Background? ? The Complex Disease Genetics Unit (CDGU) of the Genetics and Genomics Branch was established to identify genes conferring susceptibility to genetically complex rheumatic and inflammatory diseases. This often entails nonparametric linkage analysis of large numbers of sibling pairs concordant for the disease in question and/or association studies on collections of independent cases and controls using a dense map of markers drawn from a given chromosomal region or the whole genome. The CDGU has focused on rheumatoid arthritis (RA), which affects as much as 1% of the population worldwide. This work builds on our long-standing participation in the North American Rheumatoid Arthritis Consortium (NARAC), a large collaborative group that has collected samples from over 1000 sibling pairs concordant for RA, as well as from cohorts of singleton RA cases and ethnically-matched controls. In the 2006 reporting period NARAC published a high-resolution linkage analysis on a cohort of 642 Caucasian families containing 1371 affected siblings. We analyzed approximately 5700 informative single nucleotide polymorphisms (SNPs). Even with a conservative correction for markers in linkage disequilibrium, two chromosomal regions, 2q33 (corrected LOD = 3.52) and 11p12 (corrected LOD = 3.09), emerged, both approaching the genome-wide LOD score criterion of 3.6 for significance in sib-pair studies. In the 2007 reporting period our efforts have been focused on the detailed analysis of the chromosome 2q33 linkage region, and, to a lesser degree, on a new genome-wide association study in RA. ? ? Results of the Last Year? ? Analysis of candidate genes in the chromosome 2q33 region: In the 52 Mb 2-LOD support interval, we identified 13 candidate genes of interest: NOSTRIN, WASPIP, FRZB, STAT1, STAT4, HSPD1, CFLAR, CASP10, CASP8, BMPR2, CD28, CLTA4, and ICOS. We performed an association analysis for 68 genotyped tag SNPs capturing the majority of common SNP variation, and 14 SNPs whose genotypes were imputed from multimarker combinations, in 525 independent case patients with RA, and 1165 unrelated controls. In addition to a known association with a SNP in CTLA4 (rs3087243, p = 0.008), we found an association with an unlinked imputed SNP in STAT4 (rs7574865, p = 0.002). ? ? Fine mapping of associations with rheumatoid arthritis in the STAT1-STAT4 region: Based on HapMap linkage disequilibrium data for northern and western Europeans, we selected a 209 kb genomic region subsuming the STAT1 and STAT4 genes for further association analysis. We genotyped the 525 NARAC cases and 1165 unrelated controls for 63 SNPs (average density, one SNP per 3.1 kb), capturing 87% of the common variation in the HapMap Phase II data in the region, with an r squared value of more than 0.8. Four SNPs located within the large third intron of STAT4 had associations with RA with P values less than 0.001. The most significant P value, 0.0000829, was found for rs7574865. The four disease-associated SNPs were in strong linkage disequilibrium, and all had a minor allele frequency of 0.28 in the NARAC case patients with RA, as compared with 0.22 in the unrelated controls.? ? Replication studies for STAT4 variants in RA in Caucasians: We genotyped all 63 SNPs from the STAT1-STAT4 region in a replication series of 1013 North American singleton case patients with RA who were positive for anti-cyclic citrullinated peptide (CCP) antibody, and 1326 controls. The same four variants within intron 3 of STAT4 seen in the initial NARAC series were strongly associated with RA in the replication set. We also genotyped the most significantly associated SNP from the NARAC case-control series, rs7574865, in 1529 case patients with recent-onset rheumatoid arthritis and in 881 controls from the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA), and again found a significant association at rs7574865. A meta-anlaysis of the three independent case-control series for RA yielded strong evidence of an association of the minor allele of rs7574865 with disease susceptibility (P = 0.0000000464). Genotypic odds ratios for patients as opposed to controls were 1.61 for homozygotes and 1.27 for heterozygotes. Logistic regression analysis showed that rs7574865 explained the signal across the STAT1-STAT4 region. Furthermore, after accounting for the CTLA4 SNP associated with RA, the result for the STAT4 rs7574865 remained significant.? ? Replication studies for STAT4 variants in RA in Asians: We also genotyped 1123 Korean patients with RA and 1008 ethnically-matched controls for 67 SNPs within the STAT1 and STAT4 regions. A common haplotype defined by rs7574865 and 3 other SNPs carried significant risk for RA in Koreans, present in 34% of cases versus 28% of controls, P = 0.0027, odds ratio = 1.33. By logistic regression analysis, this haplotype was an independent risk factor in addition to the classical shared epitope alleles at HLA-DRB1. There were no significant associations with age of disease onset, radiographic progression, or serologic status using either allelic or haplotypic analysis.? ? Association of STAT4 variants with systemic lupus erythematosus (SLE) in Caucasians: Since STAT4 lies within linkage peaks that have also been reported in patients with SLE, three lupus series of case and control subjects of European ancestry were also genotyped. We found that the minor allele frequency for rs7574865 was significantly increased in all three series among patients (0.29 to 0.31) as compared with controls (0.22 to 0.23) (P = 0.00000956 to P = 0.03). In a meta-analysis of the three series, we found strong evidence of association of the rs7574865 minor allele with SLE (p = 0.00000000187). The odds ratio for having the allele associated with SLE in chromosomes of patients as compared with those of controls was 1.55. Genotypic odds ratios were 2.41 for homozygotes and 1.56 for heterozygotes.? ? Whole genome-association analysis in RA among Caucasians: In a combined study, NARAC samples and EIRA samples were genotyped for 317,503 SNPs, first in a discovery set of 1522 case subjects with anti-CCP-positive rheumatoid arthritis and 1850 control subjects, and then in an independent replication set of 997 anti-CCP positive RA patients and 1777 control subjects. In this analysis, we observed associations between RA and variants of the major-histocompatibility complex (MHC) and PTPN22, both of which were recognized before this study, as well as a novel association for a SNP (rs3761847) on chromosome 9 for all samples tested. The odds ratio for the latter association was 1.32, with P = 0.00000000000004. This SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5). ? ? Conclusions and Significance? ? The data from the last year substantially advance our understanding of genetic susceptibility for RA and SLE by identifying new non-MHC risk loci, and support the notion that certain genes underly more than one autoimmune disease. During the next year, we will (1) examine the role of these loci in other autoimmune and autoinflammatory disorders; (2) resequence the STAT1-STAT4 region and attempt to find other SNPs perhaps more highly associated with RA than rs7574865; (3) collaborate with the Molecular Immunology and Inflammation Branch to understand the mechanism by which STAT4 variants predispose to RA and SLE; (4) perform additional fine-mapping and mechanistic experiments in order to determine whether C5 or STAT4 is the more important susceptibility gene for RA; (5) embark on high-resolution mapping studies of other regions of the genome showing possible association with RA; and (6) begin a whole-genome association study in Behcets disease.
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