RESUMEN
Methylmalonic aciduria cblB type (MMA cblB type, MMAB OMIM #251110), caused by a deficiency in the enzyme ATP:cob(I)alamin adenosyltransferase (ATR, E.C_2. 5.1.17), is a severe metabolic disorder with a poor prognosis despite treatment. We recently described the potential therapeutic use of pharmacological chaperones (PCs) after increasing the residual activity of ATR in patient-derived fibroblasts. The present work reports the successful generation of hepatocyte-like cells (HLCs) differentiated from two healthy and two MMAB induced pluripotent stem cell (iPSC) lines, and the use of this platform for testing the effects of PCs. The MMAB cells produced little ATR, showed reduced residual ATR activity, and had higher concentrations of methylmalonic acid compared to healthy HLCs. Differential proteome analysis revealed the two MMAB HCLs to show reproducible differentiation, but this was not so for the healthy HLCs. Interestingly, PC treatment in combination with vitamin B12 increased the amount of ATR available, and subsequently ATR activity, in both MMAB HLCs. More importantly, the treatment significantly reduced the methylmalonic acid content of both. In summary, the HLC model would appear to be an excellent candidate for the pharmacological testing of the described PCs, for analyzing the effects of new drugs, and investigating the repurposing of older drugs, before testing in animal models.
Asunto(s)
Células Madre Pluripotentes Inducidas , Ácido Metilmalónico , Proteínas Adaptadoras Transductoras de Señales , Errores Innatos del Metabolismo de los Aminoácidos , Animales , Hepatocitos , Humanos , Mutación , Proteínas Proto-Oncogénicas c-cblRESUMEN
Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones.
Asunto(s)
Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Fosfotransferasas (Fosfomutasas)/deficiencia , Proteostasis/genética , Triterpenos/farmacología , Trastornos Congénitos de Glicosilación/genética , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Fibroblastos/efectos de los fármacos , Glicosilación/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/genética , Humanos , Leupeptinas/farmacología , Mutación/genética , Triterpenos Pentacíclicos , Fosfotransferasas (Fosfomutasas)/antagonistas & inhibidores , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/metabolismo , Fosfotransferasas (Fosfomutasas)/ultraestructura , Pliegue de Proteína , Proteostasis/efectos de los fármacosRESUMEN
Propionic acidemia (PA) is a rare metabolic disease associated with mutations in genes encoding the α and ß subunits of the enzyme propionyl-CoA carboxylase. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species production and oxidative damage, which have been associated with the disease pathophysiology. Clinical symptoms are heterogeneous and include cardiac complications, mainly cardiac dysfunction and arrhythmias, which are recognized as one of the major life-threatening manifestations in patients. We aimed to investigate the molecular mechanisms underlying the cardiac phenotype using a hypomorphic mouse model (Pcca-/-(A138T)) that recapitulates some biochemical and clinical characteristics of PA. We demonstrate that Pcca-/-(A138T) mice present with depressed cardiac function along with impaired cell contractility when compared to the wild-type mice. Cardiac dysfunction in Pcca-/-(A138T) mice was associated with lower systolic Ca2+ release ([Ca2+]i transients), impairment in the sarcoplasmic reticulum (SR) Ca2+ load and decreased Ca2+ re-uptake by SR-Ca2+ ATPase (SERCA2a). These functional changes correlated well with the depressed activity of SERCA2a, the elevated ROS levels and SERCA2a oxidation rate in cardiomyocytes isolated from Pcca-/-(A138T) mice. In addition, decreased SR-Ca2+ load in Pcca-/-(A138T) cardiomyocytes was associated with increased diastolic Ca2+ release. The increase in Ca2+ sparks, Ca2+ waves and spontaneous [Ca2+]i transients in Pcca-/-(A138T) cardiomyocytes could be responsible for the induction of ventricular arrhythmias detected in these mice. Overall, our results uncover the role of impaired Ca2+ handling in arrhythmias and cardiac dysfunction in PA, and identify new targets for the development of therapeutic approaches for this devastating metabolic disease.
Asunto(s)
Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Acidemia Propiónica/metabolismo , Animales , Señalización del Calcio/fisiología , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Masculino , Ratones , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Two human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of two siblings with methylmalonic acidemia cblB type carrying mutations in the MMAB gene: c.287TâC (p.Ile96Thr) and a splicing loss-of-function variant c.584GâA affecting the last nucleotide of exon 7 in MMAB (p.Ser174Cysfs*23). Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Técnicas de Reprogramación Celular , Células Madre Pluripotentes Inducidas , Mutación Missense , Factores de Transcripción , Transferasas Alquil y Aril/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Sustitución de Aminoácidos , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Factor 4 Similar a Kruppel , Masculino , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Phenylketonuria (PKU, OMIM 261600) is an autosomal recessive disease, caused by mutations in the Phenylalanine Hydroxylase (PAH) gene situated in chromosome 12q22-q24.2. This gene has 13 exons. To date, 991 mutations have been described. The genotype is one of the main factors that determine the phenotype of this disease. OBJECTIVE: Characterize PKU genotype and phenotype seen in Chilean PKU patients. METHODS: We studied the PAH gene by restriction fragment length polymorphism (RFLP) and/or sequencing techniques to identify pathogenic mutations in 71 PKU subjects. We classified the phenotype according to Guldberg predicted value. RESULTS: We identified 26 different mutations in 134 of the 142 alleles studied (94.4%), 88.7% of the subjects had biallelic pathogenic mutations while 11.3% had only one pathogenic mutation identified. Compound heterozygous represented 85.9% of the cases. Exon 7 included the majority of mutations (26.9%) and 50% of mutations were missense. The most frequent mutations were c.1066-11G > A, c.442-?_509+?del and p.Val388Met. The majority of subjects (52.3%) had the classic phenotype. CONCLUSIONS: The most frequent mutations in our Chilean PKU population were p.Val388Met, c.442?_509+?del and c.1066-11G > A. It is possible to predict phenotype by detecting the genotype, and use this information to determine disease prognosis and adjust patient's medical and nutritional management accordingly.
RESUMEN
Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG).
Asunto(s)
Deficiencias en la Proteostasis/tratamiento farmacológico , Deficiencias en la Proteostasis/etiología , Animales , Ensayos Clínicos como Asunto , Descubrimiento de Drogas , Predisposición Genética a la Enfermedad , Glicosilación/efectos de los fármacos , Humanos , Mitocondrias , Mutación , Deficiencias en la Proteostasis/diagnóstico , Deficiencias en la Proteostasis/metabolismoRESUMEN
Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.
Asunto(s)
Metilmalonil-CoA Descarboxilasa/genética , Mitocondrias/genética , Estrés Oxidativo/genética , Acidemia Propiónica/genética , Animales , Antioxidantes/metabolismo , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Homeostasis , Humanos , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/genética , Metilmalonil-CoA Descarboxilasa/metabolismo , Ratones , Mitocondrias/metabolismo , Mitocondrias/patología , Oxidación-Reducción , Acidemia Propiónica/patología , Análisis por Matrices de Proteínas , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
Specific mitochondrial enzymatic deficiencies in the catabolism of branched-chain amino acids cause methylmalonic aciduria (MMA), propionic acidemia (PA) and maple syrup urine disease (MSUD). Disease-causing mutations were identified in nine unrelated branched-chain organic acidurias (BCOA) patients. We detected eight previously described mutations: p.Asn219Tyr, p.Arg369His p.Val553Glyfs*17 in MUT, p.Thr198Serfs*6 in MMAA, p.Ile144_Leu181del in PCCB, p.Gly288Valfs*11, p.Tyr438Asn in BCKDHA and p.Ala137Val in BCKDHB gene. Interestingly, we identified seven novel genetic variants: p.Leu549Pro, p.Glu564*, p.Leu641Pro in MUT, p.Tyr206Cys in PCCB, p.His194Arg, p.Val298Met in BCKDHA and p.Glu286_Met290del in BCKDHB gene. In silico and/or eukaryotic expression studies confirmed pathogenic effect of all novel genetic variants. Aberrant enzymes p.Leu549Pro MUT, p.Leu641Pro MUT and p.Tyr206Cys PCCB did not show residual activity in activity assays. In addition, activity of MUT enzymes was not rescued in the presence of vitamin B12 precursor in vitro which was in accordance with non-responsiveness or partial responsiveness of patients to vitamin B12 therapy. Our study brings the first molecular genetic data and detailed phenotypic characteristics for MMA, PA and MSUD patients for Serbia and the whole South-Eastern European region. Therefore, our study contributes to the better understanding of molecular landscape of BCOA in Europe and to general knowledge on genotype-phenotype correlation for these rare diseases.
Asunto(s)
3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Enfermedad de la Orina de Jarabe de Arce/genética , Metilmalonil-CoA Descarboxilasa/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Acidemia Propiónica/genética , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/química , Errores Innatos del Metabolismo de los Aminoácidos/fisiopatología , Aminoácidos de Cadena Ramificada/genética , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Enfermedad de la Orina de Jarabe de Arce/fisiopatología , Metilmalonil-CoA Descarboxilasa/química , Proteínas de Transporte de Membrana Mitocondrial/química , Mutación , Fenotipo , Acidemia Propiónica/fisiopatología , Conformación ProteicaRESUMEN
Deficiencies in glycosyltransferases, glycosidases or nucleotide-sugar transporters involved in protein glycosylation lead to congenital disorders of glycosylation (CDG), a group of genetic diseases mostly showing multisystem phenotype. Despite recent advances in the biochemical and molecular knowledge of these diseases, no effective therapy exists for most. Efforts are now being directed toward therapies based on identifying new targets, which would allow to treat specific patients in a personalized way. This work presents proof-of concept for the antisense RNA rescue of the Golgi-resident protein TMEM165, a gene involved in a new type of CDG with a characteristic skeletal phenotype. Using a functional in vitro splicing assay based on minigenes, it was found that the deep intronic change c.792+182G>A is responsible for the insertion of an aberrant exon, corresponding to an intronic sequence. Antisense morpholino oligonucleotide therapy targeted toward TMEM165 mRNA recovered normal protein levels in the Golgi apparatus of patient-derived fibroblasts. This work expands the application of antisense oligonucleotide-mediated pseudoexon skipping to the treatment of a Golgi-resident protein, and opens up a promising treatment option for this specific TMEM165-CDG.
Asunto(s)
Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/terapia , Exones/genética , Terapia Genética/métodos , Proteínas de la Membrana/genética , Mutagénesis Insercional/genética , ARN sin Sentido/genética , Análisis de Varianza , Antiportadores , Proteínas de Transporte de Catión , Cartilla de ADN/genética , ADN Complementario/genética , Fibroblastos , Aparato de Golgi/genética , Humanos , Immunoblotting , Técnicas In Vitro/métodos , Microscopía Fluorescente , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/uso terapéuticoRESUMEN
Methylmalonic aciduria (MMA) cblB type is caused by mutations in the MMAB gene, which codes for the enzyme adenosine triphosphate (ATP): cobalamin adenosyltransferase (ATR). This study reports differences in the metabolic and disease outcomes of two pairs of siblings with MMA cblB type, respectively harbouring the novel changes p.His183Leu/p.Arg190dup (P1 and P2) and the previously described mutations p.Ile96Thr/p.Ser174fs (P3 and P4). Expression analysis showed p.His183Leu and p.Arg190dup to be destabilizing mutations. Both were associated with reduced ATR stability and a shorter half-life than wild-type ATR. Analysis of several parameters related to oxidative stress and mitochondrial function showed an increase in reactive oxygen species (ROS) content, a decrease in mitochondrial respiration and changes in mitochondria morphology and structure in patient-derived fibroblasts compared to control cells. The impairment in energy production and the presence of oxidative stress and fission of the mitochondrial reticulum suggested mitochondrial dysfunction in cblB patients' fibroblasts. The recovery of mitochondrial function should be a goal in efforts to improve the clinical outcome of MMA cblB type.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Sustitución de Aminoácidos , Fibroblastos/metabolismo , Humanos , Espacio Intracelular/metabolismo , Mitocondrias/ultraestructura , Evaluación de Resultado en la Atención de Salud , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , HermanosRESUMEN
Hereditary tyrosinemia type I (HT1) is a rare disease caused by a deficiency of fumarylacetoacetate hydrolase (FAH) in the tyrosine catabolic pathway, resulting mainly in hepatic alterations due to accumulation of the toxic metabolites fumarylacetoacetate, maleylacetoacetate and succinylacetone. We have characterized using minigenes four splicing mutations affecting exonic or intronic nucleotides of the FAH gene identified in two HT1 patients. Two of the mutations are novel, c.82-1G>A and c.913G>C and the other two have been previously associated with a splicing defect (c.836A>G and c.1062+5G>A). All mutations were confirmed to affect splicing in minigenes, resulting in exon skipping or activation of a cryptic splice site. We have analyzed the effect of different compounds known to modulate splicing (valproic acid, phenyl butyrate, M344, EIPA, and resveratrol) and the overexpression of splice factors of the SR protein family on the transcriptional profile of the mutant minigenes. For the c.836A>G mutation, a partial recovery of the correctly spliced transcript was observed. These results confirm the relevance of performing functional studies for mutations potentially affecting the splicing process and open the possibility of supplementary therapeutic approaches to diseases caused by splicing defects.
Asunto(s)
Hidrolasas/genética , Mutación/genética , Empalme del ARN/genética , Tirosinemias/enzimología , Tirosinemias/genética , Secuencia de Bases , Simulación por Computador , Humanos , Lactante , Datos de Secuencia MolecularRESUMEN
Knowledge of hyperphenylalaninemia (HPA) mutational spectrum in a population allows in many cases an accurate prediction of the phenotype and tetrahydrobiopterin (BH4) responsiveness, thus selecting an adequate treatment. In this work, we have performed the molecular characterization of 105 HPA patients from Galicia, in the northwest region of Spain, evaluating their phenotype and BH4 response. The mutational spectrum analysis showed 47 distinct mutations in 89 families, 37 of them (78.7%) corresponding to missense mutations. Six mutations account for 47.2% of all the investigated alleles, each one with a frequency ≥ 5% (IVS10-11G>A, p.R261Q, p.V388M, p.R176L, p.E280K, p.A300S). The most prevalent HPA mutations in Galicia are the common Mediterranean mutation IVS10-11G>A and p.R261Q, with frequencies of 13.8% and 10.5%, respectively. One novel mutation (p.K361Q; c.1081A>C) was also reported. Although a good genotype-phenotype correlation is observed, there is no exact correlation for some genotypes involving mutations p.R261Q, p.I65T or IVS10-11G>A. Forty seven patients were monitored for post-challenge BH4, establishing genotype-based predictions of BH4-responsiveness in all of them. All phenylketonuric patients with 2 nonresponsive mutations were unresponsive to BH4 and patients with mutations previously associated with BH4 responsiveness in the two alleles had a clear positive response to the test, with the exception of 5 patients with mutations p.R261Q, p.I65T and p.R68S. Our study supports a similar degree of heterogeneity of the HPA mutation spectrum in Galicia compared to reported data from Southern Europe. Patients carrying null mutations in both alleles showed the highest degree of concordance with the most severe phenotypes. Genotype is a good predictor of BH4 response.
Asunto(s)
Biopterinas/análogos & derivados , Mutación , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/epidemiología , Fenilcetonurias/genética , Adolescente , Adulto , Biopterinas/farmacología , Niño , Preescolar , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Humanos , Masculino , Epidemiología Molecular , Linaje , Fenilalanina Hidroxilasa/deficiencia , España/epidemiología , Adulto JovenRESUMEN
Inherited metabolic diseases (IMDs) belong to the group of rare diseases due to their low individual prevalence. Most of them are inherited in autosomal recessive fashion and represent good candidates for novel therapeutical strategies aimed at recovering partial enzyme function as they lack an effective treatment, and small levels of enzymatic activity have been shown to be associated with improved outcome and milder phenotypes. Recently, a novel therapeutic approach for genetic diseases has emerged, based on the ability of aminoglycosides and other compounds in allowing translation to proceed through a premature termination codon introduced by a nonsense mutation, which frequently constitute a significant fraction of the mutant alleles in a population. In this review we summarize the essentials of what is known as suppression therapy, the different compounds that have been identified by high-throughput screens or developed using a medicinal chemistry approach and the preclinical and clinical trials that are being conducted in general and in the field of IMDs in particular. Several IMDs have shown to be good models for evaluating readthrough compounds using patients' cells carrying nonsense mutations, monitoring for an increase in functional recovery and/or enzyme activity. Overall, the positive results obtained indicate the feasibility of the approach for different diseases and although the levels of protein function reached are low, they may be enough to alleviate the consequences of the pathology. Nonsense suppression thus represents a potential therapy or supplementary treatment for a number of IMD patients encouraging further clinical trials with readthrough drugs with improved functionality and low toxicity.
RESUMEN
Development of pseudoexon exclusion therapies by antisense modification of pre-mRNA splicing represents a type of personalized genetic medicine. Here we present the cellular antisense therapy and the cell-based splicing assays to investigate the effect of two novel deep intronic changes c.1957-898A>G and c.1957-920C>A identified in the methylmalonyl-coenzyme A (CoA) mutase (MUT) gene. The results show that the nucleotide change c.1957-898A>G is a pathological mutation activating pseudoexon insertion and that antisense morpholino oligonucleotide (AMO) treatment in patient fibroblasts leads to recovery of MUT activity to levels 25 to 100% of control range. On the contrary, the change c.1957-920C>A, identified in two fibroblasts cell lines in cis with c.1885A>G (p.R629G) or c.458T>A (p.D153V), appears to be a rare variant of uncertain clinical significance. The functional analysis of c.1885A>G and c.458T>A indicate that they are the disease-causing mutations in these two patients. The results presented here highlight the necessity of scanning the described intronic region for mutations in MUT-affected patients, followed by functional analyses to demonstrate the pathogenicity of the identified changes, and extend previous work of the applicability of the antisense approach in methylmalonic aciduria (MMAuria) for a novel intronic mutation.
Asunto(s)
Exones/genética , Errores Innatos del Metabolismo/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Secuencia de Bases , Línea Celular , Genotipo , Humanos , Intrones/genética , Errores Innatos del Metabolismo/enzimología , Metilmalonil-CoA Mutasa/deficiencia , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Propionatos/metabolismo , Empalme del ARN/genéticaRESUMEN
Methylmalonic acidaemia (MMA) is a genetic disorder caused by defects in methylmalonyl-CoA mutase or in any of the different proteins involved in the synthesis of adenosylcobalamin. The aim of this work was to examine the biochemical and clinical phenotype of 32 MMA patients according to their genotype, and to study the mutant mRNA stability by real-time PCR analysis. Using cellular and biochemical methods, we classified our patient cohort as having the MMA forms mut (n = 19), cblA (n = 9) and cblB (n = 4). All the mut (0) and some of the cblB patients had the most severe clinical and biochemical manifestations, displaying non-inducible propionate incorporation in the presence of hydroxocobalamin (OHCbl) in vitro and high plasma odd-numbered long-chain fatty acid (OLCFA) concentrations under dietary therapy. In contrast, mut (-) and cblA patients exhibited a milder phenotype with propionate incorporation enhanced by OHCbl and normal OLCFA levels under dietary therapy. No missense mutations identified in the MUT gene, including mut (0) and mut (-) changes, affected mRNA stability. A new sequence variation (c.562G>C) in the MMAA gene was identified. Most of the cblA patients carried premature termination codons (PTC) in both alleles. Interestingly, the transcripts containing the PTC mutations were insensitive to nonsense-mediated decay (NMD).
Asunto(s)
Transferasas Alquil y Aril/genética , Errores Innatos del Metabolismo de los Aminoácidos/genética , Prueba de Complementación Genética , Proteínas de Transporte de Membrana/genética , Ácido Metilmalónico/sangre , Metilmalonil-CoA Mutasa/genética , Proteínas Mitocondriales/genética , Biomarcadores/análisis , Línea Celular , Estudios de Cohortes , Genotipo , Humanos , Lactante , Recién Nacido , Metilmalonil-CoA Mutasa/clasificación , Proteínas de Transporte de Membrana Mitocondrial , Mutación/fisiología , Vitamina B 12/genéticaRESUMEN
We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.
Asunto(s)
Variación Genética/genética , Intrones/genética , Metilmalonil-CoA Descarboxilasa/genética , Metilmalonil-CoA Mutasa/genética , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Acidemia Propiónica/terapia , Empalme del ARN/genética , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/terapia , Células Cultivadas , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Morfolinas , Mutación/genética , Propionatos/metabolismo , Acidemia Propiónica/enzimología , Acidemia Propiónica/genética , Precursores del ARN/genéticaRESUMEN
Methylmalonic acidaemia (MMA) is a heterogeneous group of rare genetic metabolic disorders caused by defects related to intracellular cobalamin (vitamin B(12)) metabolism. Increasing evidence has emerged suggesting that free radical generation is involved in the pathophysiology of neurodegenerative diseases, including some inborn errors of metabolism. We have previously identified in MMA patients several differentially expressed proteins involved in oxidative stress [mitochondrial superoxide dismutase (MnSOD) and mitochondrial glycerophosphate dehydrogenase (mGPDH)] and apoptosis by a proteomic approach. We have now extensively evaluated various parameters related to oxidative stress and apoptosis in cultured fibroblasts from a spectrum of patients with methylmalonic acidaemia. Fibroblasts from several MMA patients showed a significant increase in intracellular reactive oxygen species (ROS) content and in MnSOD expression level with respect to controls, suggesting a cellular response to intrinsic ROS stress. Moreover, we have demonstrated, using siRNA, that mGPDH is an important ROS generator in MMA patients. Cells from patients with MMA had a higher rate of apoptosis than those of controls and there was evidence that this process primarily involves the mitochondrial/caspase-dependent pathway. ROS level-phenotype correlation revealed that patients with severe neonatal cblB disorder had elevated intracellular ROS content. These findings support the possible role of oxidative stress in the pathophysiology of methylmalonic acidaemia.
Asunto(s)
Apoptosis , Errores Innatos del Metabolismo/metabolismo , Ácido Metilmalónico/sangre , Especies Reactivas de Oxígeno/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Células Cultivadas , Fibroblastos/enzimología , Fibroblastos/patología , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Errores Innatos del Metabolismo/patología , Mitocondrias/enzimología , Estrés Oxidativo , ARN Interferente Pequeño/genética , Piel/enzimología , Piel/patología , Superóxido Dismutasa/metabolismoRESUMEN
In this report we summarize our experience in prenatal diagnosis of propionic acidemia (PA) since 1987. Overall, we have investigated 25 pregnancies at risk from 19 unrelated families. Until genetic structure of the genes involved in PA was elucidated, prenatal diagnosis has been successfully performed by means of metabolite quantitation and/or enzymatic assays in foetal issue. Today, direct propionyl-CoA carboxylase activity assay in combination with molecular analysis in chorion villi can be regarded as a fast and reliable method of choice for prenatal diagnosis of this organic acidemia.
Asunto(s)
Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/genética , Diagnóstico Prenatal/métodos , Propionatos/sangre , Líquido Amniótico/citología , Células Cultivadas , Vellosidades Coriónicas/enzimología , Cromonas , Análisis Mutacional de ADN , Femenino , Humanos , Metilmalonil-CoA Descarboxilasa/genética , Mutación , Reacción en Cadena de la Polimerasa , Embarazo , Acidemia PropiónicaRESUMEN
Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance. To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects. A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied. For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events. Functional characterization of the mutant missense alleles has been accomplished using different prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models. For the PCCA gene, the main molecular effect of the expressed mutations is related to protein instability, except two mutations in the active site predictably affecting ATP binding. In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity. A few carboxy-terminal PCCB mutations affect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer. The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed. Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/genética , Variación Genética , Mutación , Propionatos/metabolismo , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Sitios de Unión , Ligasas de Carbono-Carbono/metabolismo , Humanos , Modelos Moleculares , Fenotipo , Conformación Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.