Identification of Susceptibility Genes Involved in Rheumatoid Arthritis and Systemic Lupus Erythematosus

Abstract

Rheumatoid arthritis (RA) is a common multifactorial and systemic autoimmune disease characterized by inflammation of synovial fluid which leads to sever pain and progressive joints and bones destruction and even disability if left untreated. The etiology of RA is very complex and is suggested to be the outcome of various environmental, genetics and hormonal factors. Several genes have been identified in which the underlining mutation and polymorphisms result in this complex trait. The incidence and prevalence of RA is increasing worldwide including Pakistan, therefore, the objective of the present work was to examine Pakistani population for identification of susceptible genes and variants which are involved in onset of RA. The progression pattern of RA may exhibit ethnic variations due to differences in environmental factors and genetic pre-disposition of the individuals. Therefore, a cross sectional study was conducted on 500 RA patients (mean age at onset of disease, 39.1±13.0 years) which was aimed to estimate the progression pattern with keeping in mind the severity of disease in the Pakistani patients. It was found that, in general, RA preferentially affects women with female to male ratio of about 3:1; however, patients with above 60 years of age have equal female to male ratio. The disease severity increases with increase in the age and reaches to its peak in patients above 60 years of age (p=0.001). Rheumatoid arthritis susceptibility loci/gene were genotyped in a sample of 366 Pakistanis that comprised related and unrelated cases and controls. Findings from this part were compared with other studies to determine whether the same biological pathways are involved in determining the RA risk in different population groups. The genotyping was performed using the TaqMan assays and the results were analyzed with family case-control (FamCC) software. Twelve of the 33 SNPs/31 loci were associated in this sample with significant p- values ranging from 7.05E-06 to 3.72E-02, the most significant being the KIF5A- PIP4K2C/rs1678542 SNP. Some loci with possible and suggestive association signals were also identified. Since, CTLA4 is one the important genes implicated in many autoimmune diseases including RA and SLE, therefore, entire CTLA4 gene including 1000bp flanking region both from 5' and 3' ends was sequenced in 95 RA and 95 SLE patients to identify functional rare variants (MAF<0.05) and common variants (MAF≥0.05). A total of 38 variants were identified in both RA and SLE patients; in which 21 were already reported in publically available databases (CHIP Bioinformatics, dbSNP) and remaining 17 were novel variants. Association of PPARG gene was tested with RA in Pakistani individuals. PPARG-Pro12Ala polymorphism was genotyped in 300 Pakistani case-control sample using ARMS-PCR method and data was analyzed through Graphpad Prism 5V software. Significantly higher frequency of homozygous genotype (GG; AlaAla) was observed in cases as compare to controls [27 (18.0%) vs 5 (3.3%); χ2 18.54; p < 0.0001] which suggests that GG genotype might contribute to RA in the Pakistanis. Furthermore, significantly higher allele frequency was observed for G allele (Ala) (a minor allele) in cases having same trend and direction of association [p < 0.0001, OR 1.991(1.412-2.808), Z-test value 4.008] . Individuals from 12 large RA families were selected for linkage analysis and were screened through commercially available automated linkage panel. HumanLinkage V Panel Set GoldenGate Assay from Illumina having 6,056 SNP markers with 0.62cM average distance between the SNPs was used. Data was checked for quality controls and is being analyzed for LOD score calculation.