Single-cell analysis for mitochondrial genetic disorder disease

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We have examined the distribution of mutant mtDNA molecules in single cells from a patient with Leber hereditary optic neuropathy (LHON) and MELAS (mitochondrial encephalopathy, myopathy, lactic acidosis and stroke-like episodes). The patient was a 46-year-old unaffected male in a large pedigree with LHON.Previous analysis of DNA from the total blood buffy coat had shown that -80 % of the mtDNA molecules carried the 11778 LHON mutation and that 20 % had the normal sequence at this point. Single lymphocytes were isolated using a micromanipulation method. Each isolated cell was used for two step PCR amplification and restriction digestion. Twenty-five samples each containing a single cell were analyzed. Sixteen cells had completely mutant mtDNA,and five cells had completely normal mtDNA (intracellular homoplasmy). Four cells contained a mixture of both. Twenty one of the 25 cells had segregated to either homoplasmic mutant or homoplasmic normal mtDNA.mtDNA heteroplasmy in peripheral lymphocytes in this patient therefore was intercellular to a large extent. However, almost all cells which showed 95 % normal mitochondrial genes in MELAS rapidly changed to the cells involved mutant genes in the homoplasmic fashion after cell culture.