Minisequencing mitochondrial DNA pathogenic mutations.
BACKGROUND
There 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.


METHODS
We 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.


RESULTS
We 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.


CONCLUSIONS
We 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.
Alvarez-Iglesias
Vanesa
V
Unidade de Xenética, Instituto de Medicina Legal, Facultad de Medicina, Universidad de Santiago de Compostela, Galicia, Spain. vaneiml@usc.es
Barros
Francisco
F
Carracedo
Angel
A
Salas
Antonio
A
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D007888:Leigh Disease / Q000235:genetics; D017241:MELAS Syndrome / Q000235:genetics; D029242:Optic Atrophy, Hereditary, Leber / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D013030:Spain
D017931:DNA Primers; D004272:DNA, Mitochondrial