Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

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

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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.
Original languageEnglish
Pages (from-to)174
JournalBMC Cardiovascular Disorders
Volume19
Issue number1
DOIs
Publication statusPublished - 23 Jul 2019

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Microfluidics
Sudden Death
Autopsy
Polymerase Chain Reaction
High-Cost Technology
Ryanodine Receptor Calcium Release Channel
Mutation
Genes
Cardiac Arrhythmias
Exons
Confidence Intervals
Sensitivity and Specificity

Cite this

Raju, H., Ware, J. S., Skinner, J. R., Hedley, P. L., Arno, G., Love, D. R., ... Behr, E. R. (2019). Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy. BMC Cardiovascular Disorders, 19(1), 174. https://doi.org/10.1186/s12872-019-1154-8
Raju, Hariharan ; Ware, James S. ; Skinner, Jonathan R. ; Hedley, Paula L. ; Arno, Gavin ; Love, Donald R. ; Van Der Werf, Christian ; Tfelt-hansen, Jacob ; Winkel, Bo Gregers ; Cohen, Marta C. ; Li, Xinzhong ; John, Shibu ; Sharma, Sanjay ; Jeffery, Steve ; Wilde, Arthur A. M. ; Christiansen, Michael ; Sheppard, Mary N. ; Behr, Elijah R. / Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy. In: BMC Cardiovascular Disorders. 2019 ; Vol. 19, No. 1. pp. 174.
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title = "Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy",
abstract = "BackgroundWe 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).MethodsWe 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.ResultsThe 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.ConclusionsMolecular 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.",
author = "Hariharan Raju and Ware, {James S.} and Skinner, {Jonathan R.} and Hedley, {Paula L.} and Gavin Arno and Love, {Donald R.} and {Van Der Werf}, Christian and Jacob Tfelt-hansen and Winkel, {Bo Gregers} and Cohen, {Marta C.} and Xinzhong Li and Shibu John and Sanjay Sharma and Steve Jeffery and Wilde, {Arthur A. M.} and Michael Christiansen and Sheppard, {Mary N.} and Behr, {Elijah R.}",
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language = "English",
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pages = "174",
journal = "BMC Cardiovascular Disorders",
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Raju, H, Ware, JS, Skinner, JR, Hedley, PL, Arno, G, Love, DR, Van Der Werf, C, Tfelt-hansen, J, Winkel, BG, Cohen, MC, Li, X, John, S, Sharma, S, Jeffery, S, Wilde, AAM, Christiansen, M, Sheppard, MN & Behr, ER 2019, 'Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy', BMC Cardiovascular Disorders, vol. 19, no. 1, pp. 174. https://doi.org/10.1186/s12872-019-1154-8

Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy. / Raju, Hariharan; Ware, James S.; Skinner, Jonathan R.; Hedley, Paula L.; Arno, Gavin; Love, Donald R.; Van Der Werf, Christian; Tfelt-hansen, Jacob; Winkel, Bo Gregers; Cohen, Marta C.; Li, Xinzhong; John, Shibu; Sharma, Sanjay; Jeffery, Steve; Wilde, Arthur A. M.; Christiansen, Michael; Sheppard, Mary N.; Behr, Elijah R.

In: BMC Cardiovascular Disorders, Vol. 19, No. 1, 23.07.2019, p. 174.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

AU - Raju, Hariharan

AU - Ware, James S.

AU - Skinner, Jonathan R.

AU - Hedley, Paula L.

AU - Arno, Gavin

AU - Love, Donald R.

AU - Van Der Werf, Christian

AU - Tfelt-hansen, Jacob

AU - Winkel, Bo Gregers

AU - Cohen, Marta C.

AU - Li, Xinzhong

AU - John, Shibu

AU - Sharma, Sanjay

AU - Jeffery, Steve

AU - Wilde, Arthur A. M.

AU - Christiansen, Michael

AU - Sheppard, Mary N.

AU - Behr, Elijah R.

PY - 2019/7/23

Y1 - 2019/7/23

N2 - BackgroundWe 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).MethodsWe 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.ResultsThe 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.ConclusionsMolecular 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.

AB - BackgroundWe 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).MethodsWe 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.ResultsThe 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.ConclusionsMolecular 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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U2 - 10.1186/s12872-019-1154-8

DO - 10.1186/s12872-019-1154-8

M3 - Article

VL - 19

SP - 174

JO - BMC Cardiovascular Disorders

JF - BMC Cardiovascular Disorders

SN - 1471-2261

IS - 1

ER -