Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling
Open Access
- 3 March 2009
- journal article
- research article
- Published by Hindawi Limited in Human Mutation
- Vol. 30 (6) , 891-898
- https://doi.org/10.1002/humu.21003
Abstract
Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301–658 bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1 hr. Hum Mutat 30,1–8, 2009.Keywords
This publication has 38 references indexed in Scilit:
- Pathogenic Mitochondrial DNA Mutations Are Common in the General PopulationAmerican Journal of Human Genetics, 2008
- Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting PlatformsClinical Chemistry, 2008
- High resolution melting analysis for rapid and sensitive EGFR and KRAS mutation detection in formalin fixed paraffin embedded biopsiesBMC Cancer, 2008
- High resolution melting applications for clinical laboratory medicineExperimental and Molecular Pathology, 2008
- High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation callingHuman Mutation, 2008
- Streamlined assessment of gene variants by high resolution melt profiling utilizing the ornithine transcarbamylase gene as a model systemHuman Mutation, 2007
- Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profilesMolecular Genetics and Metabolism, 2007
- Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in theSLC22A5 geneHuman Mutation, 2005
- Current classification of mitochondrial disordersZeitschrift für Neurologie, 2003
- Sequence and organization of the human mitochondrial genomeNature, 1981