Long-range PCR libraries and next-generation sequencing for pharmacogenetic studies of patients treated with anti-TNF drugs.

Biological therapy with anti-tumor necrosis factor-α (anti-TNF-α) monoclonal antibodies significantly increased the effectiveness of autoimmune disease treatment compared with conventional medicines. However, anti-TNF-α drugs are relatively expensive and a response to the therapy is reported in only 60-70% of patients. Moreover, in up to 5% of patients adverse drug reactions occur. The various effects of biological treatment may be a potential consequence of interindividual genetic variability. Only a few studies have been conducted in this field and which refer to single gene loci. Our aim was to design and optimize a methodology for a broader application of pharmacogenetic studies in patients undergoing anti-TNF-α treatment. Based on the current knowledge, we selected 16 candidate genes: TNFRSF1A, TNFRSF1B, ADAM17, CASP9, FCGR3A, LTA, TNF, FAS, IL1B, IL17A, IL6, MMP1, MMP3, S100A8, S100A9, and S100A12, which are potentially involved in the response to anti-TNF-α therapy. As a research model, three DNA samples from Crohn's disease (CD) patients were used. Targeted genomic regions were amplified in 23 long-range (LR) PCR reactions and after enzymatic fragmentation amplicon libraries were prepared and analyzed by next-generation sequencing (NGS). Our results indicated 592 sequence variations located in all fragments with coverage range of 5-1089. We demonstrate a highly sensitive, flexible, rapid, and economical approach to the pharmacogenetic investigation of anti-TNF-α therapy using amplicon libraries and NGS technology.

Is Polymorphism in the Apoptosis and Inflammatory Pathway Genes Associated With a Primary Response to Anti-TNF Therapy in Crohn's Disease Patients?

Anti-tumor necrosis factor (TNF) therapy is used for the induction and maintenance of remission in Crohn's disease (CD) patients. However, primary nonresponders to initial treatment constitute 20%-40% of cases. The causes of this phenomenon are still unknown. In this study, we aimed to determine the genetic predictors of the variable reactions of CD patients to anti-TNF therapy. Using long-range PCR libraries and the next-generation sequencing (NGS) method, we performed broad pharmacogenetic studies including a panel of 23 genes (TNFRSF1A, TNFRSF1B, CASP9, FCGR3A, LTA, TNF, FAS, ADAM17, IL17A, IL6, MMP1, MMP3, S100A8, S100A9, S100A12, TLR2, TLR4, TLR9, CD14, IL23R, IL23, IL1R, and IL1B) in a group of 107 diagnosed and clinically characterized CD patients following anti-TNF therapy. In the studied group, we indicated, in total, 598 single nucleotide variants for all analyzed genomic targets. Twelve patients (11.2%) did not respond to the induction therapy, which was associated with alleles in 11 loci located in FCGR3A (rs7539036, rs6672453, rs373184583, and rs12128686), IL1R (rs2041747), TNFRSF1B (rs5746053), IL1B (rs1071676, rs1143639, rs1143637, and rs1143634), and FAS (rs7896789) genes. After multiple comparison corrections, the results were not statistically significant, however for nonresponders the alleles distribution for those loci presented large differences and specified scheme compared to responders and populations. These findings require further investigation in an independent larger cohort before introducing them for a clinical setting, however, we identified an interesting direction. Polymorphism of the FCGR3A, IL1R, TNFRSF1B, IL1B, and FAS genes could be a predictor of the primary response to anti-TNF therapy in CD patients.