Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy.
Adolescent
Adult
Arrhythmias, Cardiac
/ diagnosis
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
Inherited cardiac conditions
Molecular autopsy
Next generation sequencing
Sudden arrhythmic death syndrome
Sudden unexplained death
Journal
BMC cardiovascular disorders
ISSN: 1471-2261
Titre abrégé: BMC Cardiovasc Disord
Pays: England
ID NLM: 100968539
Informations de publication
Date de publication:
23 07 2019
23 07 2019
Historique:
received:
07
04
2018
accepted:
10
07
2019
entrez:
25
7
2019
pubmed:
25
7
2019
medline:
19
5
2020
Statut:
epublish
Résumé
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). 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 31337358
doi: 10.1186/s12872-019-1154-8
pii: 10.1186/s12872-019-1154-8
pmc: PMC6651896
doi:
Types de publication
Journal Article
Multicenter Study
Research Support, Non-U.S. Gov't
Validation Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
174Subventions
Organisme : British Heart Foundation
ID : FS/11/71/28918
Pays : United Kingdom
Organisme : British Heart Foundation
ID : FS/18/28/33549
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_UP_1102/20
Pays : United Kingdom
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