Detection, Quantitation, and Distribution of the T9861C mtDNA Mutation in the Alzheimer's Disease Brain

Abstract

Deficits in oxidative phosphorylation have been observed in brain and platelet samples from patients with Alzheimer's Disease. A reduction of approximately 30% in cytochrome c oxidase activity is routinely reported in these patients. Using an electrophoretic approach (single strand conformation polymorphism) to screen multiple AD and control brain samples, we have recently identified a point mutation at np 9861 in AD brain mtDNA that alters amino acid 219 of subunit III of cytochrome c oxidase from a phenylalanine to a leucine. We have developed a mismatched PCR‐RFLP assay to rapidly screen multiple samples and combined this with a modification of the single nucleotide primer extension assay to allow quantitation of low levels of this heteroplasmic mutation. In addition, we have begun to use an allele‐specific PCR amplification assay to both detect and quantify the mutation. Of forty AD brain samples tested, six were found to possess the T9861C mutation to varying degrees. None of 40 age‐matched control brains contained the mutation. This 15% frequency occurrence rate in AD brain samples is 63‐fold higher than the frequency (0.24%) of this polymorphism reported in mtDNA databases comprised of sequence information from 2,104 human samples. The T9861C mutation is heteroplasmic. The T9861C mutation is not localized to a single region but is also found in both the parietal and temporal cortex and in the caudate but not in the hippocampus. The presence of the T9861C mutation reduces cytochrome oxidase activity by 60% compared to wild type enzyme from age‐matched controls.