Vitamin C and Charcot-Marie-Tooth 1A: Pharmacokinetic considerations.

Charcot-Marie-Tooth 1A disease (CMT1A) is a disease for which no drug treatments are available. In 2004, it was reported that ascorbic acid reduced the severity of neuropathy in transgenic mice overexpressing PMP22, an animal model of human CMT1A, compared with untreated mice. Based on those results, clinical trials were undertaken at different centers worldwide and four of them have been completed, but none of them resulted in significant improvements. Based on the pharmacokinetics of ascorbic acid, we propose that the randomized clinical trial carried out thus far confirmed the tight control of ascorbic acid's absorption and proved its tolerability at one and two years. The pharmacokinetic considerations discussed in this article might largely explain the disappointing results of the recent CMT1A trials.

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.

Ataxia with isolated vitamin E deficiency: neurological phenotype, clinical follow-up and novel mutations in TTPA gene in Italian families.

Ataxia with vitamin E deficiency (AVED) is a rare autosomal recessive neurodegenerative disorder due to mutations in the alpha-tocopherol transfer protein (TTPA) gene on chromosome 8q13. AVED patients have progressive spinocerebellar symptoms and markedly reduced plasma levels of vitamin E. We studied neurological phenotype at diagnosis, and long-term effect of vitamin E supplementation in 16 patients from 12 Italian families. The most common mutations were the 744delA and 513insTT. Two novel TTPA mutations were identified: a severe truncating mutation (219insAT) in a homozygous patient, and a Gly246Arg missense mutation (G246R) in a compound heterozygous patient. The missense mutation was associated with a mild and slowly progressive form of the disease. Vitamin E supplementation therapy allowed a stabilization of the neurological conditions in most of the patients. However, development of spasticity and retinitis pigmentosa was noted in a few patients during therapy. Prompt genetic characterization of AVED patients may allow an effective early treatment and an adequate genetic counseling.

L-2-hydroxyglutaric aciduria and brain malignant tumors: a predisposing condition?

L-2-hydroxyglutaric aciduria is a rare metabolic encephalopathy displaying a subcortical leukoencephalopathy on MRI. Diagnosis rests on detection of an abnormal accumulation of L-2-hydroxyglutaric acid in body fluids. The authors report on four patients who developed a malignant brain tumor during the course of the disease. This association points to a possible role of L-2-hydroxyglutaric aciduria in predisposing to brain tumorigenesis.

Analysis of heat shock transcription factor for suppression of polyglutamine toxicity.

Individually over-expressed chaperones can interfere with cytotoxicity and aggregation of polyglutamine proteins in disease models. As chaperones cooperate, the analysis of suppression or reversal of polyglutamine pathology may require ways to up-regulate multiple chaperone coding genes. This condition might be achieved by exogenous expression of de-repressed forms of heat shock transcription factor 1 (HSF1), which mediates induction of several genes coding cytosolic and nuclear chaperones. Here we present the rationale behind this possible approach and the caveats, and employ a non-neuronal cell system to test whether Ataxin-1 aggregation can be modulated by de-repressed HSF1 mutants through augmented expression of chaperone coding genes. In our experiments, HSF1 mutants have induced heat shock protein 70 and Human DnaJ (HDJ)-1 to intermediate levels. Cells expressing such mutants also showed partial reduction of Ataxin-1 [31Q] aggregation. A consolidated positive outcome of these tests in cellular models would encourage experiments in transgenic mice and prospects for pharmacological modulation of HSF1 activity or delivery.

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.

A case of 3-methylglutaconic aciduria misdiagnosed as cerebral palsy.

3-Methylglutaconic aciduria is a rare hereditary metabolic disorder characterized by increased urinary excretion of 3-methylglutaconic and 3-methylglutaric acids. Four clinical forms are recognized. This study presents the case of a 5-year-old male with type IV 3-methylglutaconic aciduria, initially diagnosed as "static encephalopathy." The slow evolution and other clinical characteristics, together with cerebral magnetic resonance imaging (MRI) findings, eventually directed the diagnosis to organic aciduria that was confirmed by urine test. This study proposes that the clinical criteria for childhood cerebral palsy should be rigorously respected; neuroimaging studies, particularly MRI, should be conducted to confirm the diagnosis, especially in atypical cases.

Mutation analysis of the GCDH gene in Italian and Portuguese patients with glutaric aciduria type I.

Two novel (G390V and X439W) and five already known mutations were identified in a total of 14 GA I alleles from Italy and Portugal. The substitution X439W is a rare type of mutation, which breaks the stop codon of the GCDH gene. As described in other populations, R402W was the most common mutation. Genotype R227P/R402W was found in a patient with low glutarate excretion. Haplotype studies have also been performed.

Clinical, biochemical and neuroradiological findings in L-2-hydroxyglutaric aciduria.

L-2-Hydroxyglutaric aciduria is a rare inborn error of metabolism, marked by a large and persistent increase of L-2-hydroxyglutaric acid in urine, blood and cerebrospinal fluid (CSF). We present clinical, biochemical and neuroradiological findings of seven Italian patients aged 4-19 years presenting at different stages of the disease. The disorder was characterized by a progressive neurological syndrome with cerebellar and pyramidal signs, mental deterioration, epilepsy and subcortical leukoencephalopathy on magnetic resonance imaging (MRI). We observed a good correlation between the severity of the disease and the extent of lesions on MRI. We report the result of the first positive prenatal diagnosis.

Medium-chain triglyceride loading test in carnitine-acylcarnitine translocase deficiency: insights on treatment.

The results of a medium-chain triglyceride loading test in a patient with severe carnitine-acylcarnitine translocase deficiency clearly demonstrated impaired in vivo utilization of medium-chain triglycerides. The loading test was performed at the ages of 7 and 36 months. The diet was adjusted accordingly. The clinical course has been favourable and the child is now in very good condition at age 4 years. We conclude that the utilization of medium-chain triglycerides is only partial in carnitine-acylcarnitine translocase deficiency and cannot reasonably be considered an optimal source of energy for these patients. Careful adjustment of dietetic treatment may help to improve prognosis.

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.

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.

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.

Fulminant Leigh syndrome and sudden unexpected death in a family with the T9176C mutation of the mitochondrial ATPase 6 gene.

We report an Italian family in which the T-to-C point mutation at nucleotide 9176 of the mitochondrial adenosine triphosphate synthetase (mtATPase) 6 gene is associated with an early-onset fulminant form of Leigh syndrome and with sudden unexpected death in two siblings, respectively. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing revealed that the mutation was homoplasmic in mitochondrial DNA of the proband. The T9176C mutation changes a highly conserved leucine to a proline in subunit 6 of the mtATPase gene and is maternally inherited, but the maternal relatives are asymptomatic. This point mutation was initially described in two brothers with bilateral striatal necrosis, a milder variant of Leigh syndrome.

Acute, severe cardiomyopathy as main symptom of late-onset very long-chain acyl-coenzyme A dehydrogenase deficiency.

UNLABELLED: A 5-year-old boy with late-onset very long-chain acyl-CoA-dehydrogenase (VLCAD) deficiency presented with acute cardiomyopathy, myopathy, gross myoglobinuria and normoglycaemia. The clinical course after diagnosis was favourable. CONCLUSION: late-onset VLCAD deficiency may present as acute cardiomyopathy.

Utility of multimodal evoked potential study and electroencephalography in mitochondrial encephalomyopathy.

We performed electroencephalography (EEG) and multimodal evoked potential (EP) studies in 16 patients with various forms of mitochondrial encephalomyopathy (ME). The electrophysiological investigations revealed signs of involvement of the peripheral and central nervous system (CNS) in 14 patients, with a high incidence of visual-EP (VEP) alterations, indicative of visual pathway vulnerability in mitochondrial diseases. No specific pattern of abnormalities emerged and, in particular, clinical and laboratory findings did not correlate with each other. EP (particularly VEP and electroretinogram) investigations should be part of the diagnostic work-up of patients with mitochondrial disorders in order to better characterize the clinical picture, disclose involvement of specific sensory systems of the CNS, and assess patients with atypical clinical presentations.

X-linked adrenoleukodystrophy: first report of the Italian Study Group.

In this paper we report Italian data on X-linked adrenoleukodystrophy (ALD) collected from 1985 to 1997. This disease appears to be the most common of the peroxisomal disorders and is associated with a functional defect of the peroxisomal very long chain fatty acid (VLCFA) oxidation. In Italy 117 cases have been recognized, but many cases may be unrecognized due to the heterogeneous clinical manifestations that vary from mild to very severe forms. To control the devastating course of this disease two therapeutic approaches are under evaluation: bone marrow transplantation (BMT) and dietary treatment based on a mixture of glyceroyl trioleate (GTO) and glyceroyl trierucate (GTE). Our experience of 68 subjects submitted to dietary treatment shows that almost all patients with signs of cerebral involvement at the beginning of treatment worsened or died, patients with the milder form, adrenomyeloneuropathy (AMN), remained stable, while 4 of the 15 presymptomatic subjects developed neurological signs of the disease. In recent years a more accurate selection of patients and donors for BMT has given favourable results, but some strict criteria should be respected.

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.

Autosomal dominant limb girdle myopathy with ragged-red fibers and cardiomyopathy. A pedigree study by in vivo 31P-MR spectroscopy indicating a multisystem mitochondrial defect.

We describe a late-onset autosomal dominant limb girdle myopathy, associated with dilated cardiomyopathy and mental deterioration. In two affected members of the pedigree with histochemical (ragged-red and cytocrome c oxidase - negative fibers) and ultrastructural abnormalities of muscle mitochondria, in vivo muscle phosphorus MR spectroscopy disclosed a slow rate of phosphocreatine resynthesis after exercise. Brain phosphorus MR spectroscopy revealed a defect of the energy metabolism in the two patients and in a third asymptomatic member, as shown by a significantly low phosphocreatine, increased ADP and decreased phosphorylation potential. Molecular analysis of muscle mitochondrial DNA failed to reveal any known mutation, including multiple deletions of the mtDNA which have been associated with some autosomal dominant mitochondrial diseases. The multisystem clinical involvement, the presence of ragged-red fibers and the alterations revealed by in vivo brain and muscle 31P-MRS suggest that this limb-girdle syndrome represents an unusual phenotype of mitochondrial cytopathy.

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho degrees transformants.

Defects of the respiratory chain carrying out oxidative phosphorylation (OXPHOS) are the biochemical hallmark of human mitochondrial disorders. Faulty OXPHOS can be due to mutations in either nuclear or mitochondrial genes, that are involved in the synthesis of individual respiratory subunits or in their post-translational control. The most common mitochondrial disorder of infancy and childhood is Leigh's syndrome, a severe encephalopathy, often associated with a defect of cytochrome c oxidase (COX). In order to demonstrate which genome is primarily involved in COX-deficient (COX(-))-Leigh's syndrome, we generated two lines of transmitochondrial cybrids. The first was obtained by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts, with mitochondrial DNA-less (rho degree) transformant fibroblasts derived from a patient with COX(-))-Leigh's syndrome. The second cybrid line was obtained by fusing rho degree cells derived from 143B.TK- human osteosarcoma cells, with cytoplasts derived from the same patient. The first cybrid line showed a specific and severe COX(-) phenotype, while in the second all the respiratory chain complexes, including COX, were normal. These results indicate that the COX defect in our patient is due to a mutation of a nuclear gene. The use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study of nuclear DNA-mitochondrial DNA interactions.