Two Novel Mutations in the SLC25A4 Gene in a Patient with Mitochondrial Myopathy.

In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

A simplified and reliable assay for complex I in human blood lymphocytes.

Complex I activity of the mitochondrial respiratory chain is difficult to measure in blood lymphocytes because of the limited access of substrates to the enzyme complex in these cells. The results of the present study show that permeabilization of human blood lymphocytes in the presence of protease inhibitors by three cycles of freeze-thawing enables reproducible detection of the rotenone-sensitive complex I activity. To that end, the water-soluble coenzyme Q(10) analogue CoQ(1) and a relatively high concentration of blood lymphocytes were combined in small quartz cuvettes so that the amount of blood needed for this assay remained low. The relationship between the initial rate of NADH oxidation by complex I and the protein concentration was quasi-linear. The fractional inhibition of the total NADH:CoQ(1) oxidoreductase by a saturating concentration of rotenone decreased sharply at CoQ(1) concentrations higher than 20 muM, which is indicative, but does not prove the involvement of a second CoQ(1) binding site at complex I. Since the present complex I assay requires only a small amount of blood, the functionality of this important respiratory chain complex can be assessed in an easy and reliable manner not only in adult patients but also in children suspected to have a mitochondrial disease.

Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.

Transmission and prenatal diagnosis of the T9176C mitochondrial DNA mutation.

A family presented with three affected children with Leigh syndrome, a progressive neurodegenerative disorder. Analysis of the OXPHOS complexes in muscle of two affected patients showed an increase in activity of pyruvate dehydrogenase and a decrease of complex V activity. Mutation analysis revealed the T9176C mutation in the mtATPase 6 gene (OMIM 516060) and the mutation load was above 90% in the patients. Unaffected maternal relatives were tested for carrier-ship and one of them, with a mutation load of 55% in blood, was pregnant with her first child. The possibility of prenatal diagnosis was evaluated. The main problem was the lack of data on genotype-phenotype associations for the T9176C mutation and on variation of the mutation percentage in tissues and in time. Therefore, multiple tissues of affected and unaffected carriers were analysed. Eventually, prenatal diagnosis was offered with understanding by the couple that there could be considerable uncertainty in the interpretation of the results. Prenatal diagnosis was carried out twice on cultured and uncultured chorion villi and amniotic fluid cells. The result was a mutation percentage just below the assumed threshold of expression (90%). The couple decided to continue the pregnancy and an apparently healthy child was born with an as yet unclear prognosis. This is the first prenatal diagnosis for a carrier of the T9176C mutation. Prenatal diagnosis for this mutation is technically reliable, but the prognostic predictions are not straightforward.

Increased risk for cardiorespiratory failure associated with the A3302G mutation in the mitochondrial DNA encoded tRNALeu(UUR) gene.

Screening the mitochondrial DNA of a 64-year-old woman with mitochondrial myopathy revealed 76% of the tRNA(Leu(UUR)) A3302G mutation in muscle. Muscle of her affected son carried 96% mutated mitochondrial DNA. Both patients were biopsied twice, showing isolated complex I deficiency in the son's first biopsy, additional increased (within normal range) complex II + III activities in his second biopsy, combined complex I, II + III deficiency in mothers first biopsy and additional complex IV deficiency in her second biopsy. After a stay in the mountains, the son died of cardiac arrhythmia. The A3302G mutation has been reported before and is associated with mitochondrial myopathy and cardiorespiratory failure. Pathogenesis is explained by abnormal mtRNA processing, which was also reported for the adjacent C3303T mutation associated with cardiomyopathy and/or skeletal myopathy. Our findings suggest that a high mutation load of the A3302G mutation can lead to fatal cardiorespiratory failure, likely triggered by low environmental oxygen pressure and exercise.

Pearson syndrome and the role of deletion dimers and duplications in the mtDNA.

Pearson syndrome is an often fatal multisystem disease associated with mitochondrial DNA rearrangements. Here we report a patient with a novel mtDNA deletion of 3.4 kb ranging from nucleotides 6097 to 9541 in combination with deletion dimers. The mutation percentage in different tissues (blood, muscle and liver) varied between 64% and 95%. After a remission period of about a year, the patient suddenly died at the age of 3 years owing to a severe lactic acidosis. A second patient with a previously reported deletion of 8 kb and a milder phenotype was found to have mitochondrial duplications and died at the age of 10 years. From these data and data from previous reports, we hypothesize that duplications might be beneficial in the clinical course of the disease and in life expectancy.

Efficacy of carnitine in the treatment of children with attention-deficit hyperactivity disorder.

To determine safety and the efficacy of carnitine treatment in children with attention-deficit hyperactivity disorder (ADHD). The ADHD behavior was observed by parents completing the Child Behavior Checklist (CBCL) and by teachers completing the Conners teacher-rating score, in a randomized, double-blind, placebo-controlled double-crossover trial. In 13/24 boys receiving carnitine, home behavior improved as assessed with the CBCL total score (P < 0.02). In 13/24 boys, school behavior improved as assessed with the Conners teacher-rating score (P < 0.05). Before treatment, the CBCL total and sub-scores were significantly different from those of normal Dutch boys (P < 0.0001). Responders showed a significant improvement of the CBCL total scores compared to baseline (P < 0.0001). In the majority of boys no side effects were seen. At baseline and after carnitine treatment, responders showed higher levels of plasma-free carnitine (P < 0.03) and acetylcarnitine (P < 0.05). Compared to baseline, the carnitine treatment caused in the responsive patients a decrease of 20-65% (8-48 points) as assessed by the CBCL total problem rating scale. Treatment with carnitine significantly decreased the attention problems and aggressive behavior in boys with ADHD.

Intermediates of unsaturated fatty acid oxidation are incorporated in triglycerides but not in phospholipids in tissues from patients with mitochondrial beta-oxidation defects.

The fatty acid composition was determined of liver, skeletal muscle and heart obtained post mortem from patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD), multiple acyl-CoA dehydrogenase deficiency (MADD) and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Increased amounts of 4-decenoic acid 10:1(n-6), 5-dodecenoic acid 12:1(n-7), 5-tetradecenoic acid 14:1(n-9), 5,8-tetradecadienoic acid 14:2(n-6) and 7,10-hexadecadienoic acid 16:2(n-6)--intermediates of unsaturated fatty acid oxidation--were found. Fractionation into different lipid classes showed that these fatty acids were exclusively present in the triglyceride fraction. They could not be detected in the free fatty acid fraction or in the phospholipid fraction. Our results suggest that intermediates of unsaturated fatty acid oxidation that accumulate as a consequence of MCADD, MADD and VLCADD are transported to the endoplasmic reticulum for esterification into neutral glycerolipids. The pattern of accumulation is characteristic for each disease, which makes fatty acid analysis of total lipid of post-mortem tissues a useful tool in the detection of mitochondrial fatty acid oxidation defects in patients who died unexpected, for example with sudden infant death syndrome.

Immunological phenotyping of fibroblast cultures from patients with a mitochondrial respiratory chain deficit.

Conventional approaches to the diagnosis of mitochondrial respiratory chain diseases, using enzyme assays and histochemistry, are laborious and give limited information concerning the genetic basis of a deficiency. We have evaluated the diagnostic value of 12 monoclonal antibodies to subunits of the four respiratory chain enzyme complexes and F(1)F(0)-ATP synthase. Antibodies were used in immunological studies with skin fibroblast cultures derived from patients with diverse mitochondrial diseases, including patients in which the disease was caused by a nuclear genetic defect and patients known to harbor a heteroplasmic mutation in a mitochondrial tRNA gene. Immunoblotting experiments permitted the identification of specific enzyme assembly deficits and immunocytochemical studies provided clues regarding the genetic origin of the disease. The immunological findings were in agreement with the biochemical and genetic data of the patients. Our study demonstrates that characterization of the fibroblast cultures with the monoclonal antibodies provides a convenient technique to complement biochemical assays and histochemistry in the diagnosis of mitochondrial respiratory chain disorders.

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1-2 kb, digested in fragments of 90-600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA(Leu(UUR)) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA(Leu(UUR)) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.

Functional hyperactivity of hepatic glutamate dehydrogenase as a cause of the hyperinsulinism/hyperammonemia syndrome: effect of treatment.

OBJECTIVE: The combination of persistent hyperammonemia and hypoketotic hypoglycemia in infancy presents a diagnostic challenge. Investigation of the possible causes and regulators of the ammonia and glucose disposal may result in a true diagnosis and predict an optimum treatment. PATIENT: Since the neonatal period, a white girl had been treated for hyperammonemia and postprandial hypoglycemia with intermittent hyperinsulinism. Her blood level of ammonia varied from 100 to 300 micromol/L and was independent of the protein intake. METHODS: Enzymes of the urea cycle as well as glutamine synthetase and glutamate dehydrogenase (GDH) were assayed in liver tissue and/or lymphocytes. RESULTS: The activity of hepatic GDH was 874 nmol/(min.mg protein) (controls: 472-938). Half-maximum inhibition by guanosine triphosphate was reached at a concentration of 3.9 micromol/L (mean control values:.32). The ratio of plasma glutamine/blood ammonia was unusually low. Oral supplements with N-carbamylglutamate resulted in a moderate decrease of the blood level of ammonia. The hyperinsulinism was successfully treated with diazoxide. CONCLUSION: A continuous conversion of glutamate to 2-oxoglutarate causes a depletion of glutamate needed for the synthesis of N-acetylglutamate, the catalyst of the urea synthesis starting with ammonia. In addition, the shortage of glutamate may lead to an insufficient formation of glutamine by glutamine synthetase. As GDH stimulates the release of insulin, the concomitant hyperinsulinism can be explained. This disorder should be considered in every patient with postprandial hypoglycemia and diet-independent hyperammonemia.

Characterization of a novel mitochondrial DNA deletion in a patient with a variant of the Pearson marrow-pancreas syndrome.

We have recently diagnosed a patient with anaemia, severe tubulopathy, and diabetes mellitus. As the clinical characteristics resembled Pearson marrow-pancreas syndrome, despite the absence of malfunctioning of the exocrine pancreas in this patient, we have performed DNA analysis to seek for deletions in mtDNA. DNA analysis showed a novel heteroplasmic deletion in mtDNA of 8034bp in length, with high proportions of deleted mtDNA in leukocytes, liver, kidney, and muscle. No deletion could be detected in mtDNA of leukocytes from her mother and young brother, indicating the sporadic occurrence of this deletion. During culture, skin fibroblasts exhibited a rapid decrease of heteroplasmy indicating a selection against the deletion in proliferating cells. We estimate that per cell division heteroplasmy levels decrease by 0.8%. By techniques of fluorescent in situ hybridisation (FISH) and mitochondria-mediated transformation of rho(o) cells we could show inter- as well as intracellular variation in the distribution of deleted mtDNA in a cell population of cultured skin fibroblasts. Furthermore, we studied the mitochondrial translation capacity in cybrid cells containing various proportions of deleted mtDNA. This result revealed a sharp threshold, around 80%, in the proportion of deleted mtDNA, above which there was strong depression of overall mitochondrial translation, and below which there was complementation of the deleted mtDNA by the wild-type DNA. Moreover, catastrophic loss of mtDNA occurred in cybrid cells containing 80% deleted mtDNA.

Identification of two novel mutations in OCTN2 of three patients with systemic carnitine deficiency.

Systemic carnitine deficiency is a potentially lethal, autosomal recessive disorder characterized by cardiomyopathy, myopathy, recurrent episodes of hypoketotic hypoglycemia, hyperammonemia, and failure to thrive. This form of carnitine deficiency is caused by a defect in the active cellular uptake of carnitine, and the gene encoding the high affinity carnitine transporter OCTN2 has recently been shown to be mutated in patients suffering from this disorder. Here, we report the underlying molecular defect in three unrelated patients. Two patients were homozygous for the same missense mutation 632A-->G, which changes the tyrosine at amino acid position 211 into a cysteine (Y211C). The third patient was homozygous for a nonsense mutation, 844C-->T, which converts the arginine at amino acid position 282 into a stop codon (R282X). Reintroduction of wild-type OCTN2 cDNA into fibroblasts of the three patients by transient transfection restored the cellular carnitine uptake, confirming that mutations in OCTN2 are the cause of systemic carnitine deficiency.

Myopathy in very-long-chain acyl-CoA dehydrogenase deficiency: clinical and biochemical differences with the fatal cardiac phenotype.

A 30-year-old man suffered since the age of 13 years from exercise induced episodes of intense generalised muscle pain, weakness and myoglobinuria. Fasting ketogenesis was low, while blood glucose remained normal. Muscle mitochondria failed to oxidise palmitoylcarnitine. Palmitoyl-CoA dehydrogenase was deficient in muscle and fibroblasts, consistent with deficiency of very-long-chain acyl-CoA dehydrogenase (VLCAD). The gene of this enzyme had a homozygous deletion of three base pairs in exon 9, skipping lysine residue 238. Fibroblasts oxidised myristate, palmitate and oleate at a rate of 129, 62 and 38% of controls. In contrast to patients with cardiac VLCAD deficiency, our patient had no lipid storage, a normal heart function, a higher rate of oleate oxidation in fibroblasts and normal free carnitine in plasma and fibroblasts. 31P-nuclear magnetic resonance spectroscopy of muscle showed a normal oxidative phosphorylation as assessed by phosphocreatine recovery, but a significant increase in pH and in Pi/ATP ratio.

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.

Infantile fibre type disproportion, myofibrillar lysis and cardiomyopathy: a disorder in three unrelated Dutch families.

An apparently new cardioskeletal myopathy is reported in three unrelated families. Five infants were affected by rapidly progressive generalized muscle weakness, with onset shortly after birth, and dilated cardiomyopathy. All had generalized tremor (clonus) starting in the first week of life. The disease was lethal in all cases between 4 and 6 months. Muscle biopsy, performed in four of the five patients, showed a light microscopic pattern of small type I and normal-sized type II fibres. By electron microscopy small fibres were affected by myofibrillar disruption and swelling of organelles. Findings in blood and urine suggested a disturbance in energy metabolism but an extensive search for respiratory chain disorders and disorders of mitochondrial fatty acid oxidation in frozen muscle and cultured fibroblasts was negative. The findings support a new progressive autosomal recessive infantile cardioskeletal myopathy in which type I muscle fibres are preferentially affected.

Very long chain acyl-coenzyme A dehydrogenase deficiency with adult onset.

Very long chain acyl-coenzyme A (acyl-CoA) dehydrogenase (VLCAD) deficiency is a severe disorder of mitochondrial beta-oxidation in infants. We report adult onset of attacks of painful rhabdomyolysis. Gas chromatography identified strongly elevated levels of tetradecenoic acid, 14:1(n-9), tetradecadienoic acid, 14:2(n-6), and hexadecadienoic acid, 16:2(n-6). Palmitoyl-CoA and behenoyl-CoA dehydrogenase in fibroblasts were deficient. Muscle VLCAD activity was very low. DNA analysis revealed compound heterozygosity for two missense mutations in the VLCAD gene. The relatively mild clinical course may be due to residual enzyme activity as a consequence of the two missense mutations. Treatment with L-carnitine and medium chain triglycerides in the diet did not reduce the attacks of rhabdomyolysis.