Role of TNF-α gene polymorphisms in the onset of Psoriasis and Psoriatic Arthritis using Next Generation Sequencing

Psoriasis(Ps)and Psoriatic Arthritis (PsA)are a complex immune-mediated diseases resulting from the interplay between multiple genetic and environmental factors. It has been estimated that at least one-third of the genetic contribution to PsV and PsA resides in the human major histocompatibility complex region(MHC). The tumor necrosis factor-α(TNF-α)gene, which is located in the short arm of chromosome 6 in the major histocompatibility complex class III region, has been proposed as a major candidate gene in Ps and PsA. This hypothesis is supported by studies that have found high serum, synovial fluid, and synovial membrane TNF-α levels in patients with PsA. In clinical trials for psoriatic arthritis(PsA), TNF-α blockers have been shown to have excellent clinical efficacy and to prevent further structural damage to joints. The aim of my thesis was to clarify the mechanisms through which the genetic variability of TNFα and Lymphotoxin alpha(LTA)genes play a role in the susceptibility and progression of psoriasis and psoriatic arthritis and in therapeutic response with anti-TNFα inhibitors using Next Generation Sequencing approach. 180 patients with PsV and 130 patients with PsA were included into the study. The control cohort included 120 healthy, unrelated subject of the same ethnic origin with no family history of psoriasis and PsA. We included 70 patients treated with TNF-inhibitors, with the diagnosis of psoriasis and PsA, according to clinical criteria, starting the first TNF-blocker agent, and with at least 2 years of follow-up. DNA was extracted from peripheral blood. Genomic DNA were amplified for TNF/LTα, HLA-B and HLA-C under long range PCR using the specific forward and reverse primer pairs. Subsequently, genomic DNA was prepared for the following step. The genetic analysis revealed no significant differences in the frequency of the variants between psoriasis patients and controls. Some of these variants, would be attributable to the linkage disequilibrium among the alleles found during the analysis of this region and the HLA loci traditionally associated with the psoriasis: HLA-C*06:02 in many populations and HLA-C*07:18- B*58 in the Sardinian population. To evaluate if the associations found in our samples were correlated with the presence of HLA-C*06:02 and HLA-C*07:18 - B*58, a stratification analysis was carried out. Of 180 psoriatic patients, 97 (54%) were carriers of the alleles HLAC*06:02 and HLA-C*07:18 - B*58 and 83 were not carriers. The 97 alleles resulted to be in a strong linkage disequilibrium. The analysis for the group of PsA patients compared with controls have shown no significant differences in the frequency of the variants, and stratification in the PsA group showed a strong linkage disequilibrium of all the variants identified with the HLA alleles C*06:02 C*07:18 B*58:01. Finally, a study relating the response to TNF-inhibitors was performed. 60 patients were analyzed, 17 non-responders and 53 responders. The p value for the marker rs1800750 (-376G>A) (p = 0,005445) resulted strongly associated with a frequency of 14%. In conclusion, the results based on the examination of 65 SNPs of tumor necrosis factor and lymphotoxin alpha genes suggest that at least in the Sardinian population there is no direct link between genotype distribution, alleles carriage and overall number of alleles and susceptibility for Psoriasis Vulgaris and Psoriatic Arthritis. A striking result was obtained in the evaluation of the anti-TNF-α biologic therapy in the cohort of patients treated with anti-TNF-α biologic therapy showing a significant association in responders compared to non-responders.