Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next generation sequencing

Michael V. Zaragoza, Joseph Fass, Marta Diegoli, Dawei Lin, Eloisa Arbustini

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,3006 × average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20 × while heteroplasmic variants required >200× coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

Original languageEnglish
Article numbere12295
JournalPLoS One
Volume5
Issue number8
DOIs
Publication statusPublished - 2010

Fingerprint

cardiomyopathy
Cardiomyopathies
Mitochondrial DNA
mitochondrial DNA
mutation
Mitochondrial Diseases
Mutation
mutants
Sudden Cardiac Death
Firearms
genetic disorders
heart failure
Transfer RNA
serine
Serine
Substitution reactions
Heart Failure
Genes
Genome
Tissue

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next generation sequencing. / Zaragoza, Michael V.; Fass, Joseph; Diegoli, Marta; Lin, Dawei; Arbustini, Eloisa.

In: PLoS One, Vol. 5, No. 8, e12295, 2010.

Research output: Contribution to journalArticle

Zaragoza, Michael V. ; Fass, Joseph ; Diegoli, Marta ; Lin, Dawei ; Arbustini, Eloisa. / Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next generation sequencing. In: PLoS One. 2010 ; Vol. 5, No. 8.
@article{a0d431ed794744c280bd3295f95491a6,
title = "Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next generation sequencing",
abstract = "Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We {"}shotgun{"} sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,3006 × average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98{\%} concordance in variant detection. Simulation studies showed that >95{\%} of the homoplasmic variants were detected at a minimum sequence coverage of 20 × while heteroplasmic variants required >200× coverage. Several Sanger {"}misses{"} were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.",
author = "Zaragoza, {Michael V.} and Joseph Fass and Marta Diegoli and Dawei Lin and Eloisa Arbustini",
year = "2010",
doi = "10.1371/journal.pone.0012295",
language = "English",
volume = "5",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "8",

}

TY - JOUR

T1 - Mitochondrial DNA variant discovery and evaluation in human cardiomyopathies through next generation sequencing

AU - Zaragoza, Michael V.

AU - Fass, Joseph

AU - Diegoli, Marta

AU - Lin, Dawei

AU - Arbustini, Eloisa

PY - 2010

Y1 - 2010

N2 - Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,3006 × average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20 × while heteroplasmic variants required >200× coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

AB - Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We "shotgun" sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,3006 × average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20 × while heteroplasmic variants required >200× coverage. Several Sanger "misses" were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease.

UR - http://www.scopus.com/inward/record.url?scp=77957925524&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77957925524&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0012295

DO - 10.1371/journal.pone.0012295

M3 - Article

VL - 5

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 8

M1 - e12295

ER -