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Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.

We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.

Congenital disorders of glycosylation (CDG) are a group of inherited disorders caused by defects in the synthesis and processing of the linked glycans of glycoproteins and other molecules. The first patients with COG were described in 1980. Fifteen years later, phosphomannomutase was found to be the basis of the most frequent type: CDG-1a. Over the last years, several novel types have been identified related to the N-glycosylation pathway, affecting enzymes or transporters of the cytosol, endoplasmic reticulum or the Golgi compartment. CDGs are multisystemic disorders, mainly affecting the central nervous system. Yet CDG-1b and 1h are mainly hepato-intestinal diseases. Recently, several defects involving the 0-glycosylation pathways have been described, indicating that some congenital muscular dystrophies and neuronal migration disorders are caused by congenital disorders of glycosylation.

Mucopolysaccharidosis I is a metabolic disease of autosomal recessive inheritance caused by deficient activity of alpha-L-iduronidase. The clinical phenotype presents a wide spectrum of signs in the first year of life. We report a child with clinical features and laboratory data consistent with mucopolysaccharidosis I who precociously developed hydrocephalus and flexion spasms with hypsarrythmia in the electroencephalographic registration characteristic of West's syndrome. His radiologic and biochemical data suggested vitamin D-dependent rickets. To our knowledge, this is the first report of a patient demonstrating an association among mucopolysaccharidosis 1, West's syndrome, and vitamin D-dependent rickets.

BACKGROUND: Factors related to hyperhomocystinemia in the pediatric population of our geographical area with a parental history of premature coronary disease (PCD) are not well known. OBJECTIVES: To evaluate the possible association between plasma total homocysteine (tHcy), the B vitamins involved in its metabolism (folate, vitamin B12 and B6), and 677C T polymorphism of methylenetetrahydrofolate reductase (MTHFR) in a group of children with a parental history of PCD. METHODS: A cross-sectional analytical study of 80 children (aged 5-18 years old) with a parental history of PCD was performed. Values found in these children were compared with reference values for similar age groups. Plasma tHcy and vitamin B6 were evaluated by high-performance liquid chromatography with fluorometric detection. Folate and vitamin B12 concentrations were determined by radioimmunoassay. Detection of 677C T polymorphism of MTHFR was performed using polymerase chain reaction amplification and Hinfl digestion. Statistical analysis was performed using the SPSS program, version 10.0. Concentrations of tHcy and vitamins were compared using the Mann-Whitney U-test and Spearman's correlation coefficient. The association between phenotype, hyperhomocystinemia and low vitamin concentrations was analyzed using the chi-squared test. ResultsPlasma tHcy values in the children aged more than 10 years with a parental history of PCD were significantly higher (p < 0.001) than the reference values. Vitamin B12 levels were significantly lower (p 0.015), but neither folate nor vitamin B6 levels differed from the reference values. A negative correlation (p < 0.0001) was observed between tHcy and folate (r 0.47) and between tHcy and vitamin B12 levels (r 0.51). Eighty percent of the children with the TT genotype of MTHFR showed hyperhomocystinemia. Suboptimal vitamin B levels were also associated with the TT genotype of MTHFR. CONCLUSIONS: Hyperhomocystinemia detected in children with a parental history of PCD is associated with the TT genotype of MTHFR and with low folate levels. Because hyperhomocystinemia can be corrected by vitamin B supplementation, tHcy determination is recommended in the offspring of patients with PCD.

Oxidative stress may be implied in the pathogenic mechanisms of inborn errors of intermediary metabolism (IEIM).The evaluation of the antioxidant status in IEIM by the measurement of erythrocyte antioxidant enzyme activities, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase and catalase.34 patients with IEIM: 1) eleven with organic acidurias on protein restricted diet; 2) nine without special diet; 3) five patients with aminoacidopathies on protein restriction; 4) three patients with galactosemia and six with aminoacidopathies on protein free diet. Erythrocyte antioxidant enzymes were measured by spectrometric procedures adapted to the Cobas Fara II analyser.SOD activity was significantly higher in groups 2 and 4 (p= 0.009, p= 0.001, respectively), and significantly lower in group 3 (p= 0.001) compared with age matched controls. SOD activity was significantly higher in the patients with IEIM on protein free diet (groups 2 and 4) compared with those on protein restricted diet (groups 1 and 3; p= 0.002) or with controls (p= 0.003). GPx activity was found significantly lower in group 1 patients (p= 0.004), and higher in group 2 (p= 0.029) compared with controls.35% of the patients with IEIM had SOD activity above the control range, most of them with organic acidurias or homocystinuria, suggesting an induction of enzyme protein synthesis owing to an excess of free radical generation. The lower activities observed in patients on natural protein restriction may likely be due to a deficient bioavailability of antioxidant cofactors.

Introduction. Carnitine is an nonproteic nitrogenated compound acid present in all mammalian tissue and its principal activity is the long-chain fatty acid transport across the mitochondrial membrane for beta-oxidation. Development. Carnitine levels ale maintained by absorption from dietary, endogenous synthesis in the liver and venal reabsorption. An alteration in concentration could be due to an abnormality in some of mentioned mechanism or to inherited errors in metabolism. A reduction in serum carnitine levels have been reported in patients with antiepileptic drugs (AED) use. While valproic acid as monotheraphy or in combination with other AED have been associated principally, some contradictory studies demonstrated that other AED specifically carbamazepine, phenytoin and phenobarbital, also cause carnitine deficiency in about 21% of the patients whom they are administered Conclusion. This study was designed to investigated the relationship between the carnitine levels and the presence of symptoms in patients with seizures whom ave treated with a AED alone or in combination.

Introduction. The existence of neuropathy has been described in mitochondrial disorders such as MELAS, MERRF, Leigh's syndrome, the Kearns-Saye syndrome, myoneurogastro-intestinal encephalopathy and progressive external ophthalmoplegia and constitutes a basic component of the NARP (neuropathy, ataxia and retinosis pigmentosa). However, the general prevalence of the neuropathy and its characteristics within the mitochondrial encephalopathies is not well understood. Objectives. To characterize the neuropathy and try to establish a genotype-phenotype correlation. Patients and methods. within study guidelines, we made a retrospective study of 27 patients, diagnosed as having mitochondrial disease, who had had neurophysiological studies (EMG-ENG). In those in whom neuropathy had been found we analysed the clinical, neurophysiological and genetic characteristics. Results. Neuropathy was present in 37% of the patients who had an average age of 13 years, ranging from I to 25 years. Syndromic diagnoses were: 7 encephalomyopathies, one MELAS, one MERRF and one NARP. Four of the patients were classified genetically. In all but two of the patients the neuropathy was asymptomatic. The biochemical alterations seen were: deficit of Complex 1 in 3 patients, of complex III in 3 patients, of complex IV in 2 and of pyruvate dehydrogenase in one patient. The type of neuropathy found was varied with predominance of axonal-type motor neuropathy but no correlation with either biochemical defects or genetic diagnosis. Conclusions. Neuropathy is a common finding in mitochondrial disorders and probably is under-diagnosed. The axonal form predominates. We hats not been able to establish correlations between phenotypes and genotypes.

PKU is an autosomal recessive disorder. There is a broad spectrum phenotype which depends mainly on residual enzymatic activity and also on other factors such as modifying genes and non-genetic factors. This fact makes us consider that a multidisciplinary study of these patients is necessary to improve knowledge of the condition.

Objective: The study of the antioxidant status in female adolescents (N = 82) with anorexia nervosa, by the measurement of erythrocyte tocopherol concentration, and the determination of activities of the main antioxidant enzymes: superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. Method: Tocopherol was measured by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, and antioxidant enzyme activities by spectrometric methods using a Cobas Fara automated centrifugal analyzer. Results: Tocopherol was significantly decreased in the anorexic patients compared to reference values (p < .02). In 21% of patients, tocopherol levels were below the reference interval. Superoxide dismutase activity was significantly decreased (p < .0001) while catalase activity was increased (p < .0001). The activity of the glutathione system enzymes did not show significant differences between patients and controls. Discussion: The deficient concentration of erythrocyte tocopherol together with the altered antioxidant enzyme activities suggest a certain degree of oxidative damage in anorexia nervosa owing to both factors deficient micronutrient intake and oxidative stress. (C) 1999 by John Wiley & Sons, Inc.

Review of ubiquinone-10 metabolism and functions in humans, focusing its implication in the pathogenesis and physiopathology of mitochondrial encephalomyopathies.

Introduction. The correct selection of pediatric patients with clinical suspicion of mitochondrial diseases is the first step to achieve a definitive diagnosis. Material and methods. The results of the initial biochemical tests obtained in 35 children diagnosed of respiratory chain or pyruvate metabolism defects were reviewed. The efficiency of basal determinations (lactate, pyruvate, ketone bodies, amino and organic acids and carnitine), cerebrospinal fluid (CSF) analysis, and dynamic tests (exercise, glucose loading and glucose oxidation by lymphocytes) was discussed. Results. Plasma lactate and alanine, and CSF metabolites were the most informative measurements in basal status. Urine organic acids were very useful to confirm the initial suspicion. Glucose loading was the most informative and reliable challenge test for pediatric population, while exercise test was especially useful for older children with fatigability or peripheral nervous system involvement. Conclusions. Glucose oxidation by lymphocytes might be applied when the other dynamic tests can not be performed or are not informative.

INTRODUCTION: The correct selection of pediatric patients with clinical suspicion of mitochondrial diseases is the first step to achieve a definitive diagnosis. MATERIAL AND METHODS: The results of the initial biochemical tests obtained in 35 children diagnosed of respiratory chain or pyruvate metabolism defects were reviewed. The efficiency of basal determinations (lactate, pyruvate, ketone bodies, amino and organic acids and carnitine), cerebrospinal fluid (CSF) analysis, and dynamic tests (exercise, glucose loading and glucose oxidation by lymphocytes) was discussed. RESULTS: Plasma lactate and alanine, and CSF metabolites were the most informative measurements in basal status. Urine organic acids were very useful to confirm the initial suspicion. Glucose loading was the most informative and reliable challenge test for pediatric population, while exercise test was especially useful for older children with fatigability or peripheral nervous system involvement. CONCLUSIONS: Glucose oxidation by lymphocytes might be applied when the other dynamic tests can not be performed or are not informative.

PDH complex deficiency is one of the most common causes of primary lactic acidosis. Its diagnosis is complicated because of heterogeneity of the clinical presentation, the variability of the pattern of metabolites in plasma and urine, and the finding of high residual activities, especially for PDH E1 alpha deficiency in some heterozygous females. In addition, methods for measurement of PDH activity have pitfalls due to the complexity of this enzyme. We have mixed ideas from different authors to obtain a radiometric method for determination of PDH and E1 activities in fibroblasts and tissues. With this method we have detected 22 patients with low PDH activities. In four of them a mutation in the coding region of the E1 alpha gene has been found: 242 del 3 bp exon 7 in a girl with lactic academia, microcephaly and corpus callosum agenesia, H113D exon 4 in a girl with cerebral lactic academia, hypotonia and spastic tetraparesia, and in two boys affected by Leigh syndrome R263G exon 8 and Y369Q exon 11 have been found respectively. In the latter patient, codon 369 (TAC) is changed to CAA; this finding of two changes in the same codon is a rather exceptional observation. Two other patients were suspected of E3 deficiency because of the urine organic acid profile, but lipoamide dehydrogenase activity was normal. Three more girls presented multiple mitochondrial enzyme deficiencies and in one of them these deficiencies were likely attributable to decreased amount of hsp60. 13 other patients presented clinical features suggesting PDH deficiency and the activities of the complex and the E1 component were decreased or low-normal, but no mutation was demonstrated in the E1 alpha gene.

Detecció de malalties; Cribratge neonatal; Catalunya; Disease detection; Neonatal screening; Catalonia; Detección de enfermedades; Cribado neonatal; Cataluña El Programa de detecció precoç neonatal, és una actuació de prevenció secundària fonamental en Salut Pública dirigida a identificar precoçment els nadons afectats per determinades condicions genètiques, endocrines, embrionàries o infeccioses que posen en perill la seva vida, és per això que una actuació sanitària en els primers dies de vida del nadó pot facilitar l’eliminació o la reducció significativa de la morbiditat, la mortalitat o les discapacitats associades.