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

doi:10.1186/s12872-019-1154-8

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 language	English
Article number	174
Journal	BMC Cardiovascular Disorders
Volume	19
Issue number	1
Number of pages	10
ISSN	1471-2261
DOIs	https://doi.org/10.1186/s12872-019-1154-8
Publication status	Published - 23 Jul 2019

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Raju, H., Ware, J. S., Skinner, J. R., Hedley, P. L., Arno, G., Love, D. R., van der Werf, C., Tfelt-Hansen, J., Winkel, B. G., Cohen, M. C., Li, X., John, S., Sharma, S., Jeffery, S., Wilde, A. A. M., Christiansen, M., Sheppard, M. N., & Behr, E. R. (2019). Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy. BMC Cardiovascular Disorders, 19(1), Article 174. https://doi.org/10.1186/s12872-019-1154-8

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, 174. https://doi.org/10.1186/s12872-019-1154-8

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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.",

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}",

year = "2019",

month = jul,

day = "23",

doi = "10.1186/s12872-019-1154-8",

language = "English",

volume = "19",

journal = "BMC Cardiovascular Disorders",

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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 - 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.

AB - 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.

U2 - 10.1186/s12872-019-1154-8

DO - 10.1186/s12872-019-1154-8

M3 - Journal article

C2 - 31337358

SN - 1471-2261

VL - 19

JO - BMC Cardiovascular Disorders

JF - BMC Cardiovascular Disorders

IS - 1

M1 - 174

ER -

Next-generation sequencing using microfluidic PCR enrichment for molecular autopsy

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