Single cell quantification of the 8993T>G NARP mitochondrial DNA mutation by fluorescent PCR.
When a mitochondrial DNA (mtDNA) mutation is identified, the reliable and sensitive quantification of the mutation load is a prerequisite for evaluating the feasibility of prenatal/pregestational diagnosis of the disease. We have developed a quantification assay of the 8993T>G NARP mutation using semi-quantitative fluorescent PCR. The test was reproducible and the experimental values were linear even at extremely low concentrations of mutant mtDNA molecules, making quantification of the mutant load in individual cells feasible (including blastomeres). Studying single circulating lymphocytes from a single NARP 8993T>G patient, we found a broad distribution of the disease causing mutation (0-44%) supporting the remarkable variability of heteroplasmy at the cellular level. This observation and the experimental approach reported here should be relevant to either prenatal or preimplantation diagnosis.
Gigarel
Nadine
N
Department of Genetics, Hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75015 Paris, France.
Ray
Pierre F
PF
Burlet
Philippe
P
Frydman
Nelly
N
Royer
Ghislaine
G
Lebon
Sophie
S
Bonnefont
Jean Paul
JP
Frydman
René
R
Munnich
Arnold
A
Steffann
Julie
J
D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
D017931:DNA Primers; D004272:DNA, Mitochondrial