- 學位論文
以次世代定序為自體顯性多囊性腎臟病之基因檢測
Next-generation sequencing-based genetic testing of autosomal dominant polycystic kidney disease
摘要
自體顯性多囊性腎臟病為最常見之遺傳性腎臟病，全球發生率約為四百分之一至千分之一。此屬晚發性腎臟疾病，患者通常於三、四十歲後出現雙側腎臟水泡等病徵，疾病惡化至末期腎衰竭時則需依靠腹膜透析或腎移植治療。已知兩個致病基因PKD1及PKD2，但由於兩基因所含外顯子的數目龐大且有六個與PKD1高度相似的同源偽基因，故為此病做基因檢測有相當的難度與挑戰。在這個研究裡我們使用目前的標準檢測方式-長片段聚合酶連鎖反應，並且利用訂製探針來捕捉基因所在區域，包含對於GC比例高的區域做兩次的基因捕捉，接著使用次世代定序來做基因檢測。基因檢測結果經由生物資訊分析及資料過濾流程後，所有疑似致病變異點都依照疾病資料庫PKDB及ACMG的準則判定，並用Sanger定序法做驗證。同時也利用生物資訊分析偽基因對於基因檢測的影響。 在61個病患家族裡，我們對於自體顯性多囊性腎臟病的診斷率為70% (41/59)，對於自體隱性多囊性腎臟病的檢出率為50% (1/2)。其中自體顯性多囊性腎臟病的家族中有16個皆帶有相同的變異點(PKD2 c.2407C>T, p.R803X)，我們發展出利用限制酶處理的快速篩檢方式，能在2.5小時內完成此變異點的篩檢。在生物資訊分析偽基因的干擾部分，利用現行次世代定序讀取的配對型，300bp片段長度，我們能將干擾降至4%以下。 本研究結果顯示，我們能利用次世代定序為自體顯性多囊性腎臟病提供一個可信靠的基因檢測平台，並有高達70%的檢出率。利用探針捕捉方式更大量的節省手工操作的人力及時間。我們並發展適合所有分生實驗室的快速篩檢，能在短時間內針對族群內常見變異點做出正確的診斷。合併快速篩檢並次世代定序，我們能為病患提供可信的基因檢測結果，更提供他們判斷家屬成員或者未發病之年輕成員帶有疾病的依據，以利他們及早確認診斷，也許之後更能依照基因的變異給予更合適的治療方式。
並列摘要
ADPKD stands for autosomal dominant polycystic kidney disease, it affects 1/400~1/1000 individuals worldwide. It is a late on-set disease and patients often require dialysis or renal transplant when progressed into ESRD (end-stage renal disease). PKD1 and PKD2 are the causative genes for ADPKD. The large number of exons and homologous pseudogenes of PKD1 makes genetic testing challenging. In this study we perform both LRPCR (long-range polymerase chain reaction), the gold standard testing for PKD1, and probe capture as target enrichment methods, combined with region enhancement to improve coverage on high GC-content exons for NGS (next-generation sequencing) testing. All possible pathogenic variants were classified according to PKDB (PKD database) or ACMG guideline followed by Sanger sequencing for validation. We also perform bioinformatics analysis to estimate the interference of the pseudogenes. In the 61-family cohorts, we gave 70% diagnostic rate (41/59) for ADPKD patients and 50% (1/2) for ARPKD patients. With 27% (16/59) of the ADPKD patients in our study carried the same variant, we also developed a screening method of RFLP for ADPKD hotspot (PKD2 c.2407C>T, p.R803X), that allowed us to perform fast diagnostic in less than 2.5 hours. The bioinformatics analysis also showed that with longer read length with paired-end reads, we can minimize the interference of pseudogenes to as low as 4% of total reads. In conclusion, we are able to provide confident genetic testing result utilizing NGS with diagnostic rate of 70% that requires no intensive labor work prior or additional sequencing after NGS, great coverage for high GC-content with double capture for region enhancement. Finally, we develop a speedy screening test that can be performed within 2.5 hours targeting PKD2 hotspot in our population that greatly reduces costs and may be carried in most testing laboratories.
參考文獻
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