使用微流控 PCR 富集的下一代测序进行分子尸检。
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy.
机构信息
Cardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George's University of London, London, SW17 0RE, UK.
Health Cardiology, Faculty of Medicine & Health Sciences, Macquarie University, Suite 203, 2 Technology Place, Sydney, NSW, 2109, Australia.
出版信息
BMC Cardiovasc Disord. 2019 Jul 23;19(1):174. doi: 10.1186/s12872-019-1154-8.
BACKGROUND
We aimed to determine the mutation yield and clinical applicability of "molecular autopsy" following sudden arrhythmic death syndrome (SADS) by validating and utilizing low-cost high-throughput technologies: Fluidigm Access Array PCR-enrichment with Illumina HiSeq 2000 next generation sequencing (NGS).
METHODS
We validated and optimized the NGS platform with a subset of 46 patients by comparison with Sanger sequencing of coding exons of major arrhythmia risk-genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, RYR2). A combined large multi-ethnic international SADS cohort was sequenced utilizing the NGS platform to determine overall molecular yield; rare variants identified by NGS were subsequently reconfirmed by Sanger sequencing.
RESULTS
The NGS platform demonstrated 100% sensitivity for pathogenic variants as well as 87.20% sensitivity and 99.99% specificity for all substitutions (optimization subset, n = 46). The positive predictive value (PPV) for NGS for rare substitutions was 16.0% (27 confirmed rare variants of 169 positive NGS calls in 151 additional cases). The overall molecular yield in 197 multi-ethnic SADS cases (mean age 22.6 ± 14.4 years, 68% male) was 5.1% (95% confidence interval 2.0-8.1%), representing 10 cases carrying pathogenic or likely pathogenic risk-mutations.
CONCLUSIONS
Molecular autopsy with Fluidigm Access Array and Illumina HiSeq NGS utilizing a selected panel of LQTS/BrS and CPVT risk-genes offers moderate diagnostic yield, albeit requiring confirmatory Sanger-sequencing of mutational variants.
背景
我们旨在通过验证和利用低成本高通量技术，即 Fluidigm Access Array PCR 富集与 Illumina HiSeq 2000 下一代测序（NGS），确定猝死后心律失常综合征（SADS）后“分子尸检”的突变率和临床适用性。
方法
我们通过与主要心律失常风险基因（KCNQ1、KCNH2、SCN5A、KCNE1、KCNE2、RYR2）的编码外显子的 Sanger 测序比较，对 NGS 平台进行了验证和优化。利用 NGS 平台对一个大型多民族国际 SADS 队列进行测序，以确定总体分子产量；通过 NGS 鉴定的稀有变异随后通过 Sanger 测序进行再次确认。
结果
NGS 平台对致病性变异的敏感性为 100%，对所有替代物的敏感性为 87.20%，特异性为 99.99%（优化子集，n=46）。NGS 对稀有替代物的阳性预测值（PPV）为 16.0%（在 151 例额外病例中，169 个阳性 NGS 检测中，有 27 个确认的稀有变异）。197 例多民族 SADS 病例（平均年龄 22.6±14.4 岁，68%为男性）的总体分子产量为 5.1%（95%置信区间 2.0-8.1%），代表携带致病性或可能致病性风险突变的 10 例。
结论
利用 Fluidigm Access Array 和 Illumina HiSeq NGS 进行分子尸检，利用 LQTS/BrS 和 CPVT 风险基因的选定面板提供了中等的诊断产量，尽管需要对突变变异进行 Sanger 测序确认。
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