Comprehensive molecular diagnosis of mitochondrial disorders: qualitative and quantitative approach.

Mitochondrial disorders can be caused by mutations in nuclear or mitochondrial encoded genes. Point mutations and large deletions in mitochondrial DNA (mtDNA) are responsible for a small portion of the molecular defects in the mitochondrial oxidative phosphorylation system. A significant number of molecular defects of respiratory chain disorders are probably due to mutations in nuclear genes. Molecular diagnosis of mitochondrial disorders has been difficult because of broad genetic and clinical heterogeneity. Mutational analysis of common point mutations of mtDNA such as A3243G, A8344G, and T8993G/C is routinely performed. However, many patients who clearly have clinical manifestations and muscle pathology consistent with oxidative phosphorylation deficiency do not have detectable common mtDNA point mutations. A more comprehensive mutation screening method, temporal temperature gradient gel electrophoresis, was used to scan for unknown mutations in the entire mitochondrial genome. Novel mutations have been discovered but only account for a small portion of patients with suspected mitochondrial RC disorders. Real-time quantitative PCR analysis was used to measure cellular mtDNA content. Abnormal levels of mtDNA were found in many patients with respiratory chain disorders. Molecular analysis revealed that mutations in the thymidine phosphorylase gene are not seen in young patients with severe mtDNA depletion who do not demonstrate clinical features of mitochondrial neurogastrointestinal encephalomyopathy. It was also noted that an increase in the size or number of mitochondria was not necessarily associated with an increase in mtDNA content. On the contrary, in some cases the mtDNA was depleted. Respiratory activity in patients with a defective mitochondrial genome due to either point mutations or deletions may be compensated by amplification of mtDNA. Therefore, a comprehensive molecular analysis of mitochondrial respiratory chain disorders should include qualitative identification of the mutation and quantitative measurement of both the degree of mutant heteroplasmy and the total amount of mtDNA.