Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

Hariharan Raju,Steve Jeffery,Christian Van Der Werf,Shibu John,Marta C Cohen,Michael Christiansen,Jonathan R Skinner,Donald R Love,Gavin Arno,Arthur A M Wilde,Sanjay Sharma,Mary N Sheppard,Bo Gregers Winkel,Elijah R Behr,Xinzhong Li,Paula L Hedley,James S Ware,Jacob Tfelt-Hansen

doi:10.1186/s12872-019-1154-8

Abstract

BackgroundWe 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).MethodsWe 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.ResultsThe 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.ConclusionsMolecular 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.

Highlights

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 Polymerase chain reaction (PCR)-enrichment with Illumina HiSeq 2000 generation sequencing (NGS)
A yield of 19–26% mutations in long QT syndrome (LQTS), Brugada syndrome (BrS) and catecholaminergic polymorphic ventricular tachycardia (CPVT) risk-genes has been established by 2 large molecular autopsy SADS series from USA [9, 10]; population-based Australasian studies identified yields of up to 27% using exome-based methodology which included analysis of less common arrhythmia and cardiomyopathy-risk genes [4, 11]
Multiplexed PCR-based candidate gene enrichment seen in the Access Array (Fluidigm Corporation, San Francisco) may be appropriate for the moderate genetic target size recommended for molecular autopsy following SADS [14]

Summary

Introduction

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 generation sequencing (NGS). Multiplexed PCR-based candidate gene enrichment seen in the Access Array (Fluidigm Corporation, San Francisco) may be appropriate for the moderate genetic target size recommended for molecular autopsy following SADS [14]. This can be coupled with NGS by the HiSeq 2000 (Illumina Inc., San Diego) to provide a low-cost high-throughput candidate gene NGS platform

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