Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

• Main Authors: 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
• Format: Article
• Language: English
• Published: BMC 2019-07-01
• Series: BMC Cardiovascular Disorders
• Subjects: Molecular autopsy; Sudden arrhythmic death syndrome; Sudden unexplained death; Inherited cardiac conditions; Next generation sequencing
• Online Access: http://link.springer.com/article/10.1186/s12872-019-1154-8

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.