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.

Tratamiento y control de los pacientes con fenilcetonuria: resultados del Grupo Colaborativo de Unidades de Seguimiento en España / Treatment and control of patients with phenylketonuria: results from the Collaborative Group of Spanish Follow-up Units

Fundamento y objetivo: Conocer el control de la fenilcetonuria (PKU) en las unidades de seguimiento españolas y realizar un registro de pacientes. Pacientes y método:Pacientes con PKU diagnosticados y/o seguidos en España, con fenilalanina previa al tratamiento > 360μmol/L. Cuestionarios: datos anonimizados incluidos, aquellos aportados por las unidades durante el año 2010. Resultados: Se han recogido datos de las 18 unidades de seguimiento españolas. El 83% muestran una composición multidisciplinaria y todas controlan pacientes de todas las edades, con criterios de tratamiento, en general, uniformes. Se han registrado datos de 688 pacientes con PKU, con una mediana de edad de 14 años (extremos 1 mes-53 años); un 41,5% eran mayores de 18 años. Un 71,8% se diagnosticaron precozmente. Un 15,8% tienen una PKU leve, el 26% una forma moderada y el 51,5% la forma clásica. Un 78,6% de los pacientes son tratados con dieta restringida en proteínas, el 9,3% con tetrahidrobiopterina (BH4) y dieta libre y un 7,8% con BH4 y dieta. El control dietético es bueno en el 58,6% de pacientes, regular en el 26% y malo en un 15,4%. La mediana del coeficiente intelectual del total de pacientes con PKU es de 97 (extremos 25-145). El porcentaje de pacientes de diagnóstico tardío con complicaciones neurológicas y conductuales es significativamente mayor al de los diagnosticados precozmente. El 13,3% de adultos han cursado estudios universitarios y el 37,5% tiene pareja estable.Conclusiones: Este estudio permite evaluar el funcionamiento de las unidades de seguimiento de la PKU en España, así como registrar y analizar, por primera vez, los datos de los pacientes con PKU controlados en ellas. Se demuestra la necesidad de unidades de enfermedades metabólicas de adultos y el valor del diagnóstico precoz en el pronóstico de los pacientes con PKU (AU)

Background and objective: To evaluate the management of phenylketonuria (PKU) in Spanish metabolic units and to develop a patients registry. Patients and methods: PKU patients diagnosed and/or followed up in Spain, with phenylalanine values before treatment > 360μmol/L. Registered anonimous data are those yielded by the units during 2010. Results: Data from the 18 Spanish Follow-up Units were collected. Eighty-three per cent of Units are multidisciplinary, all of them corresponding to control patients of all ages, with uniform management criteria. Data of 688 PKU patients were registered (median: 14 years [1 month-53 years], 41.5% are presently > 18-year-old. 71.8% patients came from neonatal screening; 15.8% have mild-PKU, 26% moderate-PKU and 51.5% classic-PKU. 78.6% patients are treated with protein-restricted diet, 9.3% with BH4 and free diet and 7.8% with BH4 and diet. Dietary control was good in 58.6% patients, intermediate in 26% and poor in 15.3%. Median (range) intellectual quotients was 97 (25-145). The number of neurological complications in late diagnosed patients was three-times higher than those of neonatal screening patients. 13.3% of adults had university studies and 37.5% had a stable couple. Conclusions: This study allows for the first time the evaluation of the PKU management by Spanish PKU Follow-up Units, as well as the analysis and registry of controlled PKU patients. The study makes evident the need of adult Follow-up Units and the importance of neonatal screening for PKU patients prognosis

Clinical, genetic, and therapeutic diversity in 2 patients with severe mevalonate kinase deficiency.

Mevalonic aciduria (MA) represents the severest form of mevalonate kinase deficiency due to recessively inherited, loss-of-function MVK mutations. MA is an early-onset disorder characterized by a marked failure to thrive, diverse neurologic symptoms, dysmorphic features, and recurrent febrile episodes. However, significant clinical differences have been reported in the few cases published to date. Here we describe 2 unrelated Spanish patients with MA, emphasizing the clinical heterogeneity observed. One patient presented with the severe classic MA phenotype due to the homozygous p.Ile-268-Thr MVK genotype, with a poor response to conventional treatments. However, the anti-interleukin 1 agent anakinra in this patient resulted in improvement in many clinical and laboratory parameters. The second patient presented with an atypical milder phenotype because of an older age at disease onset, mild neurologic symptoms, absence of febrile episodes and dysmorphic features, and moderate-to-good response to conventional treatments. The novel p.Arg-241-Cys MVK mutation, associated with the already known p.Ser-135-Leu mutation, detected in this patient expands the genetic diversity of mevalonate kinase deficiency. This atypical presentation of MA suggests that it should be included in the differential diagnosis of unclassified patients with psychomotor retardation, failure to thrive or ataxia, even in the absence of febrile episodes.

[Treatment and control of patients with phenylketonuria: results from the Collaborative Group of Spanish Follow-up Units]. / Tratamiento y control de los pacientes con fenilcetonuria: resultados del Grupo Colaborativo de Unidades de Seguimiento en España.

BACKGROUND AND OBJECTIVE: To evaluate the management of phenylketonuria (PKU) in Spanish metabolic units and to develop a patients registry. PATIENTS AND METHODS: PKU patients diagnosed and/or followed up in Spain, with phenylalanine values before treatment > 360 µmol/L. Registered anonymous data are those yielded by the units during 2010. RESULTS: Data from the 18 Spanish Follow-up Units were collected. Eighty-three per cent of Units are multidisciplinary, all of them corresponding to control patients of all ages, with uniform management criteria. Data of 688 PKU patients were registered (median: 14 years [1 month-53 years], 41.5% are presently > 18-year-old. 71.8% patients came from neonatal screening; 15.8% have mild-PKU, 26% moderate-PKU and 51.5% classic-PKU. 78.6% patients are treated with protein-restricted diet, 9.3% with BH4 and free diet and 7.8% with BH4 and diet. Dietary control was good in 58.6% patients, intermediate in 26% and poor in 15.3%. Median (range) intellectual quotients was 97 (25-145). The number of neurological complications in late diagnosed patients was three-times higher than those of neonatal screening patients. 13.3% of adults had university studies and 37.5% had a stable couple. CONCLUSIONS: This study allows for the first time the evaluation of the PKU management by Spanish PKU Follow-up Units, as well as the analysis and registry of controlled PKU patients. The study makes evident the need of adult Follow-up Units and the importance of neonatal screening for PKU patients prognosis.

Undetectable levels of CSF amyloid-ß peptide in a patient with 17ß-hydroxysteroid dehydrogenase deficiency.

17ß-hydroxysteroid dehydrogenase 10 (HSD10) deficiency is a rare X-linked inborn error of isoleucine catabolism. Although this protein has been genetically implicated in Alzheimer's disease pathogenesis, studies of amyloid-ß peptide (Aß) in patients with HSD10 deficiency have not been previously reported. We found, in a severely affected child with HSD10 deficiency, undetectable levels of Aß in the cerebrospinal fluid, together with low expression of brain-derived neurotrophic factor, α-synuclein, and serotonin metabolites. Confirmation of these findings in other patients would help elucidating mechanisms of synaptic dysfunction in this disease, and highlight the role of Aß in both early and late periods of life.

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.

Neurocognitive function in mild hyperphenylalaninemia.

AIM: The purpose of this review was to provide an update on cognitive function in individuals with mild hyperphenylalaninemia (mHPA), the most clinically and biochemically benign form of phenylketonuria. METHOD: A review was conducted of the existing literature on mHPA. Individuals with mHPA, whose plasma phenylalanine concentration had always remained lower than 360 µmol/L without dietary restriction, were considered. RESULTS: The review of the literature indicated that there is no consensus concerning the definition of mHPA. There are few studies regarding the cognitive functions of individuals with mHPA, results are contradictory, and samples are difficult to compare from one study to another. Most studies focus only on descriptions of IQ when assessing cognitive functions. The existing literature indicates that, in general, children with mHPA do not show significant cognitive impairments, but usually achieve scores between those of individuals with phenylketonuria and those of comparison groups with regard to the cognitive functions assessed. INTERPRETATION: When assessing cognitive functions in individuals with hyperphenylalaninemia, it is not enough to measure only IQ, as deficits in executive functions can be present even when an individual's IQ is within a normal range. Further studies are needed of individuals with mHPA, using consistent selection criteria, in order to make it possible to exclude the presence of cognitive impairment and to establish a consensus regarding the level of phenylalanine that necessitates dietary treatment.

Dihydrolipoamide dehydrogenase (DLD) deficiency in a Spanish patient with myopathic presentation due to a new mutation in the interface domain.

We present a 32-year-old patient who, from age 7 months, developed photophobia, left-eye ptosis and progressive muscular weakness. At age 7 years, she showed normal psychomotor development, bilateral ptosis and exercise-induced weakness with severe acidosis. Basal blood and urine lactate were normal, increasing dramatically after effort. PDHc deficiency was demonstrated in muscle and fibroblasts without detectable PDHA1 mutations. Ketogenic diet was ineffective, however thiamine gave good response although bilateral ptosis and weakness with acidosis on exercise persisted. Recently, DLD gene analysis revealed a homozygous missense mutation, c.1440 A>G (p.I480M), in the interface domain. Both parents are heterozygous and DLD activity in the patient's fibroblasts is undetectable. The five patients that have been reported with DLD-interface mutations suffered fatal deteriorations. Our patient's disease is milder, only myopathic, more similar to that due to mutation p.G229C in the NAD(+)-binding domain. Two of the five patients presented mutations (p.D479V and p.R482G) very close to the present case (p.I480M). Despite differing degrees of clinical severity, all three had minimal clues to DLD deficiency, with occasional minor increases in α-ketoglutarate and branched-chain amino acids. In the two other patients, hypertrophic cardiomyopathy was a significant feature that has been attributed to moonlighting proteolytic activity of monomeric DLD, which can degrade other mitochondrial proteins, such as frataxin. Our patient does not have cardiomyopathy, suggesting that p.I480M may not affect the DLD ability to dimerize to the same extent as p.D479V and p.R482G. Our patient, with a novel mutation in the DLD interface and mild clinical symptoms, further broadens the spectrum of this enzyme defect.

Cerebellar hemorrhage in a patient with propionic acidemia.

Cerebellar hemorrhage (CH) is a well-known complication in newborns. Among metabolic patients, it has been classically described but rarely reported. This is the first description of a patient with propionic acidemia in whom magnetic resonance imaging (MRI) allowed diagnosis of asymptomatic CH. Due to the usual silent presentation of CH at early ages, we suggest the possibility of including a brain MRI study as part of the routine neurological evaluation in metabolic patients, especially when neurological signs appear.

Seizures versus dystonia in encephalopathic crisis of glutaric aciduria type I.

In more than two thirds of cases, glutaric aciduria type I begins in the first 3 years of life with an acute encephalopathic crisis with hypotonia or generalized rigidity, neurologic depression, irritability, seizures, and dystonia. The clinical histories were reviewed for 13 glutaric aciduria type I patients (9 male, 4 female; mean age, 8.7 months; range, 3-15 months) with encephalopathic crisis seen at Sant Joan de Déu Hospital, to describe the clinical features and the initial electroencephalographic (EEG) findings. Twelve of the patients (92%) had paroxysmal episodes at onset. Other clinical features included irritability (12/13), neurologic depression (11/13), and hypotonia (7/13). All patients evolved to dystonic tetraparesis. Thirty-five EEGs were recorded in the acute stage and during the first year of follow-up. Spike discharges on EEG were observed in only 2 of the 13 patients, and 8 had slow background activity. No patient developed seizures during follow-up. Seizures may be part of the symptomatology at the onset of glutaric aciduria type I, but most paroxysmal movements appear to be dystonic episodes. This hypothesis is supported by four facts: seizures do not occur after dystonic tetraparesis is noticed, EEG paroxysms are infrequent in the acute stage, antiepileptic drugs are not needed in the long term, and epilepsy is rare in the follow-up.

Cognitive functions and the antioxidant system in phenylketonuric patients.

The authors studied the relationship between the antioxidant system and cognitive functions in a group of 36 early and continuously treated phenylketonuric (PKU) patients (mean age=9.7 years) and 29 controls. The authors measured antioxidant cofactors and free radical damage markers in plasma (selenium, retinol, tocopherol, coenzyme Q10, malondialdehide) and antioxidant enzymes in red blood cells (glutathione peroxidase, catalase, superoxide dismutase). The authors used neuropsychological tests to screen for several cognitive functions. PKU patients showed significantly lower values of selenium, coenzyme Q10, and catalase, and significantly higher levels of malondialdehide. PKU patients showed a significantly negative correlation between plasma selenium concentrations and several Conner's Continuous Performance Test measures (more omission errors, fluctuating attention and inconsistency of response times, and slowing reaction time as the test progressed). Selenium deficiency was thus associated with a worsened performance on the Conner's Continuous Performance Test among PKU patients. In conclusion, it is important not only to control blood Phe levels in PKU but also other nutritional components such as selenium. Selenium status seems to be associated with attention functions in these PKU patients.

A new fatal case of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency.

We present a patient with severe pyridox(am)ine 5'-phosphate oxidase deficiency and homozygosity for a novel nonsense-mutation, p.A174X, in the PNPO gene who died with pyridoxal phosphate (PLP) treatment despite initial clinical recovery. He presented neonatally, with the classical clinical symptoms of the disease. Increase of urinary vanillactate was the first biochemical factor of alert. Amino acid and neurotransmitter analysis in CSF indicated reduced activity of several PLP-dependent enzymes. The diagnosis was confirmed by mutational studies. From this and the other reported patients it may be concluded that the administration of PLP should not be delayed until the complete biochemical evidence is obtained.

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.

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

In this article, one of the novel mutations, c.208_209+ 8del10, was incorrectly given as c.69_70+8del10. It corresponds to patient 64 in Table 4.

Methods for the diagnosis of creatine deficiency syndromes: a comparative study.

The increasing number of patients with creatine deficiency syndromes (CDS) stresses the need to develop screening procedures for the identification these inherited disorders. Guanidinoacetate (GAA) and creatine (Cr) are reliable biochemical markers of CDS and several analytical methods to measure both metabolites have been developed. High-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is quick and sensitive but, unlike HPLC and gas chromatography-mass spectrometry (GC/MS), it is unavailable in most laboratories. Thus, we decided to evaluate comparatively HPLC-MS/MS, GC/MS and HPLC methods, as well as to establish reference values in a healthy paediatric population. According to our results, these three methods may be suitable for analysing GAA in urine. Furthermore, Passing-Bablock plots showed good agreement among all three. However, when comparing the Cr/Crn ratio, our results revealed that while HPLC-MS/MS data were in agreement with those of GC/MS, a constant and proportional error was observed when compared with those of HPLC. Consequently, the Cr/Crn ratio obtained by the last method should be evaluated with caution. Our reference values for GAA and Cr/Crn ratio in urine negatively correlate with age. Concerning GAA and Cr measurements in plasma, it is interesting to note that in contrast to what was occurring in urine, GAA concentration increased significantly with age, while we did not find any significant difference for Cr values within the same age group.

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.