Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

Research output: Contribution to journalJournal articleResearchpeer-review


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

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.

Original languageEnglish
Article number174
JournalBMC Cardiovascular Disorders
Issue number1
Number of pages10
Publication statusPublished - 23 Jul 2019

ID: 225424041