Point mutations in mitochondrial DNA (mtDNA) are increasingly being recognized as important causes of maternally-inherited mental retardation or psychomotor regression in childhood. Accurate molecular diagnosis of known and new mtDNA mutations is of central importance for the Program Project as a whole. Thus, our first goal is to keep searching for mtDNA mutations, known and unknown, in children with mental retardation and evidence of maternally-inherited mitochondrial dysfunction. Well- established polymerase chain reaction (PCR)-based assay- will be employed for the diagnosis of known mutations, whereas new mutations will be identified through single strand conformation polymorphism (SSCP) analysis followed by mtDNA sequencing. In contrast to the rapid progress of molecular studies in mtDNA-related diseases, definition of the biochemical consequences has been slow and remains controversial. We will conduct comparative biochemical studies primarily in tissue cultures (including fibroblast and muscle cultures and p/o cybrids) from patients with similar clinical phenotypes but different mutations. Specifically, we will study ATP synthesis and ATPase activity in cells harboring the more common T->G mutation and a novel T- >C mutation at nt-8993 in the Atpase 6 gene; both mutations are associated with maternally-inherited Leigh's syndrome (MILS). We will also study the activities of all five respiratory chain complexes in cells harboring four different mutations associated with MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), two of which are in the tRNA/Leu(UUR) gene (at nt-3243 and at nt-3271) and two in protein-coding genes (at nt-9957 in the COXIII gene and at nt- 11084 in the ND4 gene). Biochemical alterations in complexes I, IV, and V will be further analyzed by immunoblot and immunotitration studies, whenever possible.
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