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

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

“…The amplification of the TNFRSF1A, TNFRSF1B, ADAM17, CASP9, FCGR3A, LTA, TNF, FAS, IL1B, IL17A, IL6, MMP1, MMP3, S100A8, S100A9, and S100A12 genes was performed in 23 fragments using previously described primers (Walczak et al, 2019). Additionally, the primers for TLR2, TLR4, TLR9, CD14, IL23, IL23R, and IL1R genes containing exons, splice junctions, and promoters, as well as 5' and 3' flanking sequences, were designed ( Table 1).…”

“…According to the clinical procedures (see patients' characteristics), 12 patients were classified as primary nonresponders to anti-TNF therapy. The NGS analysis results were aligned to the hg19 reference genome and analyzed as previously described (Walczak et al, 2019).…”

NGS study of glucocorticoids response genes in inflammatory bowel disease patients

“…Therefore, as an additional goal of this work, we developed a rapid, flexible and economical application of NGS by combining it with LR-PCR as target enrichment for pharmacogenetic studies on GC drugs. A similar solution was used successfully in earlier studies [ 19 , 20 ]. In bioinformatic analysis for our scientific purposes, in contrast to diagnostic standards of minimum coverage 30× [ 26 ], we carefully applied a reads cover filter of 10 to avoid omission of any variants at this stage ( Figure 2 ).…”

“…Primers were designed as part of this work using the NCBI (National Center for Biotechnology Information) Primer-BLAST tool in reference to the human genomic sequence (GRCh37/hg19). The only exceptions were the primers and amplification conditions for the IL-1B, TNF and ABCB1 genes, which originated from our previous studies [19][20][21]. Due to the limitation of the LR-PCR method in the length of efficiently amplified DNA fragments, the LR-PCR of short genes such as IL-1A (11.48 kb), IL-1B (7.02 kb) and IL-2 (5.25 kb) was performed in one fragment, but the long genes, e.g.…”

Pharmacogenomics, How to Deal with Different Types of Variants in Next Generation Sequencing Data in the Personalized Medicine Area

“…The trends led to the practice of personalized therapy and precision medicine implementation in clinical centers. The explosion of examples in the field of pre-emptive and/or patient genotyping shows the true advantages of high throughput sequencing technologies in the PGx area [ 5 , 6 , 7 , 8 ]. However, despite the common belief between the physicians and general practitioners in the effects of the genetic landscape on diverse drug responses, if they asked that they order the PGx tests for their patients, less than 15% will answer positively.…”