Klein Kathrin, Tremmel Roman, Winter Stefan, Fehr Sarah, Battke Florian, Scheurenbrand Tim, Schaeffeler Elke, Biskup Saskia, Schwab Matthias, Zanger Ulrich M
Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany.
Medical School, University of Tübingen, Tübingen, Germany.
Front Genet. 2019 Jan 31;10:7. doi: 10.3389/fgene.2019.00007. eCollection 2019.
We developed a panel-based NGS pipeline for comprehensive analysis of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs, other xenobiotics, and endogenous substances. The 340 genes comprised phase I and II enzymes, drug transporters and regulator/modifier genes within their entire coding regions, adjacent intron regions and 5' and 3'UTR regions, resulting in a total panel size of 1,382 kbp. We applied the ADME NGS panel to sequence genomic DNA from 150 Caucasian liver donors with available comprehensive gene expression data. This revealed an average read-depth of 343 (range 27-811), while 99% of the 340 genes were covered on average at least 100-fold. Direct comparison of variant annotation with 363 available genotypes determined independently by other methods revealed an overall accuracy of >99%. Of 15,727 SNV and small INDEL variants, 12,022 had a minor allele frequency (MAF) below 2%, including 8,937 singletons. In total we found 7,273 novel variants. Functional predictions were computed for coding variants ( = 4,017) by three algorithms (Polyphen 2, Provean, and SIFT), resulting in 1,466 variants (36.5%) concordantly predicted to be damaging, while 1,019 variants (25.4%) were predicted to be tolerable. In agreement with other studies we found that less common variants were enriched for deleterious variants. -eQTL analysis of variants with (MAF ≥ 2%) revealed significant associations for 90 variants in 31 genes after Bonferroni correction, most of which were located in non-coding regions. For less common variants (MAF < 2%), we applied the SKAT-O test and identified significant associations to gene expression for and . Moreover, our data allow comparison of functional predictions with additional phenotypic data to prioritize variants for further analysis.
我们开发了一种基于面板的二代测序流程，用于全面分析340个参与药物、其他外源性物质和内源性物质吸收、分布、代谢及排泄（ADME）过程的基因。这340个基因包括Ⅰ相和Ⅱ相酶、药物转运体以及其整个编码区、相邻内含子区域和5'及3'非翻译区（UTR）内的调节/修饰基因，从而使整个面板大小达到1382千碱基对。我们将ADME二代测序面板应用于对150名有可用全面基因表达数据的白种人肝脏供体的基因组DNA进行测序。结果显示平均测序深度为343（范围为27 - 811），而340个基因中有99%平均至少被覆盖100倍。将变异注释与通过其他方法独立确定的363个可用基因型进行直接比较，结果显示总体准确率>99%。在15727个单核苷酸变异（SNV）和小插入缺失变异中，12022个的次要等位基因频率（MAF）低于2%，其中包括8937个单例。我们总共发现了7273个新变异。通过三种算法（Polyphen 2、Provean和SIFT）对编码变异（= 4017）进行功能预测，结果显示1466个变异（36.5%）被一致预测为有害变异，而1019个变异（25.4%）被预测为可耐受变异。与其他研究一致，我们发现罕见变异中有害变异更为富集。对MAF≥2%的变异进行表达定量性状位点（eQTL）分析，经Bonferroni校正后，在31个基因中的90个变异显示出显著关联，其中大多数位于非编码区。对于罕见变异（MAF < 2%），我们应用SKAT - O检验，并确定了与基因表达的显著关联。此外，我们的数据允许将功能预测与其他表型数据进行比较，以便对变异进行优先级排序，用于进一步分析。
