Effect of various agents on adenosine triphosphate synthesis in mitochondrial complex I deficiency.

The effect of agents commonly used in the therapy of mitochondrial complex I deficiency was examined in fibroblasts from a patient. Marked improvement was observed with riboflavin, which nearly normalized the adenosine triphosphate production. The study of adenosine triphosphate production rate in fibroblasts may improve decision-making in treatment design of patients with respiratory chain defects.

The hepatic mitochondrial DNA depletion syndrome: ultrastructural changes in liver biopsies.

Mitochondrial respiratory chain disorders are an established cause of liver failure in early childhood. In some patients, the levels of mitochondrial DNA are markedly reduced, a condition referred to as mtDNA depletion syndrome (MDS). We report here on the ultrastructural changes in the livers of 10 infants with the hepatic form of this syndrome. All patients displayed progressive liver failure, neurological abnormalities, hypoglycemia, and lactic acidosis that warranted investigation of respiratory chain disorder in liver tissue, specifically expressing the disease. Decreased activity of respiratory chain complexes containing mtDNA-encoded subunits (complexes I, III, IV) was shown in 5 patients. Mitochondrial DNA depletion was confirmed by Southern blot analysis in the livers of 6 patients. We found hepatocytes filled with mitochondria having aspects of "oncocytic transformation," associated with numerous changes in shape, size, cristae, and matrix. The changes were virtually identical in all specimens. In many hepatocytes, microvesicular steatosis was the salient feature. Additional findings included cholestasis and focal cytoplasmic biliary necrosis (CBN), as well as cytosiderosis in hepatocytes and sinusoidal cells. In some hepatocytes the damage appeared extreme, but fibrosis was identified only in the few patients who died beyond 6 months of age. Although individual ultrastructural findings are not specific, when taken together, they show a diagnostic pattern highly suggestive of a respiratory chain disorder. In the appropriate clinical context, these findings can direct the clinician towards the diagnosis of hepatic MDS.

Antenatal presentation of carnitine palmitoyltransferase II deficiency.

Carnitine palmitoyl transferase (CPT) II deficiency is usually manifested around puberty by exercise induced myoglobinuria. Two Ashkenazi Jewish sibs with the rare antenatal form of CPTII deficiency are reported. On the 5th gestational month periventricular calcifications and markedly enlarged kidneys were found in both of them. The activity of CPTII in lymphocytes was undetectable and both sibs were homozygous for the 1237delAG mutation. Because of the serious consequences of homozygosity for this mutation, genotype determination of all Ashkenazi patients with the adolescent form of CPTII deficiency is warranted.

Mutation analysis of the MCM gene in Israeli patients with mut(0) disease.

Three novel mutations (IVS8+3a --> g, N219Y, and E414X) were identified in 6 unrelated patients with mut(0) methylmalonic aciduria. The presence of a wild-type along with rearranged fragments in homozygotes for the IVS8+3a --> g mutation may contribute to their later age of onset (3-11 months of age). Nonetheless, delayed onset was not associated with better neurological outcome and prolonged survival. The large number of undiagnosed dead sibs in most families suggests that the disease is largely underdiagnosed in this region.

Muscular carnitine palmitoyltransferase II deficiency in infancy.

An 8-month-old female presented with febrile myoglobinuria. The activity of carnitine palmitoyltransferase (CPT) II was decreased to 16% of the control mean, and the oxidation of the long-chain fatty acids was reduced to 25% of the mean in the fibroblasts. Homozygosity for the common mutation, S113L, was identified in the CPT II gene. Residual CPT II activity of more than 10% of the mean and homozygosity for the common mutation S113L are usually associated with a milder reduction of long-chain fatty acid oxidation to about 80% of the control and with a later age of clinical onset. The early clinical presentation in the present patient is unique and was associated with a marked impairment of long-chain fatty acid oxidation, possibly because of other genetic factors. CPT II deficiency should be included in the differential diagnosis of isolated myoglobinuria in infancy.

ATP synthesis in lipoamide dehydrogenase deficiency.

Lipoamide dehydrogenase deficiency is an inborn error of several metabolic pathways, including pyruvate metabolism, Krebs cycle, and branched-chain amino acid degradation. The clinical course is variable, ranging from infantile neurodegenerative disease to recurrent episodes of liver failure or myoglobinuria starting later in life. In contrast, residual enzymatic activity in muscle tissue spans over a narrow range. Despite the recent elucidation of the underlying molecular pathology in most patients, relationships between the genotype and the biochemical and clinical phenotype remain unclear. In order to find a suitable assay for the prediction of clinical outcome and assessment of treatment, we have evaluated enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients representing three different phenotypes and genotypes. Direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics, including lipoamide dehydrogenase activity, pyruvate dehydrogenase complex activity, and ATP production ratio in fibroblasts, were identified. Clinical parameters were not reflected by lactate/pyruvate ratio. ATP production rate was in direct relationship with the severity of the neurological involvement; the patient with reduced ATP synthesis to 30% of the control mean had a severe neurodegenerative disease, whereas ATP synthesis values above 45% were associated with a more favorable course. Incubation of the patients' fibroblasts with dichloroacetate coupled with thiamin resulted in slight but significant improvement of the cell energetic state.

Lipoamide dehydrogenase deficiency due to a novel mutation in the interface domain.

An infant with a neurodegenerative disorder accompanied by lactic acidemia is described. In muscle homogenate, the activity of lipoamide dehydrogenase (LAD), the third catalytic subunit of pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase complex (KGDHc), and branched-chain keto acid dehydrogenase complex was reduced to 15% of the control. The activity of PDHc was undetectable and the activity of KGDHc was 2% of the control mean. The immunoreactive LAD protein was reduced to about 10% of the control. Direct sequencing of LAD cDNA revealed only one mutation, substituting Asp for Val at position 479 of the precursor form. The mutation resides within the interface domain and likely perturbs stable dimerization. The phenotypic heterogeneity in LAD deficiency is not directly correlated with the residual LAD activity but rather with its impact on the multienzymatic complex activity.

Cone and rod dysfunction in the NARP syndrome.

AIMS: Description of the ophthalmic manifestations of the NARP (neuropathy, ataxia, retinitis pigmentosa) syndrome that is associated with a point mutation in position 8993 of the mitochondrial DNA (mtDNA). METHODS: A mother and her two children, all carrying the 8993 mtDNA mutation, were examined. Two had manifestations of the NARP syndrome. A complete ocular and systemic examination was performed on all three patients. RESULTS: The clinical examination, electroretinogram, and visual fields revealed a typical cone-rod dystrophy in the son, and a typical cone dystrophy in the daughter. The mother had no ocular manifestations of the disease. CONCLUSIONS: NARP is a recently described, maternally inherited mitochondrial syndrome in which a retinal dystrophy, among other abnormalities, is related to a mutation of the mtDNA at nucleotide 8993. This study demonstrates the great variability of the ocular manifestations in the NARP syndrome. It also indicates that the retinal dystrophy in at least some NARP patients affects primarily the cones.

The spectrum of mutations of the aspartoacylase gene in Canavan disease in non-Jewish patients.

Canavan disease is an infantile neurodegenerative disease that is caused by mutations in the gene encoding the enzyme aspartoacylase. It has mainly been reported in Jewish families. Genotyping of newly diagnosed patients is essential for the carrier identification and prenatal diagnosis. The sequence of the coding region was determined in 15 non-Jewish patients and 9 new mutations were identified: Y109X, P183H, V186F, M195R, P280L, P280S, A287T, 245insA, and a tentative missplicing mutation which leads to skipping of exon 5. The common pan-European mutation, A305E, was identified in 40% of the alleles and the overall detection rate was 93%.

Molecular basis of lipoamide dehydrogenase deficiency in Ashkenazi Jews.

We studied 13 patients with lipoamide dehydrogenase (LAD) deficiency, originating from seven Ashkenazi Jewish families. Their disease was characterized by recurrent attacks of vomiting, abdominal pain, and encephalopathy accompanied by elevated liver transaminases, prolonged prothrombin time, and occasionally associated with lactic and ketoacidemia or with myoglobinuria. Two patients who presented neonatally suffered from residual neurological damage with attention deficit hyperactive disorder, mild ataxia, motor incoordination, muscle hypotonia, and weakness. Nine patients who presented in early childhood or later suffered from exertional fatigue between decompensation episodes but were otherwise asymptomatic. Two patients died because of intractable metabolic acidosis and multi-organ failure. In all patients LAD activity was reduced to 8 to 21% of the control in muscle or lymphocytes. In four patients LAD protein in muscle was reduced to 20 to 60% of the control. Direct sequencing of the cDNA of the LAD gene showed that 12 of the 14 mutated alleles carried the G229C mutation and two carried an insertion mutation 105insA (Y35X). The patients who presented neonatally and had more severe sequelae were compound heterozygotes for the two mutations; patients who presented in early childhood or later were homozygous for the G229C mutation. Using an allele-specific oligonucleotide hybridization technique, nine heterozygotes for the G229C mutation were identified among 845 anonymous individuals of Ashkenazi Jewish origin disclosing a carrier rate of 1:94. Because of the significant morbidity associated with the disease, screening for the G229C mutation among Ashkenazi Jewish couples should be considered.

The molecular background of glycogen metabolism disorders.

The molecular pathology of classical glycogen storage disorders, glycogen synthase deficiency and Fanconi-Bickel syndrome is reviewed. The isolation of the respective cDNAs, the chromosomal localization of the genes and the elucidation of the genomic organization enabled mutation analysis in most disorders. The findings have shed light on the multi-protein structure of the glucose-6-phosphatase system, the phosphorylase kinase enzymatic complex and the molecular background of the differential tissue expression in debranching enzyme deficiency. The immediate practical benefit of these studies is our extending ability to predict the outcome of clinical variants and to offer genetic counseling to most families. The elucidation of the tertiary structure of these proteins and their structure-function relationship poses major challenges for the future.

Glutaryl-CoA dehydrogenase mutations in glutaric acidemia (type I): review and report of thirty novel mutations.

Glutaric acidemia type I (GA1) is caused by mutations in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCD). Sixty-three pathogenic mutations identified by several laboratories are presented, 30 of them for the first time, together with data on expression in Escherichia coli and relationship to the clinical and biochemical phenotype. In brief, many GCD mutations cause GA1, but none is common. There is little if any relationship between genotype and clinical phenotype, but some mutations, even when heterozygous, seem especially common in patients with normal or only minimally elevated urine glutaric acid.

Molecular analysis and prenatal diagnosis of human fumarase deficiency.

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle causing severe neurological impairment. The cDNA for both the rat and human enzymes has been cloned previously and shown to encode a coding region of 1.46 kb. To scan for mutations in fumarase-deficient patients we amplified the coding region of fumarase from fibroblast/lymphoblast cDNA employing the oligonucleotide primers designed from the published human and rat cDNA sequence. We then directly sequenced the polymerase chain reaction product. In seven unrelated patients, we detected four missense mutations (A265T, D383V, F269C, K187R), a nonsense mutation (W458X), a 3-bp AAA insertion that introduces an additional lysine residue at codon 435, and a spontaneous new mutation resulting in a 74-bp deletion (66del74). Seven at-risk pregnancies were monitored with one prenatal diagnosis of fumarase deficiency by molecular analysis and favorable outcome of the other pregnancies as predicted by enzyme assay of cultured fetal cells or molecular analysis.

Ineffectiveness of oral coenzyme Q10 supplementation in 3-methylglutaconic aciduria, type 3.

Coenzyme Q10 was administered under placebo controlled blinded crossover conditions to six subjects suffering from type 3 3-methylglutaconic aciduria ('optic atrophy plus'), following a report of benefit. Despite attainment of high plasma levels of coenzyme Q10, no clinical benefit was observed and there was no diminution of urinary excretion of 3-methylglutaconic acid.

Anaesthetic management of a patient with Leigh's syndrome.

PURPOSE: Leigh's syndrome, a progressive neurodegenerative disorder of infancy and childhood, is clinically characterized mainly by developmental delay, nervous system dysfunction and respiratory abnormalities such as aspiration, wheezing, breathing difficulties, gasping, hypoventilation and apnoea. Acute exacerbation and respiratory failure may follow surgery, general anaesthesia or intercurrent illnesses. Hyperlecithinemia is variably present. Histopathological findings include necrosis, vascular proliferation, astrocytosis and demyelination of several brain areas. We present a 30-month-old patient with Leigh's syndrome anaesthetized for extracorporeal shockwave lithotripsy, and describe the anaesthetic considerations. CLINICAL FEATURES: Leigh's syndrome was diagnosed at five months of age based on failure to thrive, lethargy, hypotonicity, choreo-athetosis and lactic acidaemia, with basal ganglia hypodense areas demonstrated by brain computerized tomographic scan. Muscle pyruvate dehydrogenase complex and NADH coenzyme Q oxidoreductase activity were 25% and 13% of control. No preoperative respiratory symptoms or signs were present. Preoperative fasting lasted two hours and gastric aspiration was negative. Anaesthesia was induced with ketamine and midazolam im, and N2O in oxygen, and maintained with propotol and N2O. No volatile anaesthetics were used. Intravenous fluids given were 1/2 normal saline and glucose 5% administered. Besides laryngospasm during anaesthetic induction, relieved by sublingual succinylcholine injection, the perianaesthetic course was uneventful. The lungs were mechanically ventilated and lithotripsy was performed. No adverse sequelae have occurred, and the patient was discharged one day later. CONCLUSION: Perioperative management of patients with Leigh's syndrome requires cautious attention to the metabolic, neurological and respiratory aspects of the disease, and appropriate selection of anaesthetic drugs.

Mitochondrial encephalomyopathy associated with a novel mutation in the mitochondrial tRNA(leu)(UUR) gene (A3243T).

We report a new mutation, an A-->T transition at nt 3243 in the mitochondrial tRNA(leu)(UUR) gene, in a 9-year-old girl who presented with muscle weakness of 3 years duration complicated by rapidly progressive encephalopathy. In muscle, the activity of the mitochondrial respiratory chain complexes I, III, and IV was markedly reduced. The mutation, involving a highly conserved base pair in the dihydrouridine loop, was heteroplasmic in muscle (81.4%), skin (69.3%), and blood (13.8%) and was not present in blood of 50 healthy individuals. The mitochondrial 3243 base is a "hot spot" for mutations; an A-->G transition at this position is found in a high proportion in most MELAS patients. Since the A-->T transition creates a new recognition site for the restriction enzyme TspRI, both ApaI and TspRI should be used to exclude a mutation at nt 3243.