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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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 |
Journal | BMC cardiovascular disorders
(BMC Cardiovasc Disord)
Vol. 19
Issue 1
Pg. 174
(07 23 2019)
ISSN: 1471-2261 [Electronic] England |
PMID | 31337358
(Publication Type: Journal Article, Multicenter Study, Research Support, Non-U.S. Gov't, Validation Study)
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Topics |
- Adolescent
- Adult
- Arrhythmias, Cardiac
(diagnosis, genetics, mortality)
- Australia
- Autopsy
(methods)
- Cause of Death
- Child
- Child, Preschool
- DNA Mutational Analysis
- Death, Sudden, Cardiac
(etiology)
- Europe
- Female
- Genetic Predisposition to Disease
- Heredity
- High-Throughput Nucleotide Sequencing
- Humans
- Infant
- Male
- Microfluidic Analytical Techniques
- Mutation
- New Zealand
- Pathology, Molecular
- Pedigree
- Polymerase Chain Reaction
- Predictive Value of Tests
- Reproducibility of Results
- Risk Factors
- Young Adult
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