought to be activated by G protein-coupled receptor activation and/or depletion of internal Ca2+ stores. We show that PKD2 can directly associate with TRPC1 but not TRPC3 in transfected cells and in vitro. This association is mediated by two distinct domains in PKD2. One domain involves a minimal region of 73 amino acids in the C-terminal cytoplasmic tail of PKD2 shown previously to constitute an interacting domain with PKD1. However, distinct residues within this region mediate specific interactions with TRPC1 or PKD1. The C-terminal domain is sufficient but not necessary for the PKD2-TRPC1 association. A more N-terminal domain located within transmembrane segments S2 and S5, including a putative pore helical region between S5 and S6, is also responsible for the association. Given the ability of the TRPC to form functional homo- and heteromultimeric complexes, these data provide evidence that PKD2 may be functionally related to TRPC proteins and suggest a possible role of PKD2 in modulating Ca2+ entry in response to G protein-coupled receptor activation and/or store depletion.

• Bycroft M et al.
• The structure of a PKD domain from polycystin-1: implications for polycystic kidney disease.
• EMBO J. 1999; 18: 297-305
• Display abstract

• Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell-surface glycoprotein, polycystin-1, of unknown function, which, based on its predicted domain structure, may be involved in protein-protein and protein-carbohydrate interactions. Approximately 30% of polycystin-1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a beta-sandwich fold. Although this fold is common to a number of cell-surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin-1 show extensive conservation of surface residues suggesting that this region could be a ligand-binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.

• Sappington TW, Raikhel AS
• Molecular characteristics of insect vitellogenins and vitellogenin receptors.
• Insect Biochem Mol Biol. 1998; 28: 277-300
• Display abstract

• The recent cloning and sequencing of several insect vitellogenins (Vg), the major yolk protein precursor of most oviparous animals, and the mosquito Vg receptor (VgR) has brought the study of insect vitellogenesis to a new plane. Insect Vgs are homologous to nematode and vertebrate Vgs. All but one of the insect Vgs for which we know the primary structure are cleaved into two subunits at a site [(R/K)X(R/K)R or RXXR with an adjacent beta-turn] recognized by subtilisin-like proprotein convertases. In four of the Vgs, the cleavage site is near the N-terminus, but in one insect species, it is near the C-terminus of the Vg precursor. Multiple alignments of these Vg sequences indicate that the variation in cleavage location has not arisen through exon shuffling, but through local modifications of the amino acid sequences. A wasp Vg precursor is not cleaved, apparently because the sequence at the presumed ancestral cleavage site has been mutated from RXRR to LYRR and is no longer recognized by convertases. Some insect Vgs contain polyserine domains which are reminiscent of, but not homologous to, the phosvitin domain in vertebrate Vgs. The sequence of the mosquito VgR revealed that it is a member of the low-density lipoprotein receptor (LDLR) family. Though resembling chicken and frog VgRs, which are also members of the LDLR family, it is twice as big, carrying two clusters of cysteine-rich complement-type (Class A) repeats (implicated in ligand-binding) instead of one like vertebrate VgRs and LDLRs. It is very similar in sequence and domain arrangement to the Drosophila yolk protein receptor (YPR), despite a non-vitellogenin ligand for the latter. Though vertebrate VgRs, insect VgR/YPRs, and LDLR-related proteins/megalins all accommodate one cluster of eight Class A repeats, fingerprint analysis of the repeats in these clusters indicate they are not directly homologous with one another, but have undergone differing histories of duplications, deletions, and exon shuffling so that their apparent similarity is superficial. The so-called epidermal growth factor precursor region contains two types of motifs (cysteine-rich Class B repeats and YWXD repeats) which occur independently of one another in diverse proteins, and are often involved in protein-protein interactions, suggesting that they potentially are involved in dimerization of VgRs and other LDLR-family proteins. Like the LDLR, but unlike vertebrate VgRs and the Drosophila YPR, the mosquito VgR contains a putative O-linked sugar region on the extra-cellular side of the transmembrane domain. Its function is unclear, but may protect the receptor from membrane-bound proteases. The cytoplasmic tail of insect VgR/YPRs contains a di-leucine (or leucine-isoleucine) internalization signal, unlike the tight-turn tyrosine motif of other LDLR-family proteins. The importance of understanding the details of yolk protein uptake by oocytes lies in its potential for exploitation in novel insect control strategies, and the molecular characterization of the proteins involved has made the development of such strategies a realistic possibility.

• Kuo MT, Sen S, Hittelman WN, Hsu TC
• Chromosomal fragile sites and DNA amplification in drug-resistant cells.
• Biochem Pharmacol. 1998; 56: 7-13
• Display abstract

• It has been well established that DNA amplification is one of the important mechanisms by which cultured cells acquire resistance to many cytotoxic compounds. Amplification of important genes including those encoding oncoproteins, growth factors, their receptors and cell-cycle regulators has been reported in human neoplasms. Yet, despite intensive research since the first description of DNA amplification in cultured cells about 20 years ago, the mechanisms of DNA amplification remain largely unknown. Many models have been proposed to account for the diverse manifestations of amplified DNA in many different cell sources. It is not the intention of this commentary to review these many different models. Rather, we wil focus on the recent advances in this area of research, made mainly via the fluorescence in situ hybridization technique, that have revealed a fairly common chromosomal manifestation of amplified DNA in the drug-resistant hamster cell lines and have demonstrated the association of chromosomal fragile site breakage with early events in DNA amplification. These new developments underscore the importance of future research toward understanding the molecular bases of chromosomal fragile sites, including mechanisms involved in DNA strand breakage and repair, chromosomal translocations, and deletions, which may, in turn, provide important new insights into genomic plasticity and neoplastic transformation.

• Smilenov LB et al.
• Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability.
• Oncogene. 1998; 17: 2137-42
• Display abstract

• Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.

• Debatisse M, Coquelle A, Toledo F, Buttin G
• Gene amplification mechanisms: the role of fragile sites.
• Recent Results Cancer Res. 1998; 154: 216-26
• Display abstract

• We studied the early stages of gene amplification in a Chinese hamster cell line and identified two distinct amplification mechanisms, both relying on an unequal segregation of gene copies at mitosis. In some cases, a sequence containing the selected gene is looped out, generating an acentric circular molecule, and amplification proceeds through unequal segregation of such extrachromosomal elements in successive cell cycles. In other cases, the accumulation of intrachromosomally amplified copies is driven by cycles of chromatid breakage, followed by fusion of sister chromatids devoid of a telomere, which leads to bridge formation and further break in mitosis (BFB cycles). We showed that some clastogenic drugs specifically trigger the intrachromosomal amplification pathway and strictly correlated this induction of BFB cycles to the ability of these drugs to activate fragile sites. In three model systems, we also established, that the location of centromeric and telomeric fragile sites relative to the selected genes determines the size and sequence content of the early amplicons.

• Murcia NS, Woychik RP, Avner ED
• The molecular biology of polycystic kidney disease.
• Pediatr Nephrol. 1998; 12: 721-6
• Display abstract

• In recent years there have been a number of developments in polycystic kidney disease (PKD) research. The genes associated with the predominant forms of autosomal dominant PKD have been cloned, and the gene associated with a mouse model for autosomal recessive PKD has been identified and characterized. Other studies have yielded new information regarding the role of the epidermal growth factor receptor gene in promoting renal cyst formation. In this review article we summarize recent published data on the molecular genetics of autosomal dominant and autosomal recessive PKD and provide a working model of how multiple genes participate in the PKD disease pathway.

• Kraemer C, Weil B, Christmann M, Schmidt ER
• The new gene DmX from Drosophila melanogaster encodes a novel WD-repeat protein.
• Gene. 1998; 216: 267-76
• Display abstract

• DmX is a novel gene from Drosophila melanogaster located on the X chromosome in region 5D5/6-E1. The molecular analysis of the genomic and cDNA sequences of DmX shows that the gene spans appr. 16kb and displays a mosaic structure with 15 exons. The 12kb long DmX transcript is present in Drosophila embryos, larvae and adults of both sexes. The open reading frame of DmX encodes a novel WD-repeat protein, containing at least 30 WD-repeat units. WD-repeat proteins contain a conserved motif of approximately 40 amino acids (aa), usually ending with the dipeptide Trp-Asp (WD). Homologues of the DmX gene exist in other dipteran species, in Caenorhabditis elegans and human, revealing that DmX is an evolutionarily well conserved gene. The inferred DMX amino acid sequence shows also limited, but significant similarity to a yeast ORF with unknown function. 1998 Elsevier Science B.V.

• Scott DA et al.
• Identification and mutation analysis of a cochlear-expressed, zinc finger protein gene at the DFNB7/11 and dn hearing-loss loci on human chromosome 9q and mouse chromosome 19.
• Gene. 1998; 215: 461-9
• Display abstract

• The DFNB7/11 locus for autosomal recessive non-syndromic hearing loss (ARNSHL) has been mapped to an approx. 1.5 Mb interval on human chromosome 9q13-q21. We have determined the cDNA sequence and genomic structure of a novel cochlear-expressed gene, ZNF216, that maps to the DFNB7/11 interval. The mouse orthologue of this gene maps to the murine dn (deafness) locus on mouse chromosome 19. The ZNF216 gene is highly conserved between human and mouse, and contains two regions that show homology to the putative zinc linger domains of other proteins. To determine it mutations in ZNF216 might be the cause of hearing loss at the DFNB7/11 locus, we screened the coding region of this gene in DFNB7/11 families by direct sequencing. No potential disease-causing mutations were found. In addition, Northern blot analysis showed no difference in ZNF216 transcript size or abundance between dn and control mice. These data Suggest that the ZNF216 gene is unlikely to be responsible for hearing loss at the DFNB7/11 and dn loci.

• Coquelle A, Toledo F, Stern S, Bieth A, Debatisse M
• A new role for hypoxia in tumor progression: induction of fragile site triggering genomic rearrangements and formation of complex DMs and HSRs.
• Mol Cell. 1998; 2: 259-65
• Display abstract

• Genome rearrangements including gene amplification are frequent properties of tumor cells, but how they are related to the tumor microenvironment is unknown. Here, we report direct evidence for a causal relationship between hypoxia, induction of fragile sites, and gene amplification. Recently, we showed that breaks at fragile sites initiate intrachromosomal amplification. We demonstrate here that hypoxia is a potent fragile site inducer and that, like fragile sites inducing drugs, it drives fusion of double minutes (DMs) and their targeted reintegration into chromosomal fragile sites, generating homogeneously staining regions (HSRs). This pathway operates efficiently for DMs bearing different sequences, suggesting a model of hypoxia-driven formation of the HSRs containing nonsyntenic sequences frequently observed in solid tumors.

• Glover TW et al.
• The murine Fhit gene is highly similar to its human orthologue and maps to a common fragile site region.
• Cancer Res. 1998; 58: 3409-14
• Display abstract

• The human FHIT gene is a putative tumor suppressor gene that maps to human chromosome band 3p14.2 in a region that is frequently deleted in cancers. It exhibits both genomic deletions and aberrant transcripts in a variety of tumors and spans the common fragile site FRA3B. This fragile site extends over a broad region of several hundred kb within the FHIT gene and may account for its instability in tumors. As one test of this hypothesis, we isolated the murine Fhit gene and asked whether it also contains a common fragile site and if it is unstable in mouse tumors or tumor cell lines. The Fhit gene was isolated, and the sequence was found to be 87.5% identical to that of the human FHIT gene in the open reading frame. Using fluorescence in situ hybridization, Fhit was assigned to mouse chromosome band 14A2, in a region that was previously shown to contain an aphidicolin-inducible mouse fragile site. Fluorescence in situ hybridization with genomic clones containing Fhit and flanking sequences demonstrated that gaps and breaks in the fragile site occur over a broad region within and proximal to the Fhit locus. Thus, the physical relationship of Fhit to a common fragile site is similar to that observed with the orthologous human FHIT gene and FRA3B.

• Krieg P, Schuppler M, Koesters R, Mincheva A, Lichter P, Marks F
• Repetin (Rptn), a new member of the "fused gene" subgroup within the S100 gene family encoding a murine epidermal differentiation protein.
• Genomics. 1997; 43: 339-48
• Display abstract

• We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pI of 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescence in situ hybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the "fused gene" subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.

• Sandford R et al.
• Comparative analysis of the polycystic kidney disease 1 (PKD1) gene reveals an integral membrane glycoprotein with multiple evolutionary conserved domains.
• Hum Mol Genet. 1997; 6: 1483-9
• Display abstract

• PKD1 is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease (ADPKD). Analysis of the predicted protein sequence of the human PKD1 gene, polycystin, shows a large molecule with a unique arrangement of extracellular domains and multiple putative transmembrane regions. The precise function of polycystin remains unclear with a paucity of mutations to define key structural and functional domains. To refine the structure of this protein we have cloned the genomic region encoding the Fugu PKD1 gene. Fugu PKD1 spans 36 kb of genomic DNA and has greater complexity with 54 exons compared with 46 in man. Comparative analysis of the predicted protein sequences shows a lower level of homology than in similar studies with identity of 40 and 59% similarity. However key structural motifs including leucine rich repeats (LRR), a C-type lectin and LDL-A like domains and 16 PKD repeats are maintained. A region of homology with the sea urchin REJ protein was also confirmed in Fugu but found to extend over 1000 amino acids. Several highly conserved intra- and extra-cellular regions, with no known sequence homologies, that are likely to be of functional importance were detected. The likely structure of the membrane associated region has been refined with similarity to the PKD2 protein and voltage gated Ca2+ and Na+ channels highlighted over part of this area. The overall protein structure has therefore been clarified and this comparative analysis derived structure will form the basis for the functional study of polycystin and its individual domains.

• Coquelle A, Pipiras E, Toledo F, Buttin G, Debatisse M
• Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicons.
• Cell. 1997; 89: 215-25
• Display abstract

• Drug-selected intrachromosomal gene amplification by breakage-fusion-bridge (BFB) cycles is well documented in mammalian cells, but factors governing this mechanism are not clear. Here, we show that only some clastogenic drugs induce drug resistance through intrachromosomal amplification. We strictly correlate triggering of BFB cycles to induction of fragile site expression. We demonstrate a dual role for fragile sites in intrachromosomal amplification: a site telomeric to the selected gene is involved in initiation, while a centromeric site defines the size and organization of early amplified units. The positions of fragile sites relative to boundaries of amplicons found in human cancers support the hypothesis that fragile sites play a key role in the amplification of at least some oncogenes during tumor progression.

• Paoloni-Giacobino A, Chen H, Peitsch MC, Rossier C, Antonarakis SE
• Cloning of the TMPRSS2 gene, which encodes a novel serine protease with transmembrane, LDLRA, and SRCR domains and maps to 21q22.3.
• Genomics. 1997; 44: 309-20
• Display abstract

• To contribute to the development of the transcription map of human chromosome 21 (HC21), we have used exon trapping from pools of HC21-specific cosmids. Using selected trapped exons, we have identified a novel gene (named TMPRSS2) that encodes a multimeric protein with a serine protease domain. The TMPRSS2 3.8-kb mRNA is expressed strongly in small intestine and weakly in several other tissues. The full-length cDNA encodes a predicted protein of 492 amino acids that contains the following domains: (i) A serine protease domain (aa 255-492) of the S1 family that probably cleaves at Arg or Lys residues. (ii) An SRCR (scavenger receptor cysteine-rich) domain (aa 149-242) of group A (6 conserved Cys). This type of domain is involved in the binding to other cell surface or extracellular molecules. (iii) An LDLRA (LDL receptor class A) domain (aa 113-148). This type of domain forms a binding site for calcium. (iv) A predicted transmembrane domain (aa 84-106). No typical signal peptide was recognized. The gene was mapped to 21q22.3 between markers ERG and D21S56 in the same P1 as MX1. The physiological role of TMPRSS2 and its involvement in trisomy 21 phenotypes or monogenic disorders that map to HC21 are unknown.

• Ong ST et al.
• Precise localization of the FHIT gene to the common fragile site at 3p14.2 (FRA3B) and characterization of homozygous deletions within FRA3B that affect FHIT transcription in tumor cell lines.
• Genes Chromosomes Cancer. 1997; 20: 16-23
• Display abstract

• Chromosomal or allelic losses at 3p14 are common in a variety of human tumors, including those of the lung, breast, kidney, and head and neck. This suggests the existence of a tumor suppressor gene in this band. A promising candidate is the recently cloned FHIT gene, which spans the common fragile site, FRA3B, at 3p14.2. We previously identified a region of fragility at 3p14.2 (FRA3B) of > 85 kb by cloning DNA flanking pSV2neo integrations and constructed a partial genomic contig of the region. Using probes from the contig, we tested for deletions within this region in DNA from 105 human tumor cell lines, predominantly derived from lung cancers. We identified one gastric and four lung cancer cell lines with homozygous interstitial deletions involving the FRA3B region. The deletion in one lung cancer cell line lies entirely within our contig and is < 65 kb. We have identified, cloned, and sequenced this breakpoint junction. We have also shown that our probes lie within intron S of the FHIT gene and, furthermore, that exon 5 is located approximately 1 kb from one of our probes and, thus, lies within the region of fragility. Two lines with entirely intronic deletions yield FHIT transcripts of normal size. In one of these, this was the sole transcript identified. In the other line, an FHIT transcript completely normal in sequence was accompanied by two larger abnormal transcripts. These results leave open the possibility that some homozygous deletions within the FHIT gene are without phenotypic effect and result from genetic instability of this region. However, taken together, our results provide evidence that breakage and rearrangement within the FRA3B fragile site sequences result in alterations of FHIT and are likely to be involved in carcinogenesis.

• Leung E et al.
• Cloning of novel kinectin splice variants with alternative C-termini: structure, distribution and evolution of mouse kinectin.
• Immunol Cell Biol. 1996; 74: 421-33
• Display abstract

• The analysis of cDNA clones encoding novel variant forms of mouse kinectin, an endoplasmic reticulum (ER)-bound receptor for the motor protein kinesin, is reported. Kinesin and cytoplasmic dynein are involved in mediating the anterograde and retrograde movements of intracellular vesicles along the microtubule network. The amino acid sequence deduced from kinectin cDNA isolated from mouse spleen cell and testis libraries revealed a long signal peptide or transmembrane sequence, and a 328 amino acid residue globular N-terminal domain adjacent to a much larger 858-999-residue C-terminal coiled-coil rod domain. The C-terminal domain was composed of 18 coiled-coil regions formed from multiple contiguous heptad repeats which undergo alternative splicing as evidenced by the presence of at least five small (23-33 amino acid residue) insertion sequences scattered throughout. The inserts are present in any one of a number of combinations, generating an array of novel kinectin variants. Insert 5 contains a termination codon, producing a C-terminus that is highly homologous to that of human kinectin. Three out of five mouse kinectin clones lack insert 5, generating a novel eleven amino acid C-terminus encoded by sequence that extends past the insertion site. The existence of alternative C-termini may have functional relevance given that the C-termini are exposed for interaction with kinesin, whereas the globular N-terminus is embedded in the ER membrane. Alternative C-termini represent candidate modifications that could determine specificity of binding to kinesin or cytoplasmic dynein, and the switching of directionality of movement. The cDNA hybridized to 4.5 kb transcripts expressed in all mouse cell lines and tissues examined, which provides the first indication that the kinectins are very widely distributed. Mouse kinectin is 42% similar over a 203 amino acid region to the chicken extracellular cardiac morphogen ES/130, whose canine homologue containing an inserted sequence of 10 amino acids repeated 54 times in tandem, is a ribosome receptor expressed on the ER. Mouse kinectin shares 64 and 83% identity, respectively, with its M(r) 160000 chicken and human kinectin homologues. There is a two-fold molar excess of kinectin over kinesin in unextracted vesicles, suggesting that kinectin might be a dimer. The electrostatic properties of the coiled-coil region of mouse kinectin, together with the relative frequencies of residues in particular positions within the heptad repeats support this notion.

• Musio A, Rainaldi G, Sbrana I
• Spontaneous and aphidicolin-sensitive fragile site 3cen co-localizes with the (TTAGGG)n telomeric sequence in Chinese hamster cells.
• Cytogenet Cell Genet. 1996; 75: 159-63
• Display abstract

• Aphidicolin-sensitive fragile sites were analyzed in immortalized Chinese hamster embryonal fibroblast cells (CHEF18) at three different passages along their spontaneous progression toward tumorigenicity. Five fragile sites (viz., 12q22, 3cen, 3p21, 3q31, and Xq21) were detected. Three of these sites carry spontaneous aberrations and are thus regions of chromosomal instability; however, they were not involved in the formation of the clonal rearrangements that are characteristic of CHEF 18 cells. The presence of the (TTAGGG)n telomeric sequence in chromosome bands associated with fragile sites was investigated using fluorescence in situ hybridization and primed in situ labeling. A common location of fragile sites and telomeric sequence was found at the centromere of chromosome 3.

• Moy GW, Mendoza LM, Schulz JR, Swanson WJ, Glabe CG, Vacquier VD
• The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein, PKD1.
• J Cell Biol. 1996; 133: 809-17
• Display abstract

• During fertilization, the sea urchin sperm acrosome reaction (AR), an ion channel-regulated event, is triggered by glycoproteins in egg jelly (EJ). A 210-kD sperm membrane glycoprotein is the receptor for EJ (REJ). This conclusion is based on the following data: purified REJ binds species specifically to EJ dotted onto nitrocellulose, an mAb to REJ induces the sperm AR, antibody induction is blocked by purified REJ, and purified REJ absorbs the AR-inducing activity of EJ. Overlapping fragments of REJ cDNA were cloned (total length, 5,596 bp). The sequence was confirmed by microsequencing six peptides of mature REJ and by Western blotting with antibody to a synthetic peptide designed from the sequence. Complete deglycosylation of REJ followed by Western blotting yielded a size estimate in agreement with that of the mature amino acid sequence. REJ is modular in design; it contains one EGF module and two C-type lectin carbohydrate-recognition modules. Most importantly, it contains a novel module, herein named the REJ module (700 residues), which shares extensive homology with the human polycystic kidney disease protein (PKD1). Mutations in PKD1 cause autosomal dominant polycystic kidney disease, one of the most frequent genetic disease of humans. The lesion in cellular physiology resulting from mutations in the PKD1 protein remains unknown. The homology between REJ modules of the sea urchin REJ and human PKD1 suggests that PKD1 could be involved in ionic regulation.

• Mochizuki T et al.
• PKD2, a gene for polycystic kidney disease that encodes an integral membrane protein.
• Science. 1996; 272: 1339-42
• Display abstract

• A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

• Chen MS, Featherstone T, Laszlo A
• Amplification and altered expression of the hsc70/U14 snoRNA gene in a heat resistant Chinese hamster cell line.
• Cell Stress Chaperones. 1996; 1: 47-61
• Display abstract

• We have recently demonstrated that the heat resistant phenotype of the HR-1 variant isolated from HA-1 Chinese hamster fibroblasts after a series of heat shocks is associated with the increased expression of Hsc70, the constitutive form of Hsp70 (Laszlo and Li 1985). Here, we report the cloning and characterization of the Chinese hamster hsc70 gene and its organization and expression in wild type HA-1 and permanently heat resistant HR-1 cells. DNA sequencing revealed that the structure and nucleotide sequence of the hamster hsc70 gene is highly homologous to the human and rat genes coding for Hsc70. Three of the eight introns of the hamster hsc70 gene encode U14 small nucleolar RNAs, as has been demonstrated in other species. Although putative transcriptional elements, including a TATA box, two inverted CAT boxes, and two sets of heat shock elements (HSEs) are completely conserved in the human and hamster hsc70 genes, the regulation of expression of the hamster hsc70 gene is different from that reported for its human counterpart in that the mRNA coding for Hsc70 increases at least 10-fold after a mild heat shock in Chinese hamster cells while no induction of Hsc70 by heat shock has been reported in human cell lines. In situ hybridization revealed a complex chromosomal rearrangement in HR-1 cells which results in the 4- to 5-fold amplification of the hsc70 gene as indicated by genomic Southern blots. In association with this amplification of the hsc70 gene, the levels of Hsc70 mRNA and U14 snoRNA are increased in the HR-1 cells under both normal growing conditions and after heat shock. Thus, the elevated expression of both Hsc70 and U14 snoRNA might play a role in the heat resistant phenotype of the HR-1 cells. This is the first report of the amplification of a heat shock gene and the possible induction of gene amplification by heat shock.

• Singh B, Hao W, Wu Z, Eigl B, Gupta RS
• Cloning and characterization of cDNA for adenosine kinase from mammalian (Chinese hamster, mouse, human and rat) species. High frequency mutants of Chinese hamster ovary cells involve structural alterations in the gene.
• Eur J Biochem. 1996; 241: 564-71
• Display abstract

• The enzyme adenosine kinase constitutes the major purine nucleoside phosphorylating activity in mammalian cells. In view of its central role in adenosine metabolism, which is an important physiological regulator, an understanding of the primary structure of adenosine kinase is of much interest. Using microsequence information from peptides derived from purified Syrian hamster liver enzyme, we have succeeded in isolating full length cDNA clones encoding adenosine kinase from Chinese hamster ovary cells and mouse 3T3 cells. The open reading frames in these clones consist of 334 and 335 amino acids and encode proteins of molecular masses 37364 Da and 37489 Da, respectively. In addition, the coding and upstream sequences for adenosine kinase from human (HeLa cells) and rat liver have also been cloned and sequenced. Transfection of an adenosine-kinase-deficient mutant (selected for resistance to the adenosine analog toyocamycin) of Chinese hamster ovary cells with a plasmid containing the cloned adenosine kinase cDNA, leads to regaining of adenosine kinase activity in the transformed cell. The adenosine kinase transformants also simultaneously lost their toyocamycin resistance and became similarly sensitive to the analog as the parental wild-type Chinese hamster ovary cells. The cloned adenosine kinase cDNA was also used to examine structural changes in mutants affected in adenosine kinase. In Chinese hamster ovary cells, one type of mutant that lacks adenosine kinase activity and displays high degree of resistance to various adenosine analogs, is obtained at an unusually high spontaneous frequency (10(-4)-10(-3)). Results of Southern and northern-blot analysis provide evidence that this group of mutants involves gross structural alterations affecting the adenosine kinase gene. Such structural alterations are not observed in another type of mutant which exhibits increased resistance only to C-adenosine analogs. Sequence similarity searches indicate that several of the bacterial and yeast sugar kinases (ribokinase, fructokinase and inosine-guanosine kinase) exhibit limited but significant similarity to the mammalian adenosine kinase. The sequence similarity data support the possibility that adenosine kinase shares a common evolutionary ancestor with these protein sequences.

• Rassool FV et al.
• Direct cloning of DNA sequences from the common fragile site region at chromosome band 3p14.2.
• Genomics. 1996; 35: 109-17
• Display abstract

• Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown. We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction. Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B). These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb. Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites. In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats. Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break. The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.

• Masana MI, Brown RC, Pu H, Gurney ME, Dubocovich ML
• Cloning and characterization of a new member of the G-protein coupled receptor EDG family.
• Receptors Channels. 1995; 3: 255-62
• Display abstract

• We report here the cloning of a new member of the endothelium differentiation gene (edg) subfamily of G-protein-coupled receptors. This novel cDNA sequence was cloned from the ovine pars tuberalis using a reverse transcriptase polymerase chain reaction (RT-PCR) amplification with degenerate primers homologous to the highly conserved II and VII transmembrane domains of the G-protein coupled receptor gene family. The PCR product was random primed with 32P and used as a probe to screen a size-selected cDNA ovine pars tuberalis library, which resulted in the isolation of a single clone of 2700 bp. This novel sequence was named edg-2, because its nucleic acid sequence was 55% homologous over 501nt overlap to an orphan sequence cloned from human endothelial cells, the endothelial differentiation gene, edg-1. The highest degree of aminoacid homology (42%) occurs in the seven putative transmembrane domains, particularly between the transmembrane domains III and VI (53% and 64%, respectively). The intervening hydrophilic domains are short and there are numerous putative phosphorylation sites for Ser/Thr-protein kinases in the second and third intracellular loop and in the COOH-terminal domain. Through Northern analysis of total RNA, low levels of at least four transcripts of 2.3, 2.5, 3.2 and 4 kb were found in sheep cerebral cortex and a 4.2 kb transcript was observed in NIH/3T3 fibroblasts. In addition, edg-2 transcripts (415 bp) were amplified by RT-PCR from pars tuberalis, cerebral blood vessels, hypothalamus, and retina. Serum stimulation of Chinese hamster ovary (CHO) cells expressing the edg-2 receptor resulted in increased cell proliferation, as measured by [3H]-thymidine incorporation. Edg-1 and edg-2 appear to be distinct genes that may encode protein products that bind the same or related ligand.

• Kuo MT, Vyas RC, Jiang LX, Hittelman WN
• Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.
• Mol Cell Biol. 1994; 14: 5202-11
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• Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

• Yamada K, Kato H, Kanda N, Fujii-Kuriyama Y, Utakoji T, Itoh R
• Sequence homology of Chinese hamster metallothionein genes I and II to those of the mouse and rat, and their amplification in Cd-resistant cells.
• Biochim Biophys Acta. 1994; 1219: 581-91
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• The metallothionein (MT) I and II genes were isolated from Chinese hamster cells and sequenced. The MT-II gene is located about 6 kb upstream of the MT-I gene and their arrangement is similar to those of the mouse and rat MT genes. The sequence of the Chinese hamster MT-I gene is highly homologous to those of the mouse and rat, particularly in their promoter regions of MT-I. However, the promoter region of MT-II has less homology with those of the mouse and rat due t to insertions and deletions. The MT-I and MT-II genes were equally amplified 4-8-times in the Cd-resistant Chinese hamster cells, suggesting that both genes are included in the same amplification unit. Cytogenetic analysis of Cd-resistant cells by in situ hybridization showed that they are randomly integrated into multiple sites on the chromosomes.

• Lipskaia LA et al.
• [The amplification and overexpression of mdr-family genes in ethidium bromide-resistant Chinese hamster CHO-K1 cells and in the hybrids of sensitive and resistant cells].
• Tsitologiia. 1994; 36: 1236-44
• Display abstract

• Stable mutant cells Cebr-1 and Cebr-2, resistant to ethidium bromide (EB) in concentration of 2 micrograms/ml, have been isolated by a multistep selection in Chinese hamster ovary cells. It was shown that Cebr-1 and Cebr-2 cells acquired a cross-resistance to unrelated drugs. Stable changes in the structure of chromosomes 1, 2, 5 and 8 were revealed by karyological analysis. Overexpression and amplification of mdr genes were detected in Cebr-2 cells using Northern RNA and Southern DNA blot hybridization. Two independent hybrids Hybr-1 and Hybr-2 were obtained by fusion of Cebr-2 cells with Chinese hamster lung V-79 RJK cells, sensitive to EB. Hybr-2 cells were characterized by the same level of EB-resistance as Cebr-2 cells. Hybr-1 cells have a lower level of EB-resistance than Cebr-2 cells. Hybr-2 cells have demonstrated amplification and overexpression of mdr gene, the same as in Cebr-2 cells, whereas in Hybr-1 cells no mdr gene amplification was observed, but the level of mdr gene expression was higher than in sensitive cells. The data suggest that resistance of Chinese hamster cells to EB is mediated by amplification and overexpression of mdr genes.

• Chang CC, Huh HY, Cadigan KM, Chang TY
• Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.
• J Biol Chem. 1993; 268: 20747-55
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• Accumulation of cholesterol esters as cytoplasmic lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane protein located in the endoplasmic reticulum. The ACAT protein has never been purified to homogeneity, and no antibodies directed against ACAT have been reported. The gene(s) encoding this enzyme had not been isolated. This laboratory had previously reported the isolation of Chinese hamster ovary cells expressing human ACAT activity. From DNAs of these cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macrophage cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the catalysis of acyl adenylate formation followed by acyl thioester formation and acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecular studies on ACAT.

• Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I
• Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene.
• Cell. 1993; 75: 253-61
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• Mice deficient for interleukin-2 develop normally during the first 3-4 weeks of age. However, later on they become severely compromised, and about 50% of the animals die between 4 and 9 weeks after birth. Of the remaining mice, 100% develop an inflammatory bowel disease with striking clinical and histological similarity to ulcerative colitis in humans. The alterations of the immune system are characterized by a high number of activated T and B cells, elevated immunoglobulin secretion, anti-colon antibodies, and aberrant expression of class II major histocompatibility complex molecules. The data provide evidence for a primary role of the immune system in the etiology of ulcerative colitis and strongly suggest that the disease results from an abnormal immune response to a normal antigenic stimulus.

• Grinchuk TM, Lipskaia LA, Vasiukhim VI, Sorokin EA, Artsybasheva IV, Ignatova TN
• [The amplification and overexpression of the mdr genes in Chinese hamster CHLV-79 RJK cells resistant to ethidium bromide correlate with the presence of karyotypic markers of the amplification].
• Tsitologiia. 1993; 35: 86-90
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• Evidence is provided that selection of the Chinese hamster cells CHLV-79 RJK with ethidium bromide results in amplification and overexpression of mdr family genes, one of which is encoding a transmembrane P-glycoprotein reducing the intracellular drug concentration. It is likely that the amplified copies are located at or near the sites of resident mdr gene localization to look as an abnormal chromosomal banding pattern in chromosome 1q26. In the following selection steps to higher drug concentration, the cells are keeping the degree of amplification, but mdr gene expression increases by many times. The data suggest that the resistance of the Chinese hamster cells CHLV-79 RJK to higher concentrations of ethidium bromide may be achieved via a variety of mechanisms, including as well mdr gene amplification as transcriptional regulation of these genes.

• Tanizawa A, Beitrand R, Kohlhagen G, Tabuchi A, Jenkins J, Pommier Y
• Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance.
• J Biol Chem. 1993; 268: 25463-8
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• A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that contains a catalytically altered and camptothecin (CPT)-resistant DNA topoisomerase I (top 1) (Tanizawa, A., and Pommier, Y. (1992) Cancer Res. 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs. Northern blot analysis showed a single 4.1-kilobase band without quantitative reduction between the two cell lines. We have cloned and sequenced top 1 cDNAs. DC3F and DC3F/C-10 top 1 c-DNA are 3591 and 3626 base pair long, respectively, and encode 767 amino acids. The homology of deduced amino acid sequences between Chinese hamster and mouse or human top 1 are 98.1 and 96.7, respectively. cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly505-->Ser). G505 corresponds to Gly503 of human top 1 cDNA and is located 220 amino acids away from the presumed catalytic Tyr725. The point mutation in the Chinese hamster top 1 is located in a region that is highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia virus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schizosaccharomyces pombe, mouse, and Human). A mutation of Asp533 to Gly in this same region has been shown to confer CPT resistance for human top 1. Chinese hamster top 1 protein with a Gly505-->Ser mutation that was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance. Our results suggest that the highly conserved region around Gly505 plays an important role in the interactions among top 1, DNA, and CPT.

• Kopnin BP, Sokova OI, Demidova NS
• Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.
• Mutat Res. 1992; 276: 163-77
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• Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.

• Dorner AJ, Bonneville F, Kriz R, Kelleher K, Bean K, Kaufman RJ
• Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.
• J Virol. 1991; 65: 4713-9
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• We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.

• Rassool FV, McKeithan TW, Neilly ME, van Melle E, Espinosa R 3rd, Le Beau MM
• Preferential integration of marker DNA into the chromosomal fragile site at 3p14: an approach to cloning fragile sites.
• Proc Natl Acad Sci U S A. 1991; 88: 6657-61
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• Fragile sites are specific regions of chromosomes that are prone to breakage. In cells cultured under conditions that induce fragile site expression, high levels of inter- and intrachromosomal recombination have been observed involving chromosomal bands containing fragile sites. To determine whether expression of specific fragile sites would facilitate preferential integration of exogenous DNA at these recombination hot spots, the vector pSV2Neo was transfected into a Chinese hamster-human somatic cell hybrid containing a derivative chromosome 3 as its only human component. Chromosome 3 contains a common fragile site at band 3p14.2 (FRA3B) that is induced by aphidicolin. Both cells induced to express FRA3B and the uninduced control cells were transfected with the pSV2Neo selectable plasmid. In situ hybridization of a biotin-labeled pSV2Neo probe to metaphase chromosomes revealed one to three integration sites in each stably transfected clone. Four of 13 clones transfected under conditions of FRA3B induction showed integration of pSV2Neo at 3p14; these clones also showed specific integration into hamster chromosome 1 and a rearranged chromosome characteristic of CHO cells (mar2). The 7 control clones, however, showed an apparently random pattern of pSV2Neo integration. Significant hybridization of pSV2Neo to both FRA3B and Chinese hamster chromosomes 1 and mar2 was seen in 100 cells from pooled colonies transfected after treatment with aphidicolin. These results suggest that preferential integration of marker DNA into human and Chinese hamster fragile sites occurs with exposure to aphidicolin. The nature of the DNA sequences at fragile sites is unknown and, despite a number of approaches, these sequences have not yet been isolated; our procedure may represent an approach to the cloning of fragile sites.

• Devine SE, Hussain A, Davide JP, Melera PW
• Full length and alternatively spliced pgp1 transcripts in multidrug-resistant Chinese hamster lung cells.
• J Biol Chem. 1991; 266: 4545-55
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• In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts. Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product. A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e. human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function. Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165. Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein. S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines. Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.

• Scocca JR, Krag SS
• Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells.
• J Biol Chem. 1990; 265: 20621-6
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• We have isolated a portion of the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-glucosamine-1-phosphate transferase gene (GTR2) from the genome of a tunicamycin-resistant clonal Chinese hamster ovary cell line, 3E11. The genomic fragment was selected by its hybridization to the yeast ALG-7 gene at low stringency. A 2.46-kilobase cDNA was isolated from a library prepared from 3E11 mRNA and probed with GTR2. The cDNA contained an open reading frame that encodes a protein of 408 amino acids with a molecular mass of 44.9 kDa. This protein was 43% identical in amino acid sequence to the protein of 448 amino acids encoded by the ALG-7 gene. The GTR2 gene fragment contained sequences for four exons coding for the carboxyl-terminal half of the protein. Transferase DNA sequences in 3E11 cells were 12-fold elevated over wild-type cells and 25-fold elevated when 3E11 cells were grown in the presence of tunicamycin. Transferase RNA levels in 3E11 cells were also elevated over wild-type levels but appeared unchanged by the presence of tunicamycin in the medium.

• Sen S, Teeter LD, Kuo T
• Specific gene amplification associated with consistent chromosomal abnormality in independently established multidrug-resistant Chinese hamster ovary cells.
• Chromosoma. 1987; 95: 117-25
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• Multidrug-resistant (MDR) Chinese hamster ovary (CHO) cell lines were established by selection for resistance to the toxicities of vinblastine (VB) and Adriamycin (AD) in progressively increasing drug concentrations. These cell lines have amplified the DNA sequence that has previously been shown to be amplified in another MDR CHO cell line which was selected with vincristine (VC). An overproduced 4.5 kb mRNA was detected in these MDR cell lines. We report here that the levels of DNA amplification and the 4.5 kb transcript do not correlate with the levels of drug resistance, suggesting that either translational control for the expression of the amplified gene is involved or multiple genes are participating in conferring drug resistance in these cell lines. The amplified DNA sequence was used as a probe and localized by in situ hybridization to chromosome 1q 26-28 (middle portion of the long arm) in the drug-sensitive CHO line, but proximal to the telomere of chromosome 1q in both VB- and AD-selected MDR cell lines. This is consistent with results that have been previously reported for the VC-selected MDR cell lines. Cytogenetic analyses revealed abnormal chromosomal banding patterns or homogeneously staining regions (HSR) between 1q 26-28 and the 1q ter in these independently established MDR lines. These results, taken together, suggest that chromosomal rearrangements leading to gene translocation have consistently accompanied gene amplification in these MDR cell lines. The mechanisms of translocation and its implication in multidrug resistance in these cell lines are discussed.

• Gros P, Croop J, Roninson I, Varshavsky A, Housman DE
• Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells.
• Proc Natl Acad Sci U S A. 1986; 83: 337-41
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• The mechanism by which mammalian cells acquire resistance to chemotherapeutic agents has been investigated by using molecular genetic techniques. LZ and C5, two independently derived multidrug-resistant Chinese hamster cell lines, share specific amplified DNA sequences. We demonstrate that commonly amplified DNA sequences reside in a contiguous domain of approximately equal to 120 kilobases (kb). We report the isolation of this DNA domain in cosmid clones and show that the level of amplification of the domain is correlated with the level of resistance in multidrug-resistant cell lines. The organization of the amplified domain was deduced by a unique approach utilizing in-gel hybridization of cloned DNA with amplified genomic DNA. We show that the entire cloned region is amplified in adriamycin-resistant LZ cells and independently derived, colchicine-resistant C5 cells. A mRNA species of approximately equal to 5 kb is encoded by a gene located within the boundaries of this region. Genomic sequences homologous to the 5-kb mRNA span over 75 kb of the amplified DNA segment. The level of expression of this mRNA in multidrug-resistant cells is correlated with the degree of gene amplification and the degree of drug resistance. Our results strongly suggest that the 5-kb mRNA species plays a role in the mechanism of multidrug resistance common to the LZ and C5 cell lines.

• Ray PN, Siminovitch L, Andrulis IL
• Molecular cloning of a cDNA for Chinese hamster ovary asparagine synthetase.
• Gene. 1984; 30: 1-9
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• In previous reports we have described the isolation and characterization of a number of Chinese hamster ovary (CHO) cell mutants resistant to the amino acid analogue albizziin (Alb). Multistep mutants were derived which showed a high degree of drug resistance and expressed increased levels of asparagine synthetase (AS) levels up to 300-fold over that of the parental cell line. Karyotypic analysis of these mutants revealed homogeneously staining regions (HSRs) usually indicative of gene amplification. In the present work, we provide further proof for gene amplification by showing that the mutants greatly overproduce functional AS mRNA, as evidenced by in vitro translation of purified mRNA and immunoprecipitation of AS. By using these overproducing mutants as sources of mRNA coupled with velocity centrifugation, we have been able to greatly enrich for AS sequences in our mRNA preparations to the point where they represent 1-5% of the total message. This facilitated cloning and selection of the cDNA sequences complementary to the gene. Utilizing these cloned cDNAs, we have demonstrated a correlation between gene copy number and enzyme expression in the parent and Alb-resistant mutants, thus providing direct evidence that drug resistance is due to gene amplification.

• Wahl GM et al.
• Analysis of CAD gene amplification using a combined approach of molecular genetics and cytogenetics.
• Adv Exp Med Biol. 1984; 172: 319-45
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• CAD is a multifunctional protein which catalyzes the first three steps of de novo uridine biosynthesis. Rodent cells resistant to PALA, a specific inhibitor of the ATCase activity of CAD, overproduce the CAD protein and CAD mRNA as a direct result of the amplification of the CAD gene. In order to study the mechanism of CAD gene amplification, a functional Syrian hamster CAD gene was inserted into a cosmid vector using molecular cloning techniques. The cloned genes were assayed for biological function by fusing CAD-deficient Chinese hamster ovary (CHO) cell mutants with protoplasts of E. coli containing the CAD cosmids. Two clones with functional CAD genes were isolated and shown to contain inserts 40 and 45 kb long. The cloned genes could also be introduced into wild type CHO cells by selecting for cells which became resistant to high PALA concentrations in a single step. Transformations of mutant and wild type CHO cells contained multiple active copies of the donated Syrian hamster CAD genes in addition to their endogenous CHO CAD genes. The cloned genes in all transformants analyzed are integrated into host cell chromosomes at single locations defined by in situ hybridization. Independently isolated transformants contain the donated genes in different chromosomes. Co-transformation of CHO cells with two different genes by protoplast fusion is also shown to be possible.

• Konecki DS, Brennand J, Fuscoe JC, Caskey CT, Chinault AC
• Hypoxanthine-guanine phosphoribosyltransferase genes of mouse and Chinese hamster: construction and sequence analysis of cDNA recombinants.
• Nucleic Acids Res. 1982; 10: 6763-75
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• Recombinant plasmids containing DNA inserts complementary to mRNA coding for hypoxanthine-guanine phosphoribosyltransferase (HPRT) from mouse and Chinese hamster cell lines have been isolated from cDNA libraries and characterized by DNA sequence analysis. A total of 1292 nucleotides of the mouse cDNA sequence and 1301 nucleotides of the Chinese hamster cDNA sequence has been determined. Each of these sequences includes an open reading frame of 654 nucleotides (218 amino acids) corresponding to the HPRT protein coding region. The deduced amino acid sequences for the mouse and Chinese hamster enzymes are presented and compared to that of human HPRT. At least 95% of the amino acids are conserved in the three species. In addition, we present evidence that two species of HPRT mRNA, which differ in the site of polyadenylation that is utilized during processing of the RNA transcripts, exist in Chinese hamster cells.

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