Next-generation sequencing of the human TRPV1 gene and the regulating co-players LTB4R and LTB4R2 based on a custom AmpliSeq™ panel

Abstract

BackgroundTransient 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.MethodsA 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.ResultsThe 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%.ConclusionsResults 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.

Development of an AmpliSeqTM Panel for Next-Generation Sequencing of a Set of Genetic Predictors of Persisting Pain

“…Specifically, as observed previously ( Kringel et al, 2017 ), the comprehensive genetic information and the high throughput are reflected in the assay costs. Specifically, sequencing of the 77 genes in 72 DNA samples required approximately € 18,000 for the AmpliSeq TM custom panel, € 5,500 for library preparation, € 700 for template preparation and € 700 for sequencing.…”

“…The NGS assay of the proposed set of 77 human genes relevant to persisting pain was established in 72 genomic DNA samples. As applied previously ( Kringel et al, 2017 ), only exons including 25 bases of padding around all targeted coding regions for which the realized read-depths for each nucleotide was higher than 20 were contemplated as successfully analyzed. With this acceptance criterion the whole or almost whole coverage of the relevant sequences was obtained ( Table 1 ; for details on missing variants, see Supplementary Table 3 ).…”

“…Method validation was accomplished by means of Sanger sequencing ( Sanger and Coulson, 1975 ; Sanger et al, 1977 ) in an independent external laboratory (Eurofins Genomics, Ebersberg, Germany). As performed previously with different AmpliSeq TM panels ( Kringel et al, 2017 ) and other genotyping assays ( Skarke et al, 2004 , 2005 ), four DNA samples have been chosen randomly from an independent cohort of healthy subjects and sequenced with the current NGS panel. For the detected variant type, single nucleotide polymorphisms from five different genomic regions for which clinical associations have been reported ( Table 2 ), i.e., rs324420 ( FAAH ), rs333970 ( CSF1 ), rs4986790 ( TLR4) , rs4633 ( COMT ), and rs17151558 ( RELN ) were chosen for external sequencing.…”

Machine-learned analysis of the association of next-generation sequencing–based human TRPV1 and TRPA1 genotypes with the sensitivity to heat stimuli and topically applied capsaicin

“…Next-generation sequencing of TRPA1 and TRPV1 genes was based on a custom AmpliSeq library and performed using a validated assay on an Ion Torrent personal genome machine as described in detail previously. 38 In brief, genomic DNA was extracted from 200 μL venous blood on a BioRobot EZ1 workstation applying the blood and body fluid spin protocol provided in the EZ1 DNA Blood 200 μL Kit (Qiagen, Hilden, Germany). A multiplex amplification primer set for the exomic sequences of the TRP channel genes was designed online using a web tool (Ion Ampliseq Designer; Life Technologies, Darmstadt, Germany) provided by the manufacturer of the NGS device at http://www.ampliseq.com .…”

“…This accommodates increasing molecular evidence that noncoding variants can affect mRNA splicing, stability, and structure, resulting in a reduced transcriptional efficiency 22 , 23 , 77 rendering them potentially functionally relevant. Hence, a recently developed genetic panel 38 was used to address the role TRPV1 and TRPA1 genetic variants in the sensitivity to nociceptive heat and in the reaction to hypersensitization with topical capsaicin recently assessed in a cohort of healthy subjects. 46 …”