title: Single cell quantification of the 8993T>G NARP mitochondrial DNA mutation by fluorescent PCR.
abstract: 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.
authors.affiliation: Department of Genetics, Hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75015 Paris, France.
authors.forename: Nadine
authors.initials: N
authors.lastname: Gigarel
authors.forename: Pierre F
authors.initials: PF
authors.lastname: Ray
authors.forename: Philippe
authors.initials: P
authors.lastname: Burlet
authors.forename: Nelly
authors.initials: N
authors.lastname: Frydman
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Royer
authors.forename: Sophie
authors.initials: S
authors.lastname: Lebon
authors.forename: Jean Paul
authors.initials: JP
authors.lastname: Bonnefont
authors.forename: René
authors.initials: R
authors.lastname: Frydman
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.forename: Julie
authors.initials: J
authors.lastname: Steffann
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial