Identification of the Novel Molecular Markers for Rheumatoid Arthritis (RA) in Pakistani Patients

Abstract

Rheumatoid arthritis (RA) is a systemic, complex, multifactorial autoimmune syndrome. The interplay of multiple genetic and environmental factors instigates an episode of RA. In the past era, multiple Genome-wide association studies (GWASs) have identified more than 100 HLA and non-HLA RA susceptibility genetic loci in Europeans. Given the anticipated overlap of RA-relevant genes and pathways across different ethnic groups, it was sought to replicate 58 GWASimplicated SNPs reported in Europeans in the Pakistani subjects. A total of 1,959 unrelated subjects (1,222 RA cases and 737 controls) were collected from three rheumatology facilities in Pakistan. Genotyping was performed using or TaqMan® or iPLEX methods. A total of 50 quality control (QC) passed SNPs were included in the final association analysis after excluding those that failed assay design/run or post-run QC analysis. 14 SNPs (LINC00824/rs1516971, CEP57/rs4409785, PADI4/rs2240336, STAT4/rs13426947, CTLA4/rs3087243, C5orf30/rs26232, HLA-B/MICA/rs2596565, GATA3/rs2275806, CCL21/rs951005, HLA-DRB1/rs660895, VPS37C/rs595158, SPRED2/rs934734, EOMES/rs3806624, and RUNX1/rs9979383) were replicated successfully in the Pakistani RA case-control sample at false discovery rate (FDR) of <0.20 with nominal p-values ranging from 4.73E-06 to 3.48E-02. Both, RA and Type 1 diabetes (T1D) are autoimmune diseases. Specific genetic susceptibility loci and risk factors that are involved in overall autoimmunity risk can be common among multiple autoimmune diseases. To test this hypothesis, seven T1D-related SNPs that have been previously reported to be associated with RA susceptibility in a small set of mixed family-based and casecontrol Pakistani samples were selected to replicate in a relatively large and independent RA xvi case-control sample from the same population. None of the tested SNPs showed a statistically significant association with RA susceptibility, however, the only trend for association (OR= 0.88, p=7.99E-02) was observed for only one SNP (GLIS3/rs7020673). Thus, the results of this study do not support a major role of these tested T1D SNPs in affecting RA susceptibility in the Pakistani population. CTLA4 is involved in the regulation of T-cell response during an immune reaction. Due to the direct involvement in the immune response, multiple CTLA4 single nucleotide polymorphisms (SNPs) have been associated with numerous autoimmune diseases, including RA. To explore the CTLA4 variants and their association with RA susceptibility in the Pakistani population, 24 CTLA4 variants genotyped/confirmed in 350 individuals [family-based RA cases(n)=69, individual RA cases(n)=170, healthy individual controls(n)=111] from the same population. Statistical analysis revealed six common variants (rs231775, rs231779, rs231777, rs5742909, rs11571319, and rs3087243). Four (rs231775, rs5742909, rs11571319 and rs3087243) common SNPs were further genotyped in 1,736 RA case-control subjects (1,042 independent-cases, 694 healthy controls). In statistical association analysis, a significant association of rs3087243, rs5742909, and rs11571319 with increased RA risk were observed but association results for rs231775 were more compatible with no important effect on RA risk in Pakistanis. Copy number variation (CNV) are structural variations in the human genome that have been associated with multiple clinical phenotypes. To explore the association of VPREB1, FCGR3B, and C4B CNV and RA susceptibility in the Pakistani population. A total of 1,106 subjects (616 RA cases, 490 healthy controls) were selected from three rheumatology centers in Pakistan. CNV was xvii determined using TaqMan® CN assays (Hs02879734_cn, Hs04211858_cn, and Hs07226350_cn, Applied Biosystems, Foster City, CA, USA) and data were analyzed using CopyCaller® (version 2.1; Applied Biosystems, USA) software. Logistic regression was applied for the association analysis. A significant association (OR=3.92, p=0.01746) between high VPREB1 CN (>2) and RA risk was observed. Low VPREB1 CN (<2) showed a protective effect against RA risk with a marginal p-value (OR=0.66, p=0.05622). No significant association was observed between VPREB1 CNV and autoantibody status. A significant association (p=0.042, OR=1.49) was observed between high FCGR3B CN (>2) and increased RA risk. Low FCGR3B CNV (<2) did not show any association with RA susceptibility, but a strong association (p=0.00453, OR=2.56) was observed with anti-CCP production. Low C4B CN only showed a trend for association with protection against RA risk. High C4B CNV did not show any effect on RA susceptibility but a strong association (p=0.02, OR=2.33) was observed for RF production in Pakistani RA patients.