Renal amino acid transport in adults with oxidative phosphorylation diseases.

The clinical manifestations of mitochondrial DNA (mtDNA) mutations depend on a variety of factors including ratios of normal to abnormal mtDNA and tissue-specific differences in ATP production by oxidative phosphorylation (OXPHOS). In order to investigate the effects of OXPHOS defects on renal tubule function, we characterized sodium-coupled transport processes in six individuals with OXPHOS diseases. Pathogenic mtDNA mutations were identified in five of these individuals. Sodium coupled transport processes were evaluated by determining fractional excretions of amino acids, glucose, lactate, urate, and phosphate in patients and controls. Four of the six individuals had high fractional excretions of neutral amino acids, indicating abnormal renal tubule reabsorbtion of these amino acids. Abnormalities in fractional excretions of lactate, glucose, urate, and phosphate were less pronounced. These results demonstrate that sodium-coupled transport processes in the kidney are sensitive to OXPHOS impairment. When abnormalities in these processes are encountered, an OXPHOS disease should be included in the differential diagnosis.

Leber's hereditary optic neuropathy: a model for mitochondrial neurodegenerative diseases.

A number of human diseases have been attributed to defects in oxidative phosphorylation (OXPHOS) resulting from mutations in the mitochondrial DNA (mtDNA). One such disease is Leber's hereditary optic neuropathy (LHON), a neurodegenerative disease of young adults that results in blindness due to atrophy of the optic nerve. The etiology of LHON is genetically heterogeneous and in some cases multifactorial. Eleven mtDNA mutations have been associated with LHON, all of which are missense mutations in the subunit genes for the subunits of the electron transport chain complexes I, III, and IV. Molecular, biochemical, and population genetic studies have categorized these mutations as high risk (class I), low risk (class II), or intermediate risk (class I/II). Class I mutations appear to be primary genetic causes of LHON, while class II mutations are frequently found associated with class I genotypes and may serve as exacerbating genetic factors. Different LHON pedigrees can harbor different combinations of class I, II, or I/II mtDNA mutations, as shown by the complete sequence analysis of the mtDNAs of four LHON probands. The various mtDNA genotypes included an isolated class I mutation, combined class I+II mutations, and combined class I/II+II mutations. The occurrence of such genotypes supports the hypothesis that LHON may result from the additive effects of various genetic and environmental insults to OXPHOS, each of which increases the probability of blindness.

Mitochondrial DNA complex I and III mutations associated with Leber's hereditary optic neuropathy.

Four new missense mutations have been identified through restriction analysis and sequencing of the mitochondrial DNAs (mtDNA) from Leber's hereditary optic neuropathy (LHON) patients who lacked the previously identified 11778 mutation. Each altered a conserved amino acid and correlated with the LHON phenotype in population and phylogenetic analyses. The nucleotide pair (np) 13708 mutation (G to A, ND5 gene) changed an alanine to a threonine and was found in 6/25 (24%) of non-11778 LHON pedigrees and in 5.0% of controls, the np 15257 mutation (G to A, cytochrome b gene) changed an aspartate to an asparagine and was found in 4 of the 13708-positive pedigrees and 0.3% of controls, the np 15812 mutation (G to A, cytochrome b gene) changed a valine to a methionine and was detected in two of the 15257-positive pedigrees and 0.1% of controls and the np 5244 mutation (G to A, ND2 gene) changed a glycine to a serine and was found in one of the 15812-positive patients and none of 2103 controls. The 15257 mutation altered a highly conserved amino acid in an extramembrane domain of cytochrome b that is associated with the ligation of the low potential b566 heme and the 5244 mutation altered a strongly evolutionarily conserved region of the ND2 polypeptide. The 13708 and 15812 mutations changed moderately conserved amino acids. Haplotype and phylogenetic analysis of the four np 15257 mtDNAs revealed that all harbored the same rare Caucasian haplotype and that the np 13708, np 15257, np 15812 and np 5244 mutations were added sequentially along this mtDNA lineage. Since the percentage of sighted controls decreases as these mutations accumulate, it appears that they interact synergistically, each increasing the probability of blindness. The involvement of both mitochondrial complex I (np 5244, 11778, 13708) and complex III (np 15257, 15812) mutations in LHON indicates that the clinical manifestations of this disease are the product of an overall decrease in mitochondrial energy production rather than a defect in a specific mitochondrial enzyme.

Mitochondrial DNA mutations associated with neuromuscular diseases: analysis and diagnosis using the polymerase chain reaction.

A number of neuromuscular diseases are associated with molecular defects in the mitochondrial DNA (mtDNA). These include: 1) a missense mutation at nucleotide 11778 in the mtDNA of Leber's hereditary optic neuropathy patients; 2) a heterogeneous array of deletions in the mtDNA of ocular myopathy patients; and 3) small deletions and point mutations in the mtDNA of myoclonic epilepsy and ragged red fiber disease patients. We can now diagnose these diseases at the molecular level from small patient samples by amplifying the affected mtDNA regions using the polymerase chain reaction. Leber's hereditary optic neuropathy is diagnosed through loss of an SfaNI restriction site. Ocular myopathy deletions are identified by differential amplification across deletion breakpoints. Familial diseases such as myoclonic epilepsy and ragged red fiber disease might be diagnosed by identifying small deletions through amplification and electrophoretic analysis of the entire mtDNA genome or by identifying point mutations through differential oligonucleotide hybridization. As additional mtDNA molecular defects are identified, molecular analysis will likely become a primary tool for the diagnosis of these diseases.

Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies.

The mitochondrial myopathies (MM) are a heterogenous group of neuromuscular diseases associated with abnormal mitochondria and defects in mitochondrial oxidative phosphorylation (OXPHOS). Analysis of a broad spectrum of MM patients has revealed that patients with similar clinical symptoms frequently do not have the same muscle OXPHOS defect. To determine whether some of this variation was due to methodological differences between studies, we have made a detailed survey of OXPHOS enzyme analysis procedures. The coupled OXPHOS assays for Complexes I + III and II + III were found to be variable due to competing reactions and complicated interactions between complexes. These problems were resolved by utilizing specific Complex I and III assays. The muscle mitochondria isolated from surgery patients under general anesthesia and prepared by proteinase digestion were observed to give low and highly variable OXPHOS activities. Mitochondria isolated from muscle biopsies performed under local anesthesia and finely sliced prior to homogenization gave higher and more consistent OXPHOS activities. Assays for Complexes I, III and V required mitochondrial sonication to express maximal activity, but Complex IV was prone to inactivation by excessive mechanical disruption. Mitochondria isolated from frozen muscle or from patients with an OXPHOS disease are more fragile than those isolated from fresh tissue and normal individuals. Hence, Complex IV activity can be preferentially lost from frozen and sonicated myopathy patient samples. These results suggest that variation in muscle OXPHOS analysis techniques may account for some of the discrepancies between clinical manifestations and OXPHOS defects and suggest that no single protocol is sufficient to adequately define the OXPHOS defect in MM patients.

Variable genotype of Leber's hereditary optic neuropathy patients.

Leber's hereditary optic neuropathy is caused by a single nucleotide change in the mitochondrial deoxyribonucleic acid (mtDNA). Each cell contains thousands of mitochondrial DNA molecules. We demonstrated that in certain isolated instances, the proband and close maternal lineage relatives can have mixtures of mutant and normal mitochondrial DNA molecules (heteroplasmy). The proportion of mutant mitochondrial DNA molecules was found to shift markedly across generations and within the tissues of an individual. One unaffected mother had 65% mutant mitochondrial DNA molecules whereas her affected son had essentially 100% mutant mitochondrial DNA molecules. Two affected individuals had predominantly mutant mitochondrial DNA in their blood, but significant normal mitochondrial DNA in their hair. The demonstration of heteroplasmy within maternal lineages and affected individuals means that the successful determination of the mitochondrial DNA genotype of a family or patient with Leber's hereditary optic neuropathy requires testing of more than one family member and more than one tissue from each individual.

Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy.

The muscle mitochondria of a patient with Kearns-Sayre/chronic external ophthalmoplegia plus syndrome were found to be completely deficient in respiratory complex I activity and partially deficient in complex IV and V activities. Treatment of the patient with coenzyme Q10 and succinate resulted in clinical improvement of respiratory function, consistent with the respiratory deficiencies. Restriction enzyme analysis of the muscle mtDNA revealed a 4.9-kilobase deletion in 50% of the mtDNA molecules. Polymerase chain reaction analysis demonstrated that the deletion was present in the patient's muscle but not in her lymphocytes or platelets. Furthermore, the deletion was not present in the muscle or platelets of two sisters. Hence, the mutation probably occurred in the patient's somatic cells. Direct sequencing of polymerase chain reaction-amplified DNA revealed a 4977-base-pair deletion removing four genes for subunits of complex I, one gene for complex IV, two genes for complex V, and five genes for tRNAs, which paralleled the respiratory enzymes affected in the disease. A 13-base-pair direct repeat was observed upstream from both breakpoints. Relative to the direction of heavy-strand replication, the first repeat was retained and the second repeat was deleted, suggesting a slip-replication mechanism. Sequence analysis of the human mtDNA revealed many direct repeats of 10 base pairs or greater, indicating that this mechanism could account for other reported deletions. We postulate that the prevalence of direct repeats in the mtDNA is a consequence of the guanine-cytosine bias of the heavy and light strands.

Evidence in a lethal infantile mitochondrial disease for a nuclear mutation affecting respiratory complexes I and IV.

A child died at 4 months of age of a lethal infantile mitochondrial disease associated with cardiomyopathy. Detailed pathologic evaluation of this patient revealed abnormalities in the striated muscle, smooth muscle, heart, and liver, but not the central nervous system. Biochemical analysis revealed a combined complex I and IV deficiency in skeletal muscle, heart, and liver, but not in kidney and brain. Analysis of mitochondrial translation products and mitochondrial DNA failed to detect any abnormality. Parallel studies on both parents were uniformly normal. These data support the hypothesis that this disease was the result of a nuclear DNA mutation in a developmental stage-specific and tissue-specific oxidative phosphorylation-gene.