The molecular and biochemical characterization of human mitochondrial respiratory chain deficiencie

The phenotypes associated with mitochondrial disorders are often varied and complex and patients generally possess a deficiency in either a specific respiratory chain complex or show a generalized reduction in mitochondrial function. To begin to address this complex clinical presentation and gain insight into the organization and function of the mitochondrial respiratory chain, a multifaceted approach was used to characterize various disease presentations using both biochemical and molecular biological methodologies. Initially, the cloning, sequence analysis and chromosomal localization of genomic cytochrome c oxidase (COX) tissue specific sequences (COX VIa and VII-a, liver form) are presented. These sequences are representative of a class of nuclear genes that encode mitochondrial products. Next, a tissue specific cytochrome c oxidase deficiency prevalent in the Lac-St.-Jean region of Quebec was characterized. Patients with this disease showed severe cytochrome c oxidase deficiency in the brain and liver while approximately 50% residual enzyme activity was detected in muscle, kidney and cultured skin fibroblasts. Cytochrome c oxidase adivity in the heart tissue of these patients, however, was almost normal. The autosomal recessive inheritance pattern of this disease suggested a defect in one of the nuclear encoded genes. The consequences of this biochemical phenotype and a rationale for its existence are discussed.;Two pedigrees exhibiting a metabolic hypertrophic cardiomyopathy were next characterized and disease symptoms were shown to be associated with novel point mutations in the mitochondrial tRNA$rmsp{Glycine}$ (T-to-C) or tRNA$rmsp{Isoleucine}$ (A-to-G) genes, respectively. The presentation of these mitochondrial DNA associated diseases was characterized with regards to biochemical profile and percentage of heteroplasmic mutant DNA in various tissues available from family members. A detailed discussion of the possible models by which tissue specificity can be achieved in this disease is presented. In addition, a detailed characterization of clonal cybrid cell lines, selected to contain different levels of each respective mutant DNA, is presented in order to correlate each particular point mutation with the observed biochemical profile. Finally the cloning strategy and sequence analysis of the human mitochondrial elongation factor Tu cDNA are described. A discussion highlighting the conserved features of the predicted amino acid sequence ensues.