Genes and variants underlying human congenital lactic acidosis—from genetics to personalized treatment

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, los autores pertenecientes a la UAM y el nombre del grupo de colaboración, si lo hubiereCongenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group’s experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system’s workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system’s use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this conditio

Estudio diferencial de acidosis láctica congénita

Tesis doctoral inédita leida en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 10-10-199

A Novel Regulatory Defect in the Branched-Chain α-Keto Acid Dehydrogenase Complex Due to a Mutation in the PPM1K Gene Causes a Mild Variant Phenotype of Maple Syrup Urine Disease

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype-and perhaps others.MICIN; Fundación Ramón ArecesPeer Reviewe

Dataset reporting BCKDK interference in a BCAA-catabolism restricted environment

This data article contains complementary figures to the research article >Mitochondrial response to the BCKDK-deficiency: some clues to understand the positive dietary response in this form of autism> [. 1]. Herein we present data relative to the effect of knocking down BCKDK gene on the real time oxygen consumption rate of fibroblasts obtained from a Maple Syrup Urine Disease (MSUD) patient. Interference of BCKDK expression on such cells showing a reduced branched-chain α-ketoacid dehydrogenase (BCKDHc) activity; let us generate a scenario to study the direct effect of BCKDK absence in an environment of high branched-chain amino acids (BCAAs) concentrations. Data relative to the effectiveness of the knockdown together with the potentiality of the BCKDK-knockdown to increase the deficient branched-chain α-ketoacid dehydrogenase activity detected in MSUD patients are also shown.Centro de Diagnóstico de Enfermedades Moleculares (CEDEM). Irene Bravo-Alonso is Granted from Funda- ción Ramón Areces (CIVP16A1853)Peer Reviewe

Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines

Newborn screening (NBS) is justified if early intervention is effective in a disorder generally not detected early in life on a clinical basis, and if sensitive and specific biochemical markers exist. Experience with NBS for homocystinurias and methylation disorders is limited. However, there is robust evidence for the success of early treatment with diet, betaine and/or pyridoxine for CBS deficiency and good evidence for the success of early betaine treatment in severe MTHFR deficiency. These conditions can be screened in dried blood spots by determining methionine (Met), methionine-to-phenylanine (Met/Phe) ratio, and total homocysteine (tHcy) as a second tier marker. Therefore, we recommend NBS for cystathionine beta-synthase and severe MTHFR deficiency. Weaker evidence is available for the disorders of intracellular cobalamin metabolism. Early treatment is clearly of advantage for patients with the late-onset cblC defect. In the early-onset type, survival and non-neurological symptoms improve but the effect on neurocognitive development is uncertain. The cblC defect can be screened by measuring propionylcarnitine, propionylcarnitine-to-acetylcarnitine ratio combined with the second tier markers methylmalonic acid and tHcy. For the cblE and cblG defects, evidence for the benefit of early treatment is weaker; and data on performance of Met, Met/Phe and tHcy even more limited. Individuals homozygous or compound heterozygous for MAT1A mutations may benefit from detection by NBS using Met, which on the other hand also detects asymptomatic heterozygotes. Clinical and laboratory data is insufficient to develop any recommendation on NBS for the cblD, cblF, cblJ defects, glycineN-methyltransferase-, S-adenosylhomocysteinehydrolase- and adenosine kinase deficiency

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis, multi-organ failure, and interrupted aortic arch

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.This work was supported by Grants PI060547 (to M.A.M.) and PI070167 (to A.L.A. and R.G.) from Instituto de Salud Carlos III (ISCIII), Ministry of Science and Innovation (MICIN), Spain; and GEN-0269/2006 from Comunidad de Madrid (CM) (to M.A.M. and R.G.). H.R. is supported by the Grant PI060547 (ISCIII). M.J.M. is recipient of a post-doctoral fellowship from ISCIII (CD06/00031). C.U. is recipient of a “Miguel Servet” research contract from ISCIII (CI04/00011).Peer Reviewe

Novel features in the evolution of adenylosuccinate lyase deficiency

Background: Adenylosuccinate lyase (ADSL) deficiency is an autosomal recessive disorder of the purine synthesis which results in accumulation of succinylpurines (succinyladenosine (S-Ado) and succinylamino-imidazole carboxamide riboside (SAICAr)) in body fluids. Patients present developmental delay, often accompanied by epilepsy and autistic spectrum disorders. Objectives: To describe atypical neurological features in the evolution of three novel unrelated cases of ADSL deficiency. Patients: A 9-year-old boy with severe cognitive impairment and autistic behaviour received d-ribose therapy for one year. Drug withdrawal was associated with acute neurological deterioration, severe brain atrophy and demyelination on MRI. The second patient is a 5.5-year-old girl with mild developmental delay who presented a benign course with moderate cognitive impairment as the only feature in her evolution. The final patient is a 14-year-old boy with severe cognitive impairment who developed drug-resistant epilepsy and bathing reflex seizures, progressive spasticity in the lower limbs and thoracic deformity. Methods: SAICAr and S-Ado in urine were analysed by HPLC with diode array detection. Diagnosis was confirmed by molecular analysis of the ADSL gene. Results: An elevation of S-Ado and SAICAr excretion in urine was detected in all three patients. The patients were homozygous for the missence change p.I369L and for the novel change p.M389V. Conclusion: Drug-resistant epilepsy and specific therapeutic interventions may modify the neurological outcome in ADSL deficiency. d-ribose must be considered with caution as, in our experience, it returns no clinical benefit and drug withdrawal can precipitate status epilepticus and acute neurological deterioration. © 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.Caixa Girona; Koplowitz Fundation; CIBERERPeer Reviewe

Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts.

[Background] Complex I (CI) deficiency is the most frequent cause of OXPHOS disorders. Recent studies have shown increases in reactive oxygen species (ROS) production and mitochondrial network disturbances in patients' fibroblasts harbouring mutations in CI subunits.[Objectives] The present work evaluates the impact of mutations in the NDUFA1 and NDUFV1 genes of CI on mitochondrial bioenergetics and dynamics, in fibroblasts from patients suffering isolated CI deficiency.[Results] Decreased oxygen consumption rate and slow growth rate were found in patients with severe CI deficiency. Mitochondrial diameter was slightly increased in patients' cells cultured in galactose or treated with 2′-deoxyglucose without evidence of mitochondrial fragmentation. Expression levels of the main proteins involved in mitochondrial dynamics, OPA1, MFN2, and DRP1, were slightly augmented in all patients' cells lines. The study of mitochondrial dynamics showed delayed recovery of the mitochondrial network after treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (cccp) in patients with severe CI deficiency. Intracellular ROS levels were not increased neither in glucose nor galactose medium in patients' fibroblasts.[Conclusion] Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency.Conclusion: Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency.Peer reviewe

Alta prevalencia de la mutación CBS p.T191M en pacientes homocistinúricos de Colombia

8 páginasHomocystinuria is an autosomal recessive disease most commonly caused by mutationsin cystathionine β-synthase (CBS). In this study we present the mutation analysis of 36 Colombian individuals from 10 unrelated kindred, with 11 individuals clinically classified as homocystinuric. Mutation analysis of the CBS gene revealed p.T191M, aprevalent mutation in Spain and Portugal, in the homozygous state in seven of the unrelated patients. Genotype-phenotype assessment of the p.T191M homozygous patients showed a high level of variability, including different severity in one pair of affected siblings. None of the patients responded biochemically to treatment with pharmacological doses of pyridoxine and folic acid as revealed by essentially unchangedhomocysteine levels. This study offered a unique opportunity to study 18 heterozygous(p.T191M/wt) relatives of the homocystinuric patients. One atypical finding was that many of them presented with above average total homocysteine levels, putting them atan increased risk for vascular disease. Cryptorchidism was present in three of the cases, one of which presented also with Klinefelter syndrome. In addition to the previously described p.T191M mutation, a new mutation, p.A288T, was identified in a single individual. In this paper we present the first characterization, at a molecular level, of patients with homocystinuria from Colombia.La homocistinuria es una enfermedad autosómica recesiva causada más comúnmente por mutaciones en la cistationina β-sintasa (CBS). En este estudio presentamos el análisis de mutaciones de 36 individuos colombianos de 10 familias no relacionadas, con 11 individuos clínicamente clasificados como homocistinúricos. El análisis de mutaciones del gen CBS reveló p.T191M, una mutación prevalente en España y Portugal, en estado homocigoto en siete de los pacientes no relacionados. La evaluación del genotipo-fenotipo de los pacientes homocigotos p.T191M mostró un alto nivel de variabilidad, incluida una gravedad diferente en un par de hermanos afectados. Ninguno de los pacientes respondió bioquímicamente al tratamiento con dosis farmacológicas de piridoxina y ácido fólico como lo revelan los niveles de homocisteína esencialmente sin cambios. Este estudio ofreció una oportunidad única de estudiar 18 familiares heterocigotos (p.T191M/wt) de pacientes homocistinúricos. Un hallazgo atípico fue que muchos de ellos presentaban niveles de homocisteína total superiores al promedio, lo que los colocaba en mayor riesgo de enfermedad vascular. La criptorquidia estuvo presente en tres de los casos, uno de los cuales presentó también el síndrome de Klinefelter. Además de la mutación p.T191M descrita anteriormente, se identificó una nueva mutación, p.A288T, en un solo individuo. En este artículo presentamos la primera caracterización, a nivel molecular, de pacientes con homocistinuria en Colombia