Association of Biochemical, Immunological Parameters and Genetic Polymorphism of the Gene Encoding Protein Tyrosine Phosphatase Non Receptor 22 with Rheumatoid Arthritis in Iraqi Patients

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation and joint destruction. The aim of this study was to investigate some biochemical markers and mmunological parameters in Iraqi RA patients and effects of medical treatments on these parameters. Also, the genetic polymorphism (rs2746601) for the gene encoding protein tyrosine phosphatase non-receptor 22 (PTPN22) was investigated in these patients.Blood samples were obtained from 60 (newly diagnosed and treated) RA patients (males and females) referred to Rheumatology consultation clinic in
Baghdad teaching hospital, Medical city. The RA patients included 10 newly diagnosed patients with a mean age of 28.4±2.27 years and 50 treated RA patients with a mean age of 42.96±14.59 years. On the other hand, blood samples were obtained from 30 healthy controls with a mean age of 37.33±11.72 years.The Results showed a significant increase in erythrocyte sedimentation rate (ESR) in newly iagnosed and treated RA patients. Treatments given to these patients failed to normalize ESR level. C-Reactive protein (CRP), was detected in all serum samples taken from newly diagnosed patients (100% positive results), and in 68% of treated patients in comparison with 100% negative results in healthy controls. Also, results showed that Rheumatoid factor (RF), and Anti-CCP were detected in all serum samples taken from newly diagnosed patients (100% positive results), and 60% positive results in treated RA patients in comparison with 100% negative results in healthy controls. On the other hand, interleukins levels of IL-6 and IL-23 were significantly higher in newly diagnosed RA patients compared to control. Treatment offered to the patients managed to normalize these values. Results of oxidative stress (GSH and MDA) parameters revealed that the level of the major endogenous antioxidant, glutathione (GSH) was significantly decreased in newly diagnosed RA patients. This was associated with comparable
increase in level of lipid peroxidation byproduct, Malondialdehyde (MDA). Both parameters were normalized in RA treated patients. Genetic polymorphism in Protein Tyrosine Phosphatase non-receptor 22
(PTPN22) gene was studied for rs2476601 SNP in exon 14 within chromosome 1. This SNP (rs2476601) was regarded as a major risk factor associated with susceptibility and severity of rheumatoid arthritis. Genomic DNA was first extracted from blood samples for healthy controls and (treated and newly diagnosed) RA patients. Results showed that the concentration of extracted DNA ranged between 100-200 ng/µl with purity of 1.8-2.0. Then exon 14 was amplified by using specific primers designed to be used in this study. Results of amplification showed a single specific band of 684bp which represents the complete nucleotide sequence of exon 14 on electrophoresed agarose gel (1.8%). To examine genetic polymorphism (rs2476601) in this exon, the nucleotide sequence for this fragment was determined. Results showed that the rs2476601 SNP was non-polymorphic in both RA patients and healthy control subjects with total absence of the variant ‘T’ allele. Furthermore, the frequency of the ‘T’ allele was 0.0, with T/T, C/T and C/C genotype frequencies of 0.0, 0.0, and 1.0, respectively. In conclusion, this study shows that the rs2476601 SNP of the PTPN22 gene is non-polymorphic in Iraqi population and therefore not associated with RA. However, since variations in the rest of the gene were it is not investigated, these results do not exclude other PTPN22 polymorphisms from playing a role in RA susceptibility in Iraq.