建立次世代定序分析流程以探討臺灣族群藥物基因之對偶等位基因頻率

Abstract

細胞色素P450（CYPs）的遺傳基因變異點位對於不同藥物使用劑量、反應及療效具有高度關聯性，針對不同個體間作臨床藥物使用選擇與劑量上扮演很重要的角色。而細胞色素P450基因的基因型分布在不同種族間具有相當大的差異。而隨著次世代定序技術的發展，藥物基因檢測實現藥物治療的個人化醫療，追求更好的療效和降低副作用的風險，對患者有極大的益處。因此，如何正確推斷重要的藥物代謝基因型別與單倍體，以及探討臺灣族群的藥物代謝基因對偶基因頻率（allele frequency），對於臨床上個人化精準醫療用藥非常重要。本研究升級先前建立的藥物基因體學相關基因的次世代定序基因檢測平台 – 台大醫院藥物基因檢測平台，漸趨常見的探針捕獲方法相較於全基因組定序成本更便宜，具有更高倍率的測序覆蓋深度，更長的讀取片段。台大醫院藥物基因檢測平台更新版本2.0共有250個藥物動力學以及藥物藥效學相關基因，像是ABCB1, CACNA1S, CFTR, CYPs, DRYP, EGFR, RYR1, TPMT, UGT1A1以及VKORC1等等。以上基因清單是由FDA，CPIC，PharmGKB，PGRN，PharmVar五個重要的資料庫提供的資訊整理而成。並且建立一套針對全基因組定序或是特定基因組定序的資料結果進行藥物基因的對偶基因型鑑定分析流程，並使用可辨別基因型軟體，Aldy和Stargazer。接著，為了驗證次世代定序分析流程與軟體判讀結果，我們使用傳統實驗方法聚合酶連鎖反應（PCR），設計引子（primers）用來分辨亞洲種族中佔較高比例的相似基因型CYP2D6*10，*36以及*36+*10。我們針對77個檢體進行方法驗證，利用台大醫院藥物基因檢測平台進行特定基因定序分析以及操作傳統實驗方法聚合酶連鎖反應，其中包含8個來自國際人類基因組單體型圖計劃（The International HapMap Project）當作實驗技術和分析方法上的國際標準品；6個人類基因組測序參考材料由國家標準與技術研究所（National Institute of Standards and Technology - The Genome in a Bottle Consortium）為基因組比較和基準測試方法創建；加上30個由臺灣人體生物資料庫（Taiwan Biobank）提供的已完成全基因組定序（whole genome sequencing）的檢體；以及33個檢體來自台大醫院院內曾經對特定藥物產生副作用加入研究案的病人。同時也送36個檢體到市面上藥物基因檢測服務作比對、驗證。經由不同方法比對後結果顯示，Aldy軟體判讀結果相較於Stargazer更為準確。最後我們就利用Aldy判讀基因型軟體應用於約554筆臺灣人體生物資料庫（Taiwan Biobank）提供的全基因組定序結果進行分析。提供臺灣國人特有的臨床藥物相關基因的對偶基因頻率，期許在未來能助於學術研究以及臨床上精準醫療的參考資訊。

Inherited genetic variations of cytochrome P450 (CYP) genes play an important role in drug dosing, responses and efficacy in each individual toward a wide variety of clinically used medications. Moreover, CYP genes show marked interethnic variability with inter-population differences in allele frequency. With the development of next-generation sequencing, pharmacogenomics testing has greatly benefited patients by enabling personalization of medication management, pursuing better efficacy and decreased risk of side effects. Thus, it is important to genotype star alleles and phase ambiguous haplotypes of CYP genes and other major pharmacogenes, and also identify the allele frequencies in Taiwanese population with implications for precision medicine. In this study, we first upgraded National Taiwan University Hospital (NTUH) pharmacogenomics (PGx) testing platform using capture-based target region enrichment followed by next-generation sequencing (NGS), a common methodology based on its low cost, deep depth of coverage, long length of reads than whole genome sequencing. Our updated NTUH PGx panel covers 250 major pharmacogenomics genes including pharmacokinetics genes and pharmacodynamics genes, such as ABCB1, CACNA1S, CFTR, various CYPs, DRYP, EGFR, RYR1, TPMT, UGT1A1 and VKORC1. The above list of genes is compiled from information provided by five important resources, FDA, CPIC, PharmGKB, PGRN, PharmVar. We also set up an NGS analysis pipeline with two genotyping toolkits, Aldy and Stargazer, for genotyping star alleles of major CYP genes from whole genome sequencing data and targeted gene sequencing data. Furthermore, we designed PCR primers for validation of NGS result to distinguish similar star allele CYP2D6*10, *36 and *36+*10, which have higher frequency in Asian. Then we applied NGS analysis pipeline and PCR assays to 78 individuals, including 8 samples from The International HapMap Project and 6 reference materials from The Genome in a Bottle Consortium as technique validations; 30 samples from Taiwan Biobank (TWB) and 33 patients with adverse drug reactions recruited from NTUH. At the same time, we sent 36 samples to commercial service, Agena Bioscience MassARRAY® System using iPLEX ADME CYP2D6 Panel, for validation. As result, we confirmed that the genotypes inferred by Aldy are more accurate than Stargazer. Next, we utilized 554 samples of TWB WGS data for calculating the allele frequency in Taiwanese. In conclusion, we revealed the distribution of allele frequencies of major CYP genes in Taiwanese and provide the information in aid of clinical therapy and academic research in future.