Why the study?
The authors sought to determine the mutation yield and clinical applicability of molecular autopsy following sudden arrhythmic death syndrome using low-cost high-throughput next-generation sequencing.
Does next-generation sequencing using microfluidic PCR enrichment provide adequate mutation yield and diagnostic accuracy for molecular autopsy in sudden arrhythmic death syndrome?
Does next-generation sequencing using microfluidic PCR enrichment provide adequate mutation yield and diagnostic accuracy for molecular autopsy in sudden arrhythmic death syndrome?
NGS-based molecular autopsy for SADS offers moderate diagnostic yield (5.1%) and high sensitivity but requires Sanger sequencing confirmation due to low positive predictive value for rare variants.
NGS molecular autopsy yields moderate diagnostic results in SADS but requires Sanger confirmation; leaves open routine clinical adoption pending further validation.
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.
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Raju et al. (2019) studied this question.
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