Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency.

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients.

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.

Creatine transporter deficiency: prevalence among patients with mental retardation and pitfalls in metabolite screening.

OBJECTIVES: To report the prevalence of creatine transporter deficiency in males with mental retardation and to study whether a protein-rich food intake might be a potential diagnostic pitfall. DESIGN AND METHODS: We determined creatine/creatinine ratio in urine samples from 1600 unrelated male patients with mental retardation and/or autism. Urine creatine was analyzed by HPLC-MS/MS. RESULTS: Thirty-three of 1600 cases showed increased urine creatine/creatinine ratio. Four out of these thirty-three cases were definitively diagnosed with creatine transporter deficiency, while the other 29 were false positive results. Significantly higher values were observed for urine Cr/Crn ratio in healthy volunteers after a meal based on beef or oily fish as compared to eggs, pasta or salad (Wilcoxon test: p<0.005). CONCLUSIONS: False positive results may be observed in biochemical screening for creatine transporter deficiency, and they may be due to intake of meals rich in creatine prior to urine samples analysis.

Cranial ultrasound and chronological changes in molybdenum cofactor deficiency.

Molybdenum cofactor is essential for the function of three human enzymes: sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Molybdenum cofactor deficiency is a rare autosomal recessively inherited disease. Disturbed development and damage to the brain may occur as a result of accumulation of toxic levels of sulphite. The CT and MRI findings include severe early brain abnormalities and have been widely reported, but the cranial US imaging findings have seldom been reported. We report a chronological series of cranial US images obtained from an affected infant that show the rapid development of cerebral atrophy, calcifications and white matter cysts. Our report supports the utility of cranial US, a noninvasive bed-side technique, in the detection and follow-up of these rapidly changing lesions.

A CBS haplotype and a polymorphism at the MSR gene are associated with cardiovascular disease in a Spanish case-control study.

OBJECTIVES: The aim of this study was to evaluate the association of polymorphisms present in genes related to homocysteine (Hcy) metabolism with coronary artery disease (CAD). DESIGN AND METHODS: We examined 8 polymorphisms in the cystathionine beta-synthase (CBS), glutamate carboxypeptidase II (GCPII), methionine synthase (MS), methionine synthase reductase (MSR) and methylenetetrahydrofolate reductase (MTHFR) genes in 140 CAD patients and 113 controls, by means of Chi-square, logistic regression, ANOVA and the Mann-Whitney U test. RESULTS: The c.66 G allele of MSR conferred an odds-ratio for CAD of 1.76 (95% CI 1.12-2.77), while a CBS haplotype [c.699C-c.844wt-c.1080C] was found over-represented in CAD [OR of 2.16 (1.29-3.63)]. CONCLUSIONS: Our results not only highlight the involvement of the MSR and CBS genes in the etiology of cardiovascular disease, but also emphasize the strength of haplotype analyses in association studies.

Clinical, biochemical and molecular aspects of cerebellar ataxia and Coenzyme Q10 deficiency.

Coenzyme Q(10) (CoQ) deficiency is an autosomal recessive disorder presenting five phenotypes: a myopathic form, a severe infantile neurological syndrome associated with nephritic syndrome, an ataxic variant, Leigh syndrome and a pure myopathic form. The third is the most common phenotype related with CoQ deficiency and it will be the focus of this review. This new syndrome presents muscle CoQ deficiency associated with cerebellar ataxia and cerebellar atrophy as the main neurological signs. Biochemically, the hallmark of CoQ deficiency syndrome is a decreased CoQ concentration in muscle and/or fibroblasts. There is no molecular evidence of the enzyme or gene involved in primary CoQ deficiencies associated with cerebellar ataxia, although recently a family has been reported with mutations at COQ2 gene who present a distinct phenotype. Patients with primary CoQ deficiency may benefit from CoQ supplementation, although the clinical response to this therapy varies even among patients with similar phenotypes. Some present an excellent response to CoQ while others show only a partial improvement of some symptoms and signs. CoQ deficiency is the mitochondrial encephalomyopathy with the best clinical response to CoQ supplementation, highlighting the importance of an early identification of this disorder.

The p.T191M mutation of the CBS gene is highly prevalent among homocystinuric patients from Spain, Portugal and South America.

Classical homocystinuria is due to cystathionine beta-synthase (CBS) deficiency. More than 130 mutations, which differ in prevalence and severity, have been described at the CBS gene. Mutation p.I278T is very prevalent, has been found in all European countries where it has been looked for with the exception of the Iberian peninsula, and is known to respond to vitamin B6. On the other hand, mutation p.T191M is prevalent in Spain and Portugal and does not respond to B6. We analysed 30 pedigrees from Spain, Portugal, Colombia and Argentina, segregating for homocystinuria. The p.T191M mutation was detected in patients from all four countries and was particularly prevalent in Colombia. The number of p.T191M alleles described in this study, together with those previously published, is 71. The prevalence of p.T191M among CBS mutant alleles in the different countries was: 0.75 in Colombia, 0.52 in Spain, 0.33 in Portugal, 0.25 in Venezuela, 0.20 in Argentina and 0.14 in Brazil. Haplotype analyses suggested a double origin for this mutation. No genotype-phenotype correlation other than the B6-nonresponsiveness could be established for the p.T191M mutation. Additionally, three new mutations, p.M173V, p.I429del and c.69_70+8del10, were found. The p.M173V was associated with a mild, B6-responsive, phenotype.

Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations.

In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non-denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild-type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild-type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild-type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non-denaturing PAGE revealed that the wild-type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations.

Spectrum of CBS mutations in 16 homocystinuric patients from the Iberian Peninsula: high prevalence of T191M and absence of I278T or G307S.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency has been extensively studied, but to date, no spectrum of CBS mutations of Spanish homocystinuric patients has been reported. Here we present a mutation analysis of thirteen Spanish and three Portuguese unrelated homocystinuric patients. Ten mutations were found to account for the thirty-two mutant alleles and five of these (C275Y, L338P, S349N, R379Q, and L456P) are reported here for the first time. All five novel mutations were found to affect evolutionarily conserved residues suggesting that they may impair enzyme function. Interestingly, neither of the two common CBS mutations, I278T and G307S, was detected in this series, and no patient was found to respond to pyrodoxine. Enzyme activities in cultured fibroblasts from 10 of the patients were assayed, and they ranged from 0 to 13 % of controls analyzed in parallel. The T191M mutation (which has only ever been reported once before in a Spanish patient) accounted for 50% of the mutant alleles. Comparison of the clinical data of seven patients homozygous for T191M indicated that this genotype is a poor predictor of the phenotype. A common haplotype was identified in all the T191M chromosomes of Spanish origin, while a different one was present in the four T191M chromosomes from Portuguese patients.

Lipophilic antioxidants in patients with phenylketonuria.

BACKGROUND: Low serum ubiquinone-10 concentrations have been described in phenylketonuric patients fed natural-protein-restricted diets. Such low concentrations may be related to increased free radical damage. OBJECTIVE: We evaluated the relation between low serum ubiquinone-10 concentrations and other lipophilic antioxidants (tocopherol and retinol), selenium, glutathione peroxidase activity, and malondialdehyde concentrations as a marker of lipid peroxidation. DESIGN: This was a cross-sectional study of 58 patients with phenylketonuria (aged 2-36 y; median: 13 y) under dietary treatment, 58 age-matched control subjects, and 30 children with moderate hyperphenylalaninemia fed unrestricted diets (aged 3-17 y; median: 7.5 y). Serum ubiquinone-10 concentrations were analyzed by HPLC with electrochemical detection. Serum retinol, serum tocopherol, and plasma malondialdehyde were analyzed by HPLC with ultraviolet detection. RESULTS: A significant positive correlation was observed between ubiquinone-10 and tocopherol (r = 0.510, P < 0.001) in the patients with phenylketonuria. After the patients were stratified into 2 groups according to ubiquinone-10 values, significantly lower concentrations of tocopherol were observed in group 1 (low ubiquinone values) than in group 2 (normal ubiquinone values), the hyperphenylalaninemic children, and the control group. Plasma malondialdehyde concentrations were significantly higher in group 1 than in the other groups. No significant differences between groups 1 and 2 were observed in daily intakes of selenium, ascorbate, tocopherol, or retinol. CONCLUSIONS: Plasma lipid peroxidation seems to be increased in phenylketonuria. Low concentrations of ubiquinone-10 could be associated with either excessive tocopherol consumption or high malondialdehyde concentrations in patients with phenylketonuria.