Abstract
BackgroundThere are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA) diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations.MethodsWe here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease.ResultsWe have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain) patients carrying haplogroup J lineages (Fisher's Exact test, P-value < 0.01). The assay performs well in mixture experiments of wild:mutant DNAs that emulate heteroplasmic conditions in mtDNA diseases.ConclusionWe here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.
Highlights
There are a number of well-known mutations responsible of common mitochondrial DNA diseases
There are over 100 point mutations putatively associated with human mitochondrial DNA (mtDNA) diseases [1]; only a small percentage of these mutations were properly confirmed http://www.mitomap.org/rimtab2.html
The pathogenicity of some mutations is still under question due to the fact that the conclusions claimed in a substantial number of studies do not rest on solid grounds or are partially or completely flawed [2,3,4,5]. During this last two decades, many different techniques were designed for the screening of mtDNA pathogenic mutations, including restriction fragment length polymorphism (RFLP) analysis, heteroduplex analysis (HDA), single strand conformation polymorphisms (SSCP), etc [6,7,8,9,10,11]
Summary
There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA) diseases. The pathogenicity of some mutations is still under question due to the fact that the conclusions claimed in a substantial number of studies do not rest on solid grounds or are partially or completely flawed [2,3,4,5]. During this last two decades, many different techniques were designed for the screening of mtDNA pathogenic mutations, including restriction fragment length polymorphism (RFLP) analysis, heteroduplex analysis (HDA), single strand conformation polymorphisms (SSCP), etc [6,7,8,9,10,11]. Screening methods are difficult to standardize and some of them are not suitable for replication assays due to their dependence on for instance electrophoretic conditions
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