Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy
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 gener...
Main Authors: | , , , , , , , , , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
BMC
2019-07-01
|
Series: | BMC Cardiovascular Disorders |
Subjects: | |
Online Access: | http://link.springer.com/article/10.1186/s12872-019-1154-8 |
id |
doaj-dcacc9ba179d4d699a6d0ada9f7f32ac |
---|---|
record_format |
Article |
spelling |
doaj-dcacc9ba179d4d699a6d0ada9f7f32ac2020-11-25T03:54:28ZengBMCBMC Cardiovascular Disorders1471-22612019-07-0119111010.1186/s12872-019-1154-8Next-generation sequencing using microfluidic PCR enrichment for molecular autopsyHariharan Raju0James S. Ware1Jonathan R. Skinner2Paula L. Hedley3Gavin Arno4Donald R. Love5Christian van der Werf6Jacob Tfelt-Hansen7Bo Gregers Winkel8Marta C. Cohen9Xinzhong Li10Shibu John11Sanjay Sharma12Steve Jeffery13Arthur A. M. Wilde14Michael Christiansen15Mary N. Sheppard16Elijah R. Behr17Cardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonNIHR Royal Brompton Cardiovascular Biomedical Research Unit, Royal Brompton HospitalGreenlane Paediatric and Congenital Cardiac Services, Starship Childrens HospitalDepartment for Congenital Disorders, Statens Serum InstitutCardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonDiagnostic Genetics, Auckland City HospitalAmsterdam UMC, Heart Centre, and Department of Clinical and Experimental Cardiology, Amsterdam Cardiovascular Sciences, University of AmsterdamDepartment of Cardiology, Copenhagen University HospitalDepartment of Cardiology, Copenhagen University HospitalHistopathology Department, Sheffield Children’s HospitalNIHR Royal Brompton Cardiovascular Biomedical Research Unit, Royal Brompton HospitalNIHR Royal Brompton Cardiovascular Biomedical Research Unit, Royal Brompton HospitalCardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonCardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonAmsterdam UMC, Heart Centre, and Department of Clinical and Experimental Cardiology, Amsterdam Cardiovascular Sciences, University of AmsterdamDepartment for Congenital Disorders, Statens Serum InstitutCardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonCardiovascular Sciences Research Centre, Molecular and Clinical Sciences Research Institute, St George’s University of LondonAbstract 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.http://link.springer.com/article/10.1186/s12872-019-1154-8Molecular autopsySudden arrhythmic death syndromeSudden unexplained deathInherited cardiac conditionsNext generation sequencing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
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 |
spellingShingle |
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 Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy BMC Cardiovascular Disorders Molecular autopsy Sudden arrhythmic death syndrome Sudden unexplained death Inherited cardiac conditions Next generation sequencing |
author_facet |
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 |
author_sort |
Hariharan Raju |
title |
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy |
title_short |
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy |
title_full |
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy |
title_fullStr |
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy |
title_full_unstemmed |
Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy |
title_sort |
next-generation sequencing using microfluidic pcr enrichment for molecular autopsy |
publisher |
BMC |
series |
BMC Cardiovascular Disorders |
issn |
1471-2261 |
publishDate |
2019-07-01 |
description |
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. |
topic |
Molecular autopsy Sudden arrhythmic death syndrome Sudden unexplained death Inherited cardiac conditions Next generation sequencing |
url |
http://link.springer.com/article/10.1186/s12872-019-1154-8 |
work_keys_str_mv |
AT hariharanraju nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT jamessware nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT jonathanrskinner nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT paulalhedley nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT gavinarno nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT donaldrlove nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT christianvanderwerf nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT jacobtfelthansen nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT bogregerswinkel nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT martaccohen nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT xinzhongli nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT shibujohn nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT sanjaysharma nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT stevejeffery nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT arthuramwilde nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT michaelchristiansen nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT marynsheppard nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy AT elijahrbehr nextgenerationsequencingusingmicrofluidicpcrenrichmentformolecularautopsy |
_version_ |
1724473552685498368 |