Shestak Anna G, Bukaeva Anna A, Saber Siamak, Zaklyazminskaya Elena V
Medical Genetics Laboratory, Petrovsky National Research Center of Surgery, Moscow, Russia.
Cardiac Electrophysiology Research Center, Rajaie Cardiovascular Medical and Research Center, Iran University of Medical Sciences, Tehran, Iran.
Front Genet. 2021 Feb 1;12:620337. doi: 10.3389/fgene.2021.620337. eCollection 2021.
Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of "marker" SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons.
原发性心肌病（CMPs）是单基因但多等位基因疾病，有数十个基因参与其发病机制。下一代测序（NGS）方法的应用使得心肌病的DNA诊断在时间和成本上更具效率。然而，每种CMP亚型的基因检测诊断率未能超过60%。本研究的目的是证明等位基因脱扣（ADO）是一种常见现象，它会降低基于Ion Torrent平台检测的靶向基因panel对原发性心肌病进行基因检测的诊断率。我们使用三个定制的靶向基因panel进行突变筛查，这些panel基于使用AmpliSeq Designer®自动设计的寡核苷酸引物集，包含针对37个基因的1049对引物，总长度为153 kb。用这些靶向基因panel中的至少一个对232例患者的DNA样本进行筛查。我们在IonTorrent PGM数据（3例）和毛细管Sanger测序数据（3例）中均检测到6例ADO事件，并在所有病例中鉴定出导致ADO的变异。所有ADO事件均由于寡核苷酸引物结合位点中的常见或罕见单核苷酸变异（SNV）而发生，并且由于目标DNA片段中存在“标记”SNV而被检测到。我们最终确定基于PCR的NGS存在ADO风险，这就需要使用Sanger测序来验证NGS结果。我们推测，没有ADO数据的寡核苷酸引物设计会影响高达0.77%的扩增子的扩增效率。
