Kringel Dario, Sisignano Marco, Zinn Sebastian, Lötsch Jörn
Institute of Clinical Pharmacology, Goethe - University, Frankfurt am Main, Germany.
Fraunhofer Institute of Molecular Biology and Applied Ecology - Project Group Translational Medicine and Pharmacology (IME-TMP), Frankfurt am Main, Germany.
PLoS One. 2017 Jun 28;12(6):e0180116. doi: 10.1371/journal.pone.0180116. eCollection 2017.
Transient receptor potential cation channel subfamily V member 1 (TRPV1) are sensitive to heat, capsaicin, pungent chemicals and other noxious stimuli. They play important roles in the pain pathway where in concert with proinflammatory factors such as leukotrienes they mediate sensitization and hyperalgesia. TRPV1 is the target of several novel analgesics drugs under development and therefore, TRPV1 genetic variants might represent promising candidates for pharmacogenetic modulators of drug effects.
A next-generation sequencing (NGS) panel was created for the human TRPV1 gene and in addition, for the leukotriene receptors BLT1 and BLT2 recently described to modulate TRPV1 mediated sensitisation processes rendering the coding genes LTB4R and LTB4R2 important co-players in pharmacogenetic approaches involving TRPV1. The NGS workflow was based on a custom AmpliSeq™ panel and designed for sequencing of human genes on an Ion PGM™ Sequencer. A cohort of 80 healthy subjects of Western European descent was screened to evaluate and validate the detection of exomic sequences of the coding genes with 25 base pair exon padding.
The amplicons covered approximately 97% of the target sequence. A median of 2.81 x 106 reads per run was obtained. This identified approximately 140 chromosome loci where nucleotides deviated from the reference sequence GRCh37 hg19 comprising the three genes TRPV1, LTB4R and LTB4R2. Correspondence between NGS and Sanger derived nucleotide sequences was 100%.
Results suggested that the NGS approach based on AmpliSeq™ libraries and Ion Personal Genome Machine (PGM) sequencing is a highly efficient mutation detection method. It is suitable for large-scale sequencing of TRPV1 and functionally related genes. The method adds a large amount of genetic information as a basis for complete analysis of TRPV1 ion channel genetics and its functional consequences.
瞬时受体电位阳离子通道亚家族V成员1（TRPV1）对热、辣椒素、刺激性化学物质及其他有害刺激敏感。它们在疼痛通路中发挥重要作用，与白三烯等促炎因子协同作用，介导敏化和痛觉过敏。TRPV1是几种正在研发的新型镇痛药的靶点，因此，TRPV1基因变异可能是药物效应的药物遗传学调节剂的有前景的候选者。
为人类TRPV1基因创建了一个下一代测序（NGS）面板，此外，还为最近描述的可调节TRPV1介导的敏化过程的白三烯受体BLT1和BLT2创建了该面板，使得编码基因LTB4R和LTB4R2成为涉及TRPV1的药物遗传学方法中的重要协同参与者。NGS工作流程基于定制的AmpliSeq™面板，并设计用于在Ion PGM™测序仪上对人类基因进行测序。对80名西欧血统的健康受试者进行队列筛查，以评估和验证对编码基因外显子序列的检测，外显子填充为25个碱基对。
扩增子覆盖了约97%的目标序列。每次运行获得的读数中位数为2.81×10⁶。这确定了大约140个染色体位点，其中核苷酸与包含TRPV1、LTB4R和LTB4R2这三个基因的参考序列GRCh37 hg19不同。NGS与桑格法获得的核苷酸序列之间的对应率为100%。
结果表明，基于AmpliSeq™文库和离子个人基因组机器（PGM）测序的NGS方法是一种高效的突变检测方法。它适用于TRPV1及其功能相关基因的大规模测序。该方法增加了大量遗传信息，为全面分析TRPV1离子通道遗传学及其功能后果奠定了基础。
