Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

Abstract

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.

Keywords: Inherited cardiac conditions; Molecular autopsy; Next generation sequencing; Sudden arrhythmic death syndrome; Sudden unexplained death.

Fig. 1

Age Distribution of SADS Cases. Histograms demonstrating bimodal age (years) distribution of all SADS cases included, plotted by gender ([a] Male and [b] Female). Mean ages indicated by vertical dotted gold lines

Fig. 2

Circumstances of Death amongst SADS Cases. Pie charts demonstrating activity and circumstances at time of death plotted by [a] gender and [b] age group (children are aged under 18 years)

Fig. 3

Mutation Carriers amongst SADS cases. Bar charts demonstrating pathogenic and likely pathogenic mutation carriers plotted by [a] gender and [b] age group (children are aged under 18 years). Abbreviations: BrS = Brugada syndrome; CPVT = catecholaminergic polymorphic ventricular tachycardia; LQTS = long QT syndrome