Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing
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 o...
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Published in | PloS one Vol. 5; no. 8; p. e12295 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
20.08.2010
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
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Summary: | 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 1,300x 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 20x while heteroplasmic variants required >200x 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. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Undefined-1 ObjectType-Feature-3 content type line 23 ObjectType-Feature-1 Conceived and designed the experiments: MVZ JF DL EA. Performed the experiments: MVZ JF MD. Analyzed the data: MVZ JF MD DL EA. Contributed reagents/materials/analysis tools: MVZ JF MD DL EA. Wrote the paper: MVZ. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0012295 |