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. The strategy 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. Genome-wide linkage data from previous reporting periods have confirmed a major genetic effect in the HLA region, and also showed evidence of linkage (p less than 0.005) for chromosomes 1p13, 1q43, 6q21, 10q21, 12q12, 17p13, and 18q21. Because the 18q21 region has been replicated in independent French and Canadian cohorts, we have concentrated association studies for linkage disequilibrium (LD) on this segment of the genome. NARAC previously conducted a dense single-nucleotide polymorphism (SNP) analysis of the chromosome 18q21 candidate region using a bead-based optical technology (Illumina), examining 460 cases and 460 controls. The average marker density for the SNPs successfully typed was 4.3 kb over the 10 Mb region. Two LD clusters were identified, each with several SNPs with evidence for linkage to RA (p value less than or equal to 0.001). ? ? Results of the Last Year? ? Genome-wide analyses: During the past year we completed a second genome-wide analysis of the NARAC sibling pairs using >5700 SNPs (Illumina) (the previous genome-wide linkage analysis used 379 microsatellite markers). This more informative analysis identified two new regions on 11p12 and 2q33 with strong evidence for linkage to a disease susceptibility locus (LOD > 3.0). Furthermore, new regions with suggestive evidence for linkage were found on chromosomes 4q25, 5p12, and 10q21, and additional support was obtained for previously identified regions on 18q21, 20p13, and 1q41-42.? ? Analysis of the chromosome 18q21 candidate region: During the past year, we further examined the two 18q21 regions we identified last year and also a third region, in which we found a 4-SNP haplotype with evidence for association with an RA susceptibility locus. Addition of 656 independent cases and 604 controls to the analysis of previously identified regions strengthened the statistical significance to p = 0.0005 and p = 0.0009, respectively, retaining nominal significance when corrected for possible stratification among European subpopulations. The most strongly associated SNPs in region 1 are located between a transcription factor gene, ONECUT2, and the gene encoding ferrochelatase, FECH. The minor allele frequency of the most strongly-associated SNP in this region has a higher frequency in RA cases than in controls (0.35 in RA cases vs. 0.29 in controls). The most strongly-associated SNPs in region 2 are distal to the TCF4 gene in a partially characterized transcript expressed in lung, cerebellum, and primary cells cultured from cartilage. One of these SNPs would result in the substitution of glutamine for arginine at residue 95 (R95Q) of the predicted protein (p = 0.002). In a third, newly identified 18q region, we found a relatively rare 4-SNP haplotype with evidence for association with RA (the haplotype was found at 1.6% frequency in 460 controls and 4.4% frequency in 459 independent cases p=0.0006). We genotyped additional samples for a total of 1386 controls and 667 independent cases and found the haplotype frequency was 2.1% in controls and 3.9% in cases (Chi square analysis p = 0.001, permutation analysis, p = 0.008). The genomic region containing this haplotype was located distal to region 1 and the 4 SNPs were within FECH, encoding ferrochelatase, a mitochondrial enzyme responsible for the final step in the heme synthesis pathway, and which is mutated in erythropoietic protoporphyria. Further evaluation of these three regions entails the genotyping of an additional 1000 cases and controls that is currently underway.? ? Association of variants within the peptidyl arginine deiminase, type IV (PADI4) gene: Earlier studies from another group indicated that a specific variant and haplotype of the peptidyl arginine deiminase, type IV (PADI4) gene are associated with RA among the Japanese. During the previous reporting period we found that a different PADI4 haplotype associated with RA among Caucasians. In order to thoroughly evaluate the variants within the PADI4 gene region we genotyped the HapMap CEU samples for 69 polymorphic SNPs located within this 75 kb gene region. We then selected 22 tag SNPs that non-redundantly represented all 69 SNPs and genotyped the NARAC cases and controls for these 22 SNPs. We found two of the tag SNPs to be more strongly associated with RA than padi4-94, the most strongly associated SNP reported in the Japanese population. We genotyped all the SNPs in strong LD with the two disease associated SNPs and identified a total of 3 SNPs, all located in the first intron of PADI4, with minor allele frequencies of 5-6 percent in controls and 10-11 percent in cases (p equals 0.00006 to 0.00009). When we limited the analysis to the 453 rheumatoid factor positive cases, the p value was 0.000005, suggesting that the association was even stronger in this phenotypically more homogeneous group. Current studies are focused on determining whether these non-coding SNPs are associated with a difference in transcriptional efficiency.? ? Analysis of the type I diabetes-associated variant in the interferon-induced helicase gene: A nonsynonymous variant, A946T, in the interferon-induced helicase gene (IFIH1) was recently reported to be associated with type I diabetes. We genotyped this variant in the NARAC cohort and controls and found results similar to those observed in type I diabetes patients, that the minor allele (T) was reduced in cases compared with healthy controls. In the type I diabetes cases the minor allele frequency was 0.35 in cases and 0.39 in controls. We found the minor allele frequency was 0.37 in RA cases and 0.41 in controls (p = 0.02 in 653 independent cases and 1344 controls, p = 0.005 in an analysis including all affecteds that discounts sibs for their relatedness, i.e., the effective number of cases = 824). Replication studies are underway.? ? Other candidate genes evaluated: Additional candidate genes evaluated included FCRL3 (Fc receptor-like 3), which is associated with RA in the Japanese, NFKB1 (encoding the p50 NF kappa B subunit), and FCGR3A, which is associated with RA in the UK. In the NARAC cohort none of the polymorphisms we evaluated were associated with RA.? ? Conclusions and Significance? ? The data of the last year support the notion that multiple non-HLA genes confer modest levels of risk for RA, and continue to support the likelihood of an RA susceptibility locus on chromosome 18q. During the next year, we plan to continue studies of this region. We will also continue studies of genes not on chromosome 18, including PADI4 and IFIH1. As a part of the NARAC collaboration, we will also continue to provide in-depth analysis of regions within the newly identified linkage peaks and new regions identified by more global analyses, such as whole-genome association studies. Finally, we plan replication studies for those genomic regions that currently give equivocal results.
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