Nancy G. Kennaway

Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2–(2–nitro–4–trifluoro–methylbenzyol)–1,3–cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH−/− mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system.

Mutations in COX15 Produce a Defect in the Mitochondrial Heme Biosynthetic Pathway, Causing Early-Onset Fatal Hypertrophic Cardiomyopathy

Deficiencies in the activity of cytochrome c oxidase (COX), the terminal enzyme in the respiratory chain, are a frequent cause of autosomal recessive mitochondrial disease in infants. These patients are clinically and genetically heterogeneous, and all defects so far identified in this group have been found in genes coding for accessory proteins that play important roles in the assembly of the COX holoenzyme complex. Many patients, however, remain without a molecular diagnosis. We have used a panel of retroviral vectors expressing human COX assembly factors in these patients to identify the molecular basis for the COX deficiency by functional complementation. Here we show that overexpression of COX15, a protein involved in the synthesis of heme A, the heme prosthetic group for COX, can functionally complement the isolated COX deficiency in fibroblasts from a patient with fatal, infantile hypertrophic cardiomyopathy. Mutation analysis of COX15 in the patient identified a missense mutation (C700T) on one allele, changing a conserved arginine to tryptophan (R217W), and a splice-site mutation in intron 3 on the other allele (C447-3G), resulting in a deletion of exon 4. This splicing error introduces a frameshift and a premature stop codon, resulting in an unstable mRNA and, likely, a null allele. Mitochondrial heme A content was reduced in the patients heart and fibroblast mitochondria, and levels of heme O were increased in the patients heart. COX activity and the total amount of fully assembled enzyme were reduced by 50%-70% in patient fibroblasts. Expression of COX15 increased heme A content and rescued COX activity. These results suggest that reduced availability of heme A stalls the assembly of COX. This study establishes COX15 as an additional cause, along with SCO2, of fatal infantile, hypertrophic cardiomyopathy associated with isolated COX deficiency.

A microdeletion in cytochrome c oxidase (COX) subunit III associated with COX deficiency and recurrent myoglobinuria

We have identified a 15-bp microdeletion in a highly conserved region of the mitochondrially encoded gene for cytochrome c oxidase (COX) subunit III in a patient with severe isolated COX deficiency and recurrent myoglobinuria. The mutant mitochondrial DMA (mtDNA) comprised 92% of the mtDNA in muscle and 0.7% in leukocytes. Immunoblots and immunocytochemistry suggested a lack of assembly or instability of the complex. Microdissected muscle fibres revealed significantly higher proportions of mutant mtDNA in COX-negative than in COX-positive fibres. This represents the first case of isolated COX deficiency to be defined at the molecular level.

Identification and Characterization of a Common Set of Complex I Assembly Intermediates in Mitochondria from Patients with Complex I Deficiency

Deficiencies in the activity of complex I (NADH: ubiquinone oxidoreductase) are an important cause of human mitochondrial disease. Complex I is composed of at least 46 structural subunits that are encoded in both nuclear and mitochondrial DNA. Enzyme deficiency can result from either impaired catalytic efficiency or an inability to assemble the holoenzyme complex; however, the assembly process remains poorly understood. We have used two-dimensional Blue-Native/SDS gel electrophoresis and a panel of 11 antibodies directed against structural subunits of the enzyme to investigate complex I assembly in the muscle mitochondria from four patients with complex I deficiency caused by either mitochondrial or nuclear gene defects. Immunoblot analyses of second dimension denaturing gels identified seven distinct complex I subcomplexes in the patients studied, five of which could also be detected in nondenaturing gels in the first dimension. Although the abundance of these intermediates varied among the different patients, a common constellation of subcomplexes was observed in all cases. A similar profile of subcomplexes was present in a human/mouse hybrid fibroblast cell line with a severe complex I deficiency due to an almost complete lack of assembly of the holoenzyme complex. The finding that diverse causes of complex I deficiency produce a similar pattern of complex I subcomplexes suggests that these are intermediates in the assembly of the holoenzyme complex. We propose a possible assembly pathway for the complex, which differs significantly from that proposed for Neurospora, the current model for complex I assembly.

Aerobic conditioning in patients with mitochondrial myopathies: Physiological, biochemical, and genetic effects

Aerobic training has been shown to increase work and oxidative capacity in patients with mitochondrial myopathies, but the mechanisms underlying improvement are not known. We evaluated physiological (cycle exercise, 31P‐MRS), biochemical (enzyme levels), and genetic (proportion of mutant/wild‐type genomes) responses to 14 weeks of bicycle exercise training in 10 patients with heteroplasmic mitochondrial DNA (mtDNA) mutations. Training increased peak work and oxidative capacities (20–30%), systemic arteriovenous O2 difference (20%), and 31P‐MRS indices of metabolic recovery (35%), consistent with enhanced muscle oxidative phosphorylation. Mitochondrial volume in vastus lateralis biopsies increased significantly (50%) and increases in deficient respiratory chain enzymes were found in patients with Complex I (36%) and Complex IV (25%) defects, whereas decreases occurred in 2 patients with Complex III defects (‐20%). These results suggest that the cellular basis of improved oxygen utilization is related to training‐induced mitochondrial proliferation likely resulting in increased levels of functional, wild‐type mtDNA. However, genetic analysis indicated the proportion of wild‐type mtDNA was unchanged (3/9) or fell (6/9), suggesting a trend toward preferential proliferation of mutant genomes. The long‐term implications of training‐induced increases in mutant relative to wild‐type mtDNA, despite positive physiological and biochemical findings, need to be assessed before aerobic training can be proposed as a general treatment option.

MELAS syndrome with mitochondrial tRNA Leu[UUR] mutation: Correlation of clinical state, nerve conduction, and muscle 31P magnetic resonance spectroscopy during treatment with nicotinamide and riboflavin

We report a patient with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes treated with riboflavin and nicotinamide for 18 months, during which time previously frequent encephalopathic spells ceased. To confirm clinical benefit, we withdrew treatment and monitored response with muscle 31P magnetic resonance spectroscopy (MRS) and sural nerve conduction studies. Of three prospectively chosen MRS variables, two changed coincidentally with clinical end points; phosphocreatine (PCr)/adenosine triphosphate recovery rates fell in parallel with sural nerve sensory amplitudes, and a drop in muscle bioenergetic efficiency (relationship of inorganic phosphate/PCr to the accelerating force of contracting muscle) coincided with development of encephalopathy. Investigations revealed a deficiency of respiratory complex I and mutation of the mitochondrial tRNALeu(UUR). We suggest that a defective cellular energy state in mitochondrial disease may be partially treatable and that changes seen in appropriate muscle spectroscopy studies may parallel improvement in brain and peripheral nerve function.

Mitochondrial Encephalomyopathy and Complex III Deficiency Associated with a Stop-Codon Mutation in the Cytochrome b Gene

We have reinvestigated a young woman, originally reported by us in 1983, who presented with exercise intolerance and lactic acidosis associated with severe deficiency of complex III and who responded to therapy with menadione and ascorbate. Gradually, she developed symptoms of a mitochondrial encephalomyopathy. Immunocytochemistry of serial sections of muscle showed a mosaic of fibers that reacted poorly with antibodies to subunits of complex III but reacted normally with antibodies to subunits of complexes I, II, or IV, suggesting a mutation of mtDNA. These findings demonstrate the diagnostic value of immunocytochemistry in identifying specific respiratory-chain deficiencies and, potentially, distinguishing between nuclear- or mtDNA-encoded defects. Sequence analysis revealed a stop-codon mutation (G15242A) in the mtDNA-encoded cytochrome b gene, resulting in loss of the last 215 amino acids of cytochrome b. PCR-RFLP analysis indicated that the G15242A mutation was heteroplasmic and was present in a high percentage (87%) of affected tissue (skeletal muscle) and a low percentage (0.7%) of unaffected tissue (blood) but was not detected in controls. Analysis of microdissected muscle fibers showed a significant correlation between the immunoreactivity toward the Rieske protein of complex III and the percentage of mutant mtDNA: immunopositive fibers had a median value of 33% of the G15242A mutation, whereas immunonegative, ragged-red fibers had a median value of 89%, indicating that the stop-codon mutation was pathogenic in this patient. The G15242A mutation was also present in several other tissues, including hair roots, indicating that it must have arisen either very early in embryogenesis, before separation of the primary germ layers, or in the maternal germ line. The findings in this patient are contrasted with other recently described patients who have mutations in the cytochrome b gene.

Mitochondrial myopathy with succinate dehydrogenase and aconitase deficiency. Abnormalities of several iron-sulfur proteins.

Recently, we described a patient with severe exercise intolerance and episodic myoglobinuria, associated with marked impairment of succinate oxidation and deficient activity of succinate dehydrogenase and aconitase in muscle mitochondria (1). We now report additional enzymatic and immunological characterization of mitochondria. In addition to severe deficiency of complex II, manifested by reduction of succinate dehydrogenase and succinate:coenzyme Q oxidoreductase activities to 12 and 22% of normal, respectively, complex III activity was reduced to 37% and rhodanese to 48% of normal. Furthermore, although complex I activity was not measured, immunoblot analysis of complex I showed deficiency of the 39-, 24-, 13-, and 9-kD peptides with lesser reductions of the 51- and 18-kD peptides. Immunoblots of complex III showed markedly reduced levels of the mature Rieske protein in mitochondria and elevated levels of its precursor in the cytosol, suggesting deficient uptake into mitochondria. Immunoreactive aconitase was also low. These data, together with the previous documentation of low amounts of the 30-kD iron-sulfur protein and the 13.5-kD subunit of complex II, compared to near normal levels of the 70-kD protein suggest a more generalized abnormality of the synthesis, import, processing, or assembly of a group of proteins containing iron-sulfur clusters.

Lactic acidosis and mitochondrial myopathy associated with deficiency of several components of complex III of the respiratory chain

Summary: We have studied a 17-year-old girl with lactic acidosis (3-18 mEq/liter) and progressive muscle weakness since 9 years of age. Morphological findings in muscle were of a typical ragged red myopathy with multiple collections of bizarre mitochondria, some containing paracrystalline inclusions.The carnitine content of serum and muscle was normal, as were the activities of carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase in the patients muscle. Measurement of the enzymes of oxidative phosphorylation in both crude muscle homogenates and mitochondrial fractions showed close to normal activities of cytochrome c oxidase, succinate dehydrogenase, and ATPase. In contrast, succinate cytochrome c reductase activity was greatly reduced in the patient, being 0.035 μmol/min/g tissue in whole muscle (controls 1.16 ± 0.47 μmol/min/g tissue) and 8 nmol/min/mg protein in the mitochondria (control, 340 nmol/min/mg protein). Rotenonesensitive NADH-cytochrome c reductase was also undetectable in the patients mitochondria. Spectral analysis of cytochromes showed decrease of reducible cytochrome b to 16% of the control. These results indicate a defect of ubiquinol-cytochrome c reductase or the cytochrome bc1 segment (complex III) of the electron transport chain. Antibody-binding studies of the individual components of complex III showed additional deficiencies of core proteins I and II and peptide VI, indicating a more widespread defect of complex III than was evident from spectral analysis and enzyme activity measurements alone.Urine organic acid analysis after fasting and following a medium chain triglyceride load showed unusually high levels of lactate and 3-hydroxybutyrate, lower than expected levels of acetoacetate and dicarboxylic acids, and the presence of several other metabolites suggesting a disturbed citric acid cycle and redox state. Thus, the defect in this patient may be more widespread than is apparent from the clinical presentation.