LC-MS/MS-Based Serum Protein Profiling for Identification of Candidate Biomarkers in Pakistani Rheumatoid Arthritis Patients.

Rheumatoid arthritis is an autoimmune disorder of complex disease etiology. Currently available serological diagnostic markers lack in terms of sensitivity and specificity and thus additional biomarkers are warranted for early disease diagnosis and management. We aimed to screen and compare serum proteome profiles of rheumatoid arthritis serotypes with healthy controls in the Pakistani population for identification of potential disease biomarkers. Serum samples from rheumatoid arthritis patients and healthy controls were enriched for low abundance proteins using ProteoMinerTM columns. Rheumatoid arthritis patients were assigned to one of the four serotypes based on anti-citrullinated peptide antibodies and rheumatoid factor. Serum protein profiles were analyzed via liquid chromatography-tandem mass spectrometry. The changes in the protein abundances were determined using label-free quantification software ProgenesisQITM followed by pathway analysis. Findings were validated in an independent cohort of patients and healthy controls using an enzyme-linked immunosorbent assay. A total of 213 proteins were identified. Comparative analysis of all groups (false discovery rate < 0.05, >2-fold change, and identified with ≥2 unique peptides) identified ten proteins that were differentially expressed between rheumatoid arthritis serotypes and healthy controls including pregnancy zone protein, selenoprotein P, C4b-binding protein beta chain, apolipoprotein M, N-acetylmuramoyl-L-alanine amidase, catalytic chain, oncoprotein-induced transcript 3 protein, Carboxypeptidase N subunit 2, Apolipoprotein C-I and Apolipoprotein C-III. Pathway analysis predicted inhibition of liver X receptor/retinoid X receptor activation pathway and production of nitric oxide and reactive oxygen species pathway in macrophages in all serotypes. A catalogue of potential serum biomarkers for rheumatoid arthritis were identified. These biomarkers can be further evaluated in larger cohorts from different populations for their diagnostic and prognostic potential.

Exploration of shared genetic susceptibility loci between type 1 diabetes and rheumatoid arthritis in the Pakistani population.

OBJECTIVE: Type 1 diabetes (T1D) and rheumatoid arthritis (RA) are autoimmune diseases. It is known that certain genetic loci and factors that increase the overall autoimmunity risk can be shared among different autoimmune diseases. We sought to replicate seven T1D-related SNPs (single nucleotide polymorphisms) that have been previously reported to be associated with RA susceptibility in a small set of mixed family-based and case-control Pakistani sample in a relatively large and independent RA case-control sample from the same population. Seven T1D-associated SNPs (GLIS3/rs7020673, BACH2/rs11755527, SKAP2/rs7804356, GDSMB/rs2290400, C6orf173/rs9388489, LOC399716/rs947474 and DLK1-MEG2/rs941576) were genotyped in a large Pakistani RA case-control sample (n = 1959) using TaqMan® SNP genotyping assays. RESULTS: None of the tested SNPs showed statistically significant association with RA susceptibility; however, one SNP (GLIS3/rs7020673) showed a trend for association (OR = 0.88, p = 7.99E-02). Our study has failed to replicate the previously reported association of seven T1D-associated SNPs with RA risk in a large sample from the same population. Thus, our results do not support a major role of these T1D SNPs in affecting RA susceptibility in the Pakistani population.

Vitamin D as a Principal Factor in Mediating Rheumatoid Arthritis-Derived Immune Response.

Rheumatoid arthritis (RA) is a systemic multifactorial autoimmune disorder. The interactions between diverse environmental and genetic factors lead to the onset of this complex autoimmune disorder. Serum levels of vitamin D (VD) are involved in the regulation of various immune responses. Vitamin D is a key signaling molecule in the human body that maintains calcium as well as phosphate homeostasis. It also regulates the functions of the immune system and, thus, can play a substantial role in the etiology of various autoimmune disorders, including RA. Low serum VD levels have been found to be associated with a higher risk of RA, although this finding has not been replicated consistently. The molecular mechanisms by which VD influences autoimmunity need to be further explored to understand how variation in plasma VD levels could affect the pathogenesis of RA. This mini-review focuses on the influence of VD and its serum levels on RA susceptibility, RA-associated complexities, treatment, and transcriptome products of key proinflammatory cytokines, along with other cytokines that are key regulators of inflammation in rheumatoid joints.

Lack of tumor necrosis factor alpha gene polymorphism -857c/t (rs1799724) association in Pakistani rheumatoid arthritis patients.

BACKGROUND: Rheumatoid arthritis (RA) is a common systemic autoimmune disease, influenced greatly by the pro-inflammatory cytokine tumor necrosis factor- alpha (TNF-α). Single nucleotide polymorphisms (SNPs) in regulatory regions of the TNF-α gene play a significant role in disease development and pathogenesis. The aim of this study was to investigate the association of TNF-α -857C/T (rs1799724) SNP with RA activity or severity in our Pakistani study group. METHODS: The study included 134 (116 women, 18 men) patients with RA and 134 ethnically matched healthy controls (108 women, 26 men). Each patient's disease activity was measured by Disease Activity Score of 28 joints. The genotypes were determined in all included individuals following allele-specific polymerase chain reaction along with the prerequisite internal amplification controls. Statistical analysis including chi-square/Fischer exact test and one-way analysis of variance; nonparametric Kruskal-Wallis test was employed using Graphpad Prism 6 software for association study. RESULTS: The prevalence of TNF-α -857C/T (rs1799724) polymorphism was not differentially distributed between RA patients and controls in either allele frequency, with odds ratio (95% CI) of 0.9661 (0.6714-1.390) and P-value of 0.8527, or genotype frequency with χ2 of 0.5015 and P-value of 0.7782. Moreover, no correlation was found when genotype frequency distribution was analyzed with disease severity (P = 0.6321 and Kruskal-Wallis statistics of 1.098). CONCLUSION: The study demonstrated -857C/T (rs1799724) polymorphism may not have influenced RA susceptibility in our study group. However, investigations of genetic variability influence on disease outcome in large prospective cohorts are required, so the complicated interconnection of genetic and environmental elements can be emulated for better understanding.

Genetic link of type 1 diabetes susceptibility loci with rheumatoid arthritis in Pakistani patients.

Rheumatoid arthritis (RA) and type 1 diabetes (T1D) are two autoimmune disorders that have been reported to co-occur in the same subjects or in different subjects from the same family. This suggests the sharing of disease susceptibility loci between RA and T1D. This study was aimed to find out such susceptibility loci that are common in both T1D and RA in Pakistani population. A total of 366 Pakistanis comprising related and unrelated RA cases and controls were recruited. Blood samples were collected from all patients followed by DNA isolation. Thirty-one single-nucleotide polymorphisms (SNPs) previously reported to be associated with T1D were genotyped in RA cases and controls using TaqMan SNP genotyping assays. Data was analyzed using FamCC software. We have identified seven SNP associations that survived multiple testing corrections using false discovery rate: SKAP2/rs7804356 (p = 2.47E-04), GLIS3/rs7020673 (p = 2.86E-04), GSDMB/rs2290400 (p = 23.48E-04), BACH2/rs11755527 (p = 9.16E-04), C6orf173/ rs9388489 (p = 3.11E-03), PRKCQ/DKFZp667F0711/ rs947474 (p = 4.53E-03), and DLK1/ rs941576 (p = 9.51E-03). Our results support the presence of overlapping loci between RA and T1D in Pakistani patients.