Historically, a major focus of this project has been the identification of rheumatoid arthritis susceptibility genes. The GGB was one of the founding members of the North American Rheumatoid Arthritis Consortium (NARAC), which has collected large numbers of DNA samples from sibling pairs concordant for rheumatoid arthritis as well as singleton cases. During the 2007 reporting period we discovered a variant of STAT4 (which encodes the signal transducer and activator of transcription 4 protein) that is associated with both rheumatoid arthritis and systemic lupus erythematosus. We also discovered a single nucleotide polymorphism close to the C5 and TRAF1 loci that is associated with rheumatoid arthritis. During the 2008 reporting period we have focused on the following projects:? ? Variant Form of the STAT4 Gene is Associated with Sjogrens Syndrome. Sjogrens syndrome is an autoimmune disease that affects tear and salivary glands, leading to dry eyes and mouth. This syndrome is frequently found in conjunction with other autoimmune rheumatic diseases, such as rheumatoid arthritis and systemic lupus erythematosus, and it is then described as secondary Sjogrens syndrome, but it can also occur in isolation, in which case it is described as primary Sjogrens syndrome. In order to separate the effects of the gene variant on risk of Sjogrens syndrome from its effects on other autoimmune diseases, we genotyped DNAs from 124 patients with primary Sjogrens syndrome and compared them with 1143 healthy controls. We have now established that the STAT4 gene variant associated with rheumatoid arthritis and systemic lupus erythematosus is also associated with Sjogrens syndrome suggesting that STAT4 dysregulation can promote a variety of autoimmmune pathologic conditions. ? ? The STAT4 variant is specifically associated with the most severe forms of systemic lupus erythematosus. Working with Dr. Criswells and Dr. Gregersens groups, we collected 4 large series of systemic lupus erythematosus patients (a total of 1398 cases) and determined their disease manifestations from their medical records. We genotyped these cases and 2690 healthy controls for 137 single nucleotide polymorphism (SNP) markers in a broad genomic region including and surrounding the STAT4 gene and confirmed our previous findings that a variant form of the STAT4 gene was associated with disease (allele frequency in cases = 31.1% versus controls = 22.5%) and that no other markers in the region were independently associated with disease. Interestingly, the STAT4 gene variant frequency was higher in the subset of cases with kidney involvement (34.3%), and was even higher in cases with severe kidney disease (38.1%) suggesting that the variant more specifically predisposes to SLE with kidney disease. Similarly, the variant frequency was found to be increased in cases with disease onset at less than 30 years of age (33.8%) and in cases with autoantibodies to double stranded DNA (35.1%) suggesting that the variant also more strongly contributes to disease with these features. Kidney involvement, early disease onset, and autoantibodies to double stranded DNA are all correlated disease features and are associated with a poor clinical prognosis. Conversely, the STAT4 variant was not associated with disease in the subset of cases with skin disease manifestations that often occurs in systemic lupus in the absence of more severe disease manifestations. These results suggest that the STAT4 variant is specifically associated with severe systemic lupus erythematosus.? ? Additional Genetic Variants Associated with Rheumatoid Arthritis. Working with Dr. Plenges and Dr. Gregersens groups we have now expanded our original genome-wide association studies by performing meta-analysis studies combining multiple genotyped large collections of rheumatoid arthritis cases and controls and by replicating these new findings by genotyping additional large independent case-control collections. We have thus identified several reproducible new genetic variants associated with disease. These loci include: two independent loci near TNFAIP3 (a gene encoding TNF-induced protein-3 or A20, which is a potent inhibitor of NF-kB signaling and is required for termination of tumor necrosis factor (TNF)-induced signals), a variant at CD40 (a gene involved in stimulating helper activity by CD4+ T cells in immune reactions including immunoglobulin class switching, memory B-cell development and germinal center formation), and one at CCL21 (a gene involved in lymphocyte trafficking). With HLADRB1, PTPN22, TRAF1/C5, and STAT4, there are now eight genetic loci that are convincingly associated with rheumatoid arthritis. We also identified four loci that have good evidence for association, but that require further confirmation: an intronic variant in CDK6 (a cyclin-dependent kinase involved in proliferation of B cells and CD8 memory cells), a variant near PIP4K2C (which has been implicated in signaling through the B-cell antigen receptor), a variant near TNFRSF14 (which is known to bind TRAF family members and is involved in activation of the transcription factors NF-kB and AP-1), and a variant located 100 kb away from the 3 end of PRKCQ (a gene which encodes a kinase required for the activation of the transcription factors NF-kB and AP-1, and may link the T-cell receptor signaling complex to the activation of the transcription factors). All these new disease-associated variants have small effect sizes (less than or equal to 1.33). We estimate that the total percent variance explained for all known non-MHC common genetic variants is just 3.6% and therefore less than half of the genetic variation can be explained by the known rheumatoid arthritis risk alleles. This suggests that other common alleles of modest effect size should be identified with additional GWA studies and deeper replication in large autoantibody-positive rheumatoid arthritis sample collections.? ? Failure to Replicate a Female-Specific Locus in Rheumatoid Arthritis Case-Control Collection from North America. We attempted to replicate a locus on chromosome 7q that was reported to be a female-specific disease locus. By evaluating the cases and controls for stratification (using whole genome SNP genotype data), we found that the locus was susceptible to stratification problems in the North American population and after correction found no evidence for association. ? ? Rheumatoid Arthritis in the Asian Population is Genetically Distinct From the Caucasian Population. Last year we reported that the same STAT4 variant that contributes to rheumatoid arthritis in the Caucasian population was also associated with disease in a large Korean case-control collection. We therefore sought to determine whether other disease-associated loci identified in the Caucasian population were associated with disease in the Korean population. None of the disease-associated SNPs we genotyped (loci from PTPN22, TRAF1/C5, TNFAIP3, CD40 or CCL21) were associated with disease in 1123 Korean patients with rheumatoid arthritis compared with 1008 healthy Korean controls. We conclude that most of the disease-associated genetic variants have arisen independently and it is likely that different points in disease-associated pathways are influenced by genetic risk factors in these two populations.? ? During the 2008 reporting period we have also begun an initiative to study the syndrome of periodic fever with aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA). PFAPA is the most common periodic fever syndrome in children, and appears to run in families. Functional and genetic studies are under way.
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