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

The Pharmacogenomics Journal volume 19, pages 358–367 (2019)Cite this article

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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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  1. These authors contributed equally: Michal Walczak, Marzena Skrzypczak-Zielinska

Authors and Affiliations

  1. Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479, Poznan, Poland

    Michal Walczak, Marzena Skrzypczak-Zielinska, Oliwia Zakerska-Banaszak, Daria Marszalek & Ryszard Slomski

  2. Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, 61–614, Poznan, Poland

    Marianna Plucinska

  3. Department of Gastroenterology, Dietetics and Internal Diseases, University of Medical Sciences, Przybyszewskiego 49, 60-355, Poznan, Poland

    Liliana Lykowska-Szuber, Kamila Stawczyk-Eder & Agnieszka Dobrowolska

  4. Department of Biochemistry and Biotechnology, University of Life Sciences, Dojazd 11, 60-632, Poznan, Poland

    Ryszard Slomski

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  1. Michal Walczak
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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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