Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

  • Hariharan Raju (Creator)
  • James S. Ware (Creator)
  • Jonathan R. Skinner (Creator)
  • Paula L. Hedley (Creator)
  • Gavin Arno (Creator)
  • Donald R. Love (Creator)
  • Christian Van Der Werf (Creator)
  • Jacob Tfelt-hansen (Creator)
  • Bo Gregers Winkel (Creator)
  • Marta C. Cohen (Creator)
  • Xinzhong Li (Contributor)
  • Shibu John (Creator)
  • Sanjay Sharma (Creator)
  • Steve Jeffery (Creator)
  • Arthur A. M. Wilde (Creator)
  • Michael Christiansen (Creator)
  • Mary N. Sheppard (Creator)
  • Elijah R. Behr (Creator)



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.
Date made available23 Jul 2019

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