RESUMEN
Hundreds of mutations in a single gene result in rare diseases, but why mutations induce severe or attenuated states remains poorly understood. Defect in glycine decarboxylase (GLDC) causes Non-ketotic Hyperglycinemia (NKH), a neurological disease associated with elevation of plasma glycine. We unified a human multiparametric NKH mutation scale that separates severe from attenuated neurological disease with new in silico tools for murine and human genome level-analyses, gathered in vivo evidence from mice engineered with top-ranking attenuated and a highly pathogenic mutation, and integrated the data in a model of pre- and post-natal disease outcomes, relevant for over a hundred major and minor neurogenic mutations. Our findings suggest that highly severe neurogenic mutations predict fatal, prenatal disease that can be remedied by metabolic supplementation of dams, without amelioration of persistent plasma glycine. The work also provides a systems approach to identify functional consequences of mutations across hundreds of genetic diseases. Our studies provide a new framework for a large scale understanding of mutation functions and the prediction that severity of a neurogenic mutation is a direct measure of pre-natal disease in neurometabolic NKH mouse models. This framework can be extended to analyses of hundreds of monogenetic rare disorders where the underlying genes are known but understanding of the vast majority of mutations and why and how they cause disease, has yet to be realized.
Asunto(s)
Modelos Animales de Enfermedad , Glicina-Deshidrogenasa (Descarboxilante)/química , Glicina-Deshidrogenasa (Descarboxilante)/genética , Glicina/metabolismo , Hiperglicinemia no Cetósica/genética , Animales , Femenino , Genómica , Genotipo , Glicina/genética , Humanos , Hiperglicinemia no Cetósica/metabolismo , Hiperglicinemia no Cetósica/patología , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Mutación Missense , FenotipoRESUMEN
Obesity is associated with altered glycine metabolism in humans. This study investigated the mechanisms regulating glycine metabolism in obese rats. Eight-week-old Zucker diabetic fatty rats (ZDF; a type-II diabetic animal model) received either 1% glycine or 1.19% L-alanine (isonitrogenous control) in drinking water for 6 weeks. An additional group of lean Zucker rats also received 1.19% L-alanine as a lean control. Glycine concentrations in serum and liver were markedly lower in obese versus lean rats. Enteral glycine supplementation restored both serum and hepatic glycine levels, while reducing mesenteric and internal white fat mass compared with alanine-treated ZDF rats. Blood glucose and non-esterified fatty acid (NEFA) concentrations did not differ between the control and glycine-supplemented ZDF rats (P > 0.10). Both mRNA and protein expression of aminomethyltransferase (AMT) and glycine dehydrogenase, decarboxylating (GLDC) were increased in the livers of obese versus lean rats (P < 0.05). In contrast, glycine cleavage system H (GCSH) hepatic mRNA expression was downregulated in obese versus lean rats, although there was no change in protein expression. These findings indicate that reduced quantities of glycine observed in obese subjects likely results from an upregulation of the hepatic glycine cleavage system and that dietary glycine supplementation potentially reduces obesity in ZDF rats.
Asunto(s)
Tejido Adiposo Blanco/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Suplementos Dietéticos , Glicina/administración & dosificación , Hígado/efectos de los fármacos , Obesidad/tratamiento farmacológico , Tejido Adiposo Blanco/metabolismo , Alanina/administración & dosificación , Alanina/metabolismo , Aminometiltransferasa/genética , Aminometiltransferasa/metabolismo , Animales , Regulación del Apetito/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Tipo 2/metabolismo , Glicina/metabolismo , Proteína H del Complejo de la Glicina Descarboxilasa/genética , Proteína H del Complejo de la Glicina Descarboxilasa/metabolismo , Glicina-Deshidrogenasa (Descarboxilante)/genética , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Hígado/metabolismo , Masculino , Obesidad/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas ZuckerRESUMEN
Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.
Asunto(s)
Aminometiltransferasa/genética , Proteína H del Complejo de la Glicina Descarboxilasa/genética , Glicina-Deshidrogenasa (Descarboxilante)/genética , Mutación , Defectos del Tubo Neural/genética , Animales , Complejo Glicina-Descarboxilasa/metabolismo , Humanos , Ratones , Ratones Noqueados , Mutación MissenseRESUMEN
Potato (Solanum tuberosum L. cv. Désirée) plants with an antisense reduction in the P-protein of the glycine decarboxylase complex (GDC) were used to study the interaction between respiration and photorespiration. Mitochondria isolated from transgenic plants had a decreased capacity for glycine oxidation and glycine accumulated in the leaves. Malate consumption increased in leaves of GDC deficient plants and the capacity for malate and NADH oxidation increased in isolated mitochondria. A lower level of alternative oxidase protein and decreased partitioning of electrons to the alternative pathway was found in these plants. The adenylate status was altered in protoplasts from transgenic plants, most notably the chloroplastic ATP/ADP ratio increased. The lower capacity for photorespiration in leaves of GDC deficient plants was compensated for by increased respiratory decarboxylations in the light. This is interpreted as a decreased light suppression of the tricarboxylic acid cycle in GDC deficient plants in comparison to wild-type plants. The results support the view that respiratory decarboxylations in the light are restricted at the level of the pyruvate dehydrogenase complex and/or isocitrate dehydrogenase and that this effect is likely to be mediated by mitochondrial photorespiratory products.