Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

Dobrowolski, Steven F.; Hendrickx, Alexandra T.M.; Bosch, B.J.C. van den; Smeets, Hubert J.M.; Gray, Jesse; Miller, Trent; Sears, Mitch

doi:10.1002/humu.21003

Cited by 69 publications

(56 citation statements)

References 37 publications

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“…For instance, the m.4456C→T variant (Figure 2a) has also been reported as a polymorphic change in MITOMAP. 35 This variant was identified within one of the 36 short PCR amplicons tiled to cover the entire mtDNA. Close examination revealed that the mtDNA primers used for the amplification of the fragment encompassing the m.4456C→T variant were completely homologous to a NUMT sequence on chromosome 1.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Cui

Chen

et al. 2013

Genetics in Medicine

111

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1-3Pathogenic mtDNA mutations, ranging from point mutations to large deletions, are often present in mixed proportions with wildtype mtDNA molecules within the same cell, a biological phenomenon termed heteroplasmy. The degree of mtDNA mutant heteroplasmy can vary significantly across different tissues of the same individual, and the percentage of a mutation is an important contributor to the clinical phenotype.4,5 Determination of the heteroplasmic status of any mtDNA mutation is clinically important to providing a family with informative genetic counseling regarding recurrence risk. Therefore, accurate measurement of heteroplasmy is an essential component of the molecular diagnostic scheme for mtDNA-related disorders.The diagnosis of an individual with suspected mtDNA-related disorders begins with a thorough clinical evaluation and assessment of family history.6 If a disorder with matrilineal inheritance can be established, common mtDNA point mutations and large deletions are typically analyzed using PCR-based targeted assays and Southern blotting methodology. If the screening for common point mutations and large deletions is negative, then the entire mitochondrial genome is usually analyzed by a conventional PCR-based Sanger sequencing approach with multiple pairs of primers. When a mutation is identified, analysis of the degree of heteroplasmy is carried out using various methods, including the commonly used allele refractory mutation system-based quantitative PCR (ARMS qPCR) for quantitative measurement.7 Large deletions are usually confirmed by PCR using primers encompassing the deletion break points followed by sequence analysis to map the break points, or, alternatively, by oligonucleotide array comparative genome hybridization.8 These sequential approaches are time consuming and costly. In an effort to improve efficiency and sensitivity of mtDNA mutation detection, we recently developed a comprehensive one-step long range/massively parallel sequencing (LR-PCR/MPS)-based approach that uses a single pair of PCR primers to amplify the entire mitochondrial genome. This method allows the simultaneous detection of point mutations with quantified heteroplasmy as well as large mtDNA deletions with accurately mapped break points. Purpose: The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome. Methods:The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers. Results:Our results demo...

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Section: Discussionmentioning

confidence: 99%

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Cui

Chen

et al. 2013

Genetics in Medicine

111

View full text Add to dashboard Cite

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“…Rouleau et al [2009] used the possibility provided by some instruments to perform quantitative PCR and HRMA in one instrument to scan for both quantitative (deletions/duplications) and qualitative nucleotide changes in one assay. Finally, Dobrowolski et al [2009] described the use of HRMA to scan the entire 16.6 kb human mitochondrial genome (mtDNA) for sequence variants in less then 2 hr. Identification of mtDNA variants is complicated, as many are heteroplasmic, with the variant allele present at highly variable percentages.…”

Section: Applications Mutation Detectionmentioning

confidence: 99%

“…In fact, based on these figures, HRMA prescreening of a five-exon gene is already cost-effective. In addition, to detect somatic changes or heteroplasmy [Dobrowolski et al, 2009], HRMA seems more sensitive then sequencing (see Quantification).…”

Section: Pressequence Screeningmentioning

confidence: 99%

“…It should be noted that when melt probes are included resolution can be improved to below 5%. Other qHRMA applications are the determination of differences in allelic expression, based on the presence of a variant in the mRNA and the detection of heteroplasmy in mtDNA [Dobrowolski et al, 2009].…”

Section: Quantification-mosaicism and Copy Number Variant (Cnv) Confimentioning

confidence: 99%

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High-Resolution Melting Analysis (HRMA)-More than just sequence variant screening

et al. 2009

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Transition of the double-stranded DNA molecule to its two single strands, DNA denaturation or melting, has been used for many years to study DNA structure and composition. Recent technological advances have improved the potential of this technology, especially to detect variants in the DNA sequence. Sensitivity and specificity were increased significantly by the development of so-called saturating DNA dyes and by improvements in the instrumentation to measure the melting behavior (improved temperature precision combined with increased measurements per time unit and drop in temperature). Melt analysis using these new instruments has been designated high-resolution melting curve analysis (HRM or HRMA). Based on its ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity, HRMA is quickly becoming the tool of choice to screen patients for pathogenic variants. Here we will briefly discuss the latest developments in HRMA and review in particular other applications that have thus far received less attention, including presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants and mosaicism), an alternative to gel-electrophoresis and clone characterization. Together, these diverse applications make HRMA a multipurpose technology and a standard tool that should be present in any laboratory studying nucleic acids.

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“…Quantitative real-time PCR coupled with high-resolution melt (HRM) analysis fulfills these criteria (23,(28)(29)(30)(31)(32)(33)(34).…”

Section: Schreeg Et Almentioning

confidence: 99%

Rapid High-Resolution Melt Analysis of Cytauxzoon felis CytochromebTo Aid in the Prognosis of Cytauxzoonosis

et al. 2015

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Cytauxzoon felis is a virulent, tick-transmitted, protozoan parasite that infects felines. Cytauxzoonosis was previously thought to be uniformly fatal in domestic cats. Treatment combining atovaquone and azithromycin (A&A) has been associated with survival rates of over 60%. Atovaquone, a ubiquinone analogue, targets C. felis cytochrome b (cytb), of which 30 unique genotypes have been identified. The C. felis cytb genotype cytb1 is associated with increased survival rates in cats treated with A&A. The purpose of this study was to design a PCR panel that could distinguish C. felis cytb1 from other cytochrome b genotypes. Primer pairs were designed to span five different nucleotide positions at which single-nucleotide polymorphisms in the C. felis cytb gene had been identified. Through the use of high-resolution melt analysis, this panel was predicted to distinguish cytb1 from other cytb genotypes. Assays were validated using samples from 69 cats with cytauxzoonosis for which the C. felis cytb genotypes had been characterized previously. The PCR panel identified C. felis cytb1 with 100% sensitivity and 98.2% specificity. High-resolution melt analysis can rapidly provide prognostic information for clients considering A&A treatment in cats with cytauxzoonosis. Cytauxzoonosis is an emerging disease in domestic and wild felids in North and South America, caused by the tick-transmitted apicomplexan parasite Cytauxzoon felis (1-8). Cytauxzoonosis was originally thought to be uniformly fatal in domestic cats (4, 5), but our understanding of the epidemiology of C. felis is evolving. Recent evidence indicates that some cats survive C. felis infections without any evidence of clinical disease and/or history of antiprotozoal therapy (9-13). Whether this change is due to increased recognition of subclinical infections, differences in infectious doses, alternative mechanisms of transmission, or differences in virulence between strains is unclear. For cats presented to veterinary hospitals with acute cytauxzoonosis, however, mortality rates remain high. Even with advances in treatment, mortality rates ranged from 40 to 74% in a prospective randomized clinical trial (14). In that study, atovaquone and azithromycin (A&A) treatment was associated with improved survival rates compared to imidocarb dipropionate treatment (14), which was previously considered the treatment of choice (15, 16). The majority of cats with acute cytauxzoonosis that die do so within 2 to 5 days after presentation (4,14). Given this rapid clinical course, it is critical to initiate A&A therapy as soon as possible.Azithromycin is thought to target the mitochondrial ribosomes of the parasite, while atovaquone is presumed to target protozoal cytochrome b (cytb), disrupting electron transport in the parasite mitochondria (17, 18). In related parasites, mutations in the putative atovaquone-binding site of cytb have been associated with responses to A&A treatment (19-21). A recent study identified a C. felis cytb genotype (cytb1) that was associated with increase...

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Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

Cited by 69 publications

References 37 publications

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

High-Resolution Melting Analysis (HRMA)-More than just sequence variant screening

Rapid High-Resolution Melt Analysis of Cytauxzoon felis CytochromebTo Aid in the Prognosis of Cytauxzoonosis

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