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

Abstract

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.

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Acknowledgements

This work was supported by the Foundation for the Development of Biotechnology and Genetics POLBIOGEN. S-EK is recipient of a fellowship for young researcher from Poznan Medical University (Poland) grant no 502–14–02223359–10715.

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Correspondence to Marzena Skrzypczak-Zielinska.

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Walczak, M., Skrzypczak-Zielinska, M., Plucinska, M. et al. Long-range PCR libraries and next-generation sequencing for pharmacogenetic studies of patients treated with anti-TNF drugs. Pharmacogenomics J 19, 358–367 (2019). https://doi.org/10.1038/s41397-018-0058-9

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