Mitochondrial DNA sequencing/haplotyping quantification qPCR
A high number of mitochondrial DNA (mtDNA) is present in eukaryotic cells. The mtDNA is a double-stranded circular molecule of 16569 bp which encode 13 sub-units of the I, III,IV complexes of the respiratory chain and ATP synthase, 22 tRNA and 2 rRNA and is characterized by a high mutation rate and the absence of recombination. Parts of mtDNA variants are polymorphisms (mtSNPs) accumulated along the time and are used to define major monophyletic clades in the mtDNA tree: haplogroups. Those haplogroups can play a synergistic role in development of several neurological pathologies or conversely can confer survival advantage in various epidemics. Over 250 pathogenic mtDNA mutations (point mutations and rearrangements) have been characterised, shown to cause a wide variety of diseases with heterogeneity of phenotypes and a variable age of onset. A part of those mutations are present in all mtDNA copies (homoplasmic state) but the majority is not (heteroplasmic state) and the clinical phenotype depends on the percentage of mutated mtDNA copies.
We developed a sequencing-based technique to screen the whole-mtDNA. The first step consists of the PCR amplification of the mt-DNA in 8 fragments of nearly 2000 pb. Those fragments are then used as matrix for DNA sequencing with 48 overlapping fragments. All those fragments are then aligned with consensus Cambridge mtDNA sequence with Seqcape software (Applied Biosystems). The pathogenicity of all mtDNA variants is evaluated with three criteria: aminoacid change, presence in mtDNA databases (Mitomap, mtDB …) and conservation along species.
This technique enables to find common pathological mutations, to discover new mutations and to determine the haplogroup of the patient. It can be carry over on DNA extracted from blood, urines, muscle or skin biopsy notably.
After identification of a mutation, we can evaluate heteroplasmy level by a fluorescent PCR (the forward or the reverve primer is linked to a fluorescent dye) coupled to RFLP (Restriction Fragment Length Polymorphism). In this case, the mutation creates or abolishes a restriction site and cutting of the restriction enzyme will result in fluorescent fragments of different length. The RFLP product migrates in electrophoresis capillary (Applied Biosystems) and the fluorescent peaks are analyzed by Peak Scanner software (Applied Biosystems). The percentage of hétéroplasmie is calculated as the ratio of WT/muted peak intensity.
Modifications of respiratory chain functioning can also be linked to a variation in mtDNA copy number, even a decrease (depletion) or and increase (mitochondrial biogenesis). This can be evaluated by quantitative PCR by establishing a ratio between a mtDNA encoded gene and a nuclear encoded one.