BMC Cardiovascular Disorders (Jul 2019)

Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

  • 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

DOI
https://doi.org/10.1186/s12872-019-1154-8
Journal volume & issue
Vol. 19, no. 1
pp. 1 – 10

Abstract

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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.

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