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Open Access 01-12-2019 | Autopsy | Research article

Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

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

Published in: BMC Cardiovascular Disorders | Issue 1/2019

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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Title: Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy
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
Publication date: 01-12-2019
Publisher: BioMed Central
Keywords: Autopsy
Polymerase Chain Reaction
Published in: BMC Cardiovascular Disorders / Issue 1/2019
Electronic ISSN: 1471-2261
DOI: https://doi.org/10.1186/s12872-019-1154-8

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Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

Abstract

Background

Methods

Results

Conclusions

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