Identification of new mutations in the ETHE1 gene in a cohort of 14 patients presenting with ethylmalonic encephalopathy.

BACKGROUND: Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life. METHODS: 14 patients with EE were investigated for mutations in the ETHE1 gene. RESULTS: Of the 14 patients, 5 were found to carry novel mutations. CONCLUSIONS: This work expands our knowledge of the causative mutations of EE.

Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes.

Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.

Hepatic ketogenesis and muscle carnitine deficiency.

The levels of plasma free carnitine and ketone bodies have been found to fluctuate inversely in fasting individuals without muscle disease. Circulating short-chain acyl-carnitines paralleled beta-hydroxybutyrate levels. A patient with lipid storage myopathy and muscle carnitine deficiency, and his two daughters, developed exaggerated ketogenesis on fasting. The content of total carnitines in the patient's liver was normal, but free carnitine was reduced to 50 percent, and total esterified carnitines were four times greater than the mean value for the controls. The decreased muscle carnitine content in this case may have resulted from chronic hepatic ketogenesis, draining muscle carnitine. Alternatively, decreased muscle carnitine content may have initiated hepatic ketogenesis.

Fatal ataxic encephalopathy and carnitine acetyltransferase deficiency: a functional defect of pyruvate oxidation?

A 3-year 8-month-old girl died after 14 months of illness characterized by episodes of intermittent ataxia associated with oculomotor palsy, hypotonia, mental confusion, and disturbances of consciousness. In the last 4 months of life, there were signs of liver dysfunction. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase activities were normal in autopsy brain specimens and in cultured fibroblasts from the patient. Carnitine acetyltransferase was deficient in liver, brain, kidney, and cultured fibroblasts. Medium- and long-chain carnitine acyltransferase activities were normal. It is proposed that a functional defect of acetyl-coenzyme A (acetyl-CoA) utilization in brain mitochondria accompanies the carnitine acetyltransferase deficiency.

Systemic carnitine deficiency due to lack of electron transfer flavoprotein:ubiquinone oxidoreductase.

A child with myopathy and systemic carnitine deficiency died at age 8 years in an acute metabolic attack. He had glutaric aciduria type II, and his cultured fibroblasts contained normal activity of four different acyl CoA dehydrogenases, but there was deficiency of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF-QO). This enzyme is thought to reduce coenzyme Q in the respiratory chain, funneling reducing equivalents from seven flavoproteins in the beta-oxidation of acyl CoAs. There was massive urinary excretion of the short-chain acylcarnitines that accumulated in mitochondria as a result of the ETF-QO defect. Carnitine therefore acts as a buffer for excessive accumulation of intramitochondrial acyl CoAs, and defective beta-oxidation can cause carnitine insufficiency.

Fumarase deficiency is an autosomal recessive encephalopathy affecting both the mitochondrial and the cytosolic enzymes.

A 7-month-old boy died in a demented state after a clinical history characterized by generalized seizures, psychomotor deterioration, and fumaric aciduria. We found a marked deficiency of both mitochondrial and cytosolic fumarases in skeletal muscle, brain, cerebellum, heart, kidney, liver, and cultured fibroblasts. Fumarase activities were 30 to 50% compared with controls in both mitochondria and cytosol from cultured fibroblasts of the parents. Antifumarase cross-reacting material was present in negligible amounts in the patient's tissues. Our data indicate that this disease is an autosomal recessive encephalopathy, due to a single mutation affecting the gene encoding both forms of the enzyme.

Late-onset riboflavin-responsive myopathy with combined multiple acyl coenzyme A dehydrogenase and respiratory chain deficiency.

We studied the effect of riboflavin treatment on the clinical status and on the activities of beta-oxidation and respiratory chain enzymes in a 69-year-old patient with late-onset myopathy. Before treatment, she was very weak and wasted in the limbs and trunk muscles; also, she could not walk or attend to daily activities. Marked lipid storage was present in the muscle biopsy. The activities of short-chain acyl coenzyme A (acyl-CoA) dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) in isolated muscle mitochondria were reduced to less than 10% of control values. This defect in fatty acid oxidation was associated with a marked deficiency of two flavin-dependent respiratory chain complexes: complex I activity was 20% and complex II activity was 25% of control values. By contrast, the activities of the nonflavin-dependent complex III and complex IV were normal. Western blot analysis of the patient's muscle mitochondrial extracts with antibodies raised against purified SCAD, MCAD, and the alpha- and beta-subunits of the electron transfer flavoprotein (ETF) showed absence of SCAD cross-reacting material (CRM), markedly decreased MCAD-CRM, and normal amounts of both alpha- and beta-ETF-CRM. After riboflavin treatment, the patient's clinical status dramatically improved and morphologic changes in muscle disappeared. SCAD activity increased to 55% of control values, whereas MCAD, LCAD, and complex I and complex II activities normalized. SCAD and MCAD immunoreactivity was restored to normal. On the basis of our experience and the data in the literature, we concluded that some lipid storage myopathies can show dramatic response to riboflavin.

Respiratory chain and mitochondrial DNA in muscle and brain in Parkinson's disease patients.

There are several reports of a defect of complex I in the substantia nigra (SN) of Parkinson's disease (PD) patients. To evaluate whether this is specific to dopaminergic neurons or the phenotypically relevant consequence of a widespread failure of the mitochondrial oxidative phosphorylation (OXPHOS) system, we measured respiratory enzyme activities in muscle homogenates from 16 PD patients and eight age-matched controls, and in muscle isolated mitochondria of six PD patients and six age-matched controls. We found no difference between the PD and control groups. In addition, we detected, by polymerase chain reaction, the mitochondrial DNA (mtDNA) "common deletion" (CD) in muscle specimens of 14 of 17 PD patients, but we obtained similar results in age-matched controls. In both groups, the amount of CD-specific deleted (delta) mtDNA ranged from 0.0% to 0.1%. Our data suggest that PD cannot be attributed to a multisystem decline of mitochondrial OXPHOS, and that lesions of muscle mtDNA in PD are likely due to normal aging. However, there was a remarkable accumulation of delta mtDNA in the SN of a PD patient and an age-matched control, suggesting that the SN is exquisitely sensitive to age-dependent damage of the mitochondrial genome.

Systemic carnitine deficiency: clinical, biochemical, and morphological cure with L-carnitine.

A 20-year-old woman had systemic carnitine deficiency. Biochemical studies of cultured fibroblasts, skeletal muscle mitochondria, and fluids showed no evidence of other disease that might deplete tissue carnitine stores. Carnitine supplementation produced a dramatic improvement in her clinical condition: she gained weight and strength and recovered brain function, which had deteriorated slightly after repeated episodes of encephalopathy. Lipid droplets disappeared from skeletal muscle and plasma, and muscle carnitine content rose from low to normal values. On treatment, she excreted less carnitine than controls. This form of systemic carnitine deficiency may be due to defective carnitine biosynthesis.

Very-long-chain acyl-coenzyme A dehydrogenase deficiency in a child with recurrent myoglobinuria.

A 9-year-old boy had recurrent episodes of myoglobinuria and normal urinary organic acid profile. Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency was detected biochemically in cultured skin fibroblasts and confirmed by Western blot analysis. The patient had a distinctive plasma fatty-acid profile, which was present even between attacks. Early diagnosis of this disorder is important because of the apparently protective effect of an appropriate dietary regimen.

Familial cardiomyopathy, mental retardation and myopathy associated with desmin-type intermediate filaments.

The clinical and morphological findings of a familial case affected by mental retardation, severe biventricular hypertrophic cardiomyopathy and vacuolar myopathy are reported. The phenotype of this patient is similar to that described by other authors, in which a lysosomal glycogen storage disease with normal acid maltase levels was suspected. However, in our case the vacuoles were stained by several antibodies directed against various sarcolemmal proteins, such as dystrophin and spectrin, and therefore, were not of lysosomal origin. Some of these vacuoles were clearly derived from the splitting of the fibres and invagination of the extracellular space; autophagic vacuoles were not observed. The accumulation of desmin-type, intermediate filaments was demonstrated on immunocytochemistry both in the skeletal and cardiac muscles. A brother of the propositus was also affected by mental retardation, severe cardiomyopathy and died suddenly at the age of 24 yr. A cardiomyopathy and mental subnormality were also present in other male cousins of the proband, while sudden death occurred in several females relatives, whose intelligence was normal. None of these latter individuals was available for further investigation. This report expands the spectrum of desmin associated myopathy and cardiomyopathy to include a familial condition with associated mental retardation.

Lysosomal glycogen storage with normal acid maltase: a familial study with successful heart transplant.

Lysosomal glycogen storage in muscle with normal acid maltase activity is a rare inherited condition characterized by cardiomyopathy, mental retardation and mild myopathy in males, but generally only cardiomyopathy in females. Three cases (index case, his sister and her son) are described in a family with at least two other affected members. The index case underwent a successful heart transplant. The sister has cardiac involvement, myopathic changes and mental impairment--to our knowledge the first report of multisystem involvement in a female. We propose that skeletal muscle should be examined in young patients with hypertrophic cardiomyopathy. Furthermore, female relatives of males with the disease should be investigated for cardiomyopathy; they would be excellent candidates for life-saving heart transplant, since myopathy and mental retardation, if clinically evident, are mild.

Parafascicular thalamic nucleus deafferentation reduces c-fos expression induced by dopamine D-1 receptor stimulation in rat striatum.

We investigated the role played by the parafascicular thalamostriatal pathway, one of the major excitatory inputs to the striatum, in the D-1 receptor induction of c-fos messenger RNA expression in the rat striatum. The full D-1 receptor agonist, SKF-82958 (0.05, 0.1, 0.5 and 1 mg/kg, s.c., 30 min), dose-dependently induced c-fos messenger RNA in naive rat striatum as determined by northern blot analysis. One day following electrolytic lesion of the parafascicular thalamostriatal nucleus, striatal c-fos signal by itself was not altered but the stimulated expression of c-fos was strongly decreased. Sections of sham-operated and acute-lesioned dorsal striata of vehicle- or SKF-82958-treated rats were processed for in situ hybridization histochemistry at the single-cell level with an RNA probe for c-fos. The basal expression of striatal c-fos was poorly detectable in sham and lesioned groups. Sections of dorsal striata from sham-operated rats treated with SKF-82958 showed two types of labeled neurons for c-fos: the lightly and the very densely labeled neurons which are few in number. Thalamic lesion strongly reduced SKF-82958 stimulated expression of c-fos RNA in both types of labeled cells. The frequency distribution of c-fos labeling per neuron in dorsal striata of lesioned rats treated with SKF-82958 was shifted to the left, and its median was lower than in the sham-operated striata treated with the D-1 receptor agonist. The results provide evidence that the excitatory projections from the parafascicular nucleus of the thalamus, thought to operate primarily through the N-methyl-D-aspartate receptors, exert a facilitatory control over D-1 receptor-induced c-fos gene expression specifically in the dorsal striatum.

The parafascicular thalamic nucleus modulates messenger RNA encoding glutamate decarboxylase 67 in rat striatum.

We investigated whether the parafascicular thalamostriatal pathway, one of the major excitatory inputs to the striatum, regulates the expression in rat striatum of messenger RNA encoding two isoforms of glutamate decarboxylase (mol. wt 67,000: glutamate decarboxylase 67 and mol. wt 65,000: glutamate decarboxylase 65). Acute (one day) and chronic (14 days) electrolytic lesions of the parafascicular nucleus resulted in 58% and 23% decreases in glutamate decarboxylase 67 messenger RNA expression, respectively, as determined by northern blot analysis. Glutamate decarboxylase 65 messenger RNA was not modified by either lesion. Sections of sham- and acute-lesioned striata were processed for in situ hybridization histochemistry at the single cell level with an RNA probe for glutamate decarboxylase 67. Labelling of glutamate decarboxylase 67 messenger RNA was decreased in both types of cells known to be present in the striatum, i.e. the lightly and the very densely-labelled neurons. The frequency distribution of glutamate decarboxylase 67 labelling per neuron in the lesioned striata, in fact, was shifted to the left and its median was lower than in the sham-lesioned striata. In view of the excitatory nature of the thalamostriatal pathway, we examined the subtype of glutamate receptors modulating the glutamate decarboxylase 67 gene expression. The N-methyl-D-aspartate-type receptor antagonist, dizocilpine, at 0.1-0.5 mg/kg i.p., produced a marked and persistent reduction in striatal glutamate decarboxylase 67 messenger RNA. The non-N-methyl-D-aspartate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (12 nmol/side, i.c.v.) had no such effect. The results provide evidence that excitatory thalamostriatal afferents selectively modulate the gene expression of glutamate decarboxylase 67, probably through the N-methyl-D-aspartate subtype of glutamate receptors.

Increased striatal expression of glutamate decarboxylase 67 after priming of 6-hydroxydopamine-lesioned rats.

Previous single exposure (priming) to a dopamine receptor agonist greatly enhances the contralateral turning behaviour elicited by dopamine D1 receptor agonists in unilaterally 6-hydroxydopamine lesioned rats. In the present study we have investigated the levels of glutamate decarboxylase 67 and glutamate decarboxylase 65 messenger RNA in the striatum of 6-hydroxydopamine-lesioned rats primed with L-3,4-dihydroxyphenylalanine (L-DOPA) and challenged with the D1 receptor agonist SKF 38393, three days thereafter. As previously reported, levels of glutamate decarboxylase 67 messenger RNA increased in the striatum denervated by the 6-hydroxydopamine lesion as compared with the intact one. Striatal glutamate decarboxylase 67 messenger RNA levels, measured three days after priming with L-DOPA (50 mg/kg), further increased in the lesioned striatum while were not modified in the intact one. Administration of SKF 38393 (3 mg/kg) elicited a more intense contralateral turning behaviour in primed than in drug-naive 6-hydroxydopamine-lesioned rats but did not induce any change in striatal glutamate decarboxylase 67 messenger RNA. In contrast, striatal levels of glutamate decarboxylase 65 messenger RNA were not modified by either 6-hydroxydopamine lesions or priming with L-DOPA. The results show that priming with L-DOPA induces long-lasting changes in GABAergic neurons of the 6-hydroxydopamine-lesioned striatum. These changes might play a role in the increased behavioural response of striatal D1 receptors induced by priming.

Peripheral nervous system damage in experimental chronic Chagas' disease.

Electromyographic and histopathologic studies were performed in Rockland mice chronically infected with CA-I Trypanosoma cruzi strain. At 4 months post-infection the emg failed to show spontaneous activity, but a diminished interference pattern was detected in half of the infected group, while mean motor unit potential amplitude and duration were increased, compared with controls. An active denervation was observed at 6 months which persisted up to 9 months, when motor unit potential showed a significantly lower mean activity and duration. At 12 months most of the infected mice developed a reduced interference pattern, polyphasic motor unit potential increase with higher duration and amplitude than controls. Histopathologic studies showed myositis with perivascular involvement as well as intramuscular neuritis, especially at 4 and 12 months. Atrophic and hypertrophic fibers were seen. Few amastigote nests were detected. Inflammatory neuropathy with the demyelinated fibers and scanty axonal degeneration were the most common features in all infected mice. Mild myelinated fiber loss was only evident after 12 months. Endoneural parasites were seen only in the perineural macrophagic cells. These findings suggest that the neurogenic mechanism involved in the pathogenesis of muscle damage in this experimental model of chronic Chagas' disease consistently has been overlooked. The features registered here suggest that T. cruzi-infected mice developed a bimodal muscle denervation with an early acute period at any time before month 4, followed by reinnervation with a subsequent new acute denervation period by month 6, followed in turn by a slow later reinnervation.

Debrancher deficiency neuromuscular disorder with pseudohypertrophy in two brothers.

A neuromuscular disorder is reported in two brothers, aged 28 and 38 years, with glycogenosis type III. Both patients had proximal weakness, pseudohypertrophy of sternocleidomastoid, trapezius and quadriceps muscles, mild distal wasting and myopathic EMG changes. Pseudohypertrophy was more evident in the younger brother, whereas weakness was prominent in the older one. In the former, muscle biopsy revealed vacuolar myopathy and virtual absence of amylo-1,6-glucosidase enzyme. Few familial cases of debrancher deficiency neuromuscular disorder have been reported. Distal wasting has been considered a quite characteristic manifestation of the disease. It is also suggested that this particular kind of pseudohypertrophy may represent a distinctive feature of glycogenosis type III.

Recessive carnitine palmityl transferase deficiency: biochemical studies in tissue cultures and platelets.

In a new case of carnitine palmityl transferase (CPT) deficiency the defect was documented in muscle and muscle cultures with an isotope exchange reaction, using different concentrations of palmityl-DL-carnitine and a forward reaction with and without albumin. The defect was expressed in cultured skin fibroblasts only by the "reverse" and "hydroxamate" reactions. The parents and the patient's daughter had intermediate levels of the enzyme in platelets and fibroblasts, supporting the concept that CPT deficiency has an autosomal recessive pattern of inheritance. The growth pattern and development of muscle cultures in this CPT-deficient patient indicate that CPT activity may be sufficient to allow normal muscle differentiation in culture without lipid storage.

Genotype to phenotype correlations in mitochondrial encephalomyopathies associated with the A3243G mutation of mitochondrial DNA.

We studied 22 subjects carrying the A3243G point mutation of human mitochondrial DNA (mtDNA). In 14 cases the clinical phenotype was characterized by mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), while 8 patients had chronic progressive external ophthalmoplegia (CPEO). The proportion of A3243G heteroplasmy in muscle was determined by two methods; densitometry on a diagnostic restriction-fragment length polymorphism and solid-phase mini-sequencing. We found a highly significant inverse correlation between the percentage of A3243G mutation and the specific activity of complex I, the respiratory complex with the highest number of mtDNA-encoded subunits, suggesting a direct effect of the mutation on mtDNA translation. No correlation was observed between the percentage of mutated mtDNA and the presence or absence of specific clinical features, such as stroke, ophthalmoplegia and diabetes mellitus. However, in the MELAS group the percentage of mutated mtDNA molecules was strongly correlated with the age of onset, while no such correlation was found in the CPEO group, suggesting a different time-dependent evolution of the mutation in the two groups. Finally, in contrast with other mtDNA mutations associated with ragged-red fibres (RRF), in both MELAS3243 and CPEO3243 we observed a high proportion of RRF that were positive to the histochemical reaction to cytochrome c oxidase, a morphological feature that seems to be specific for the neuromuscular phenotypes associated with mutations affecting the tRNA(Leu(UUR)) gene.

The beta-oxidation of arachidonic acid and the synthesis of docosahexaenoic acid are selectively and consistently altered in skin fibroblasts from three Zellweger patients versus X-adrenoleukodystrophy, Alzheimer and control subjects.

The beta-oxidation of [3H] arachidonic acid (AA; 20:4 n-6) and the conversion of [1-14C]eicosapentaenoic acid (EPA, 20:5 n-3) to docosahexaenoic acid (DHA, 22:6 n-3) have been studied in skin fibroblasts from patients with inherited peroxisomal diseases, such as Zellweger (ZW) and X-linked adrenoleukodystrophy (X-ALD), from patients with Alzheimer's disease (AD), a non-inherited neuropathology, and from controls. EPA is not converted to DHA, while there is enhanced formation of the intermediate product 22:5 n-3 in ZW, when compared to X-ALD, AD and controls. We also confirmed that AA is not beta-oxidized to 4,7,10-hexadecatrienoic acid (16:3), a metabolite produced by peroxisomes, while being more effectively converted to the elongation product 22:4, in ZW, in comparison to X-ALD, AD and controls. The data demonstrate a defect in DHA synthesis and in AA beta-oxidation, and the occurrence of associated adaptative modifications in the metabolism of these long chain PUFA, in three Italian ZW patients.