AAV-mediated expression of mouse or human GLDC normalises metabolic biomarkers in a GLDC-deficient mouse model of Non-Ketotic Hyperglycinemia.

Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH have limited effect and are not curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using a ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one­carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one­carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.

The behavioral phenotype of children and adolescents with attenuated non-ketotic hyperglycinemia, intermediate to good subtype.

AIM: We aim to describe the behavioral phenotype of children and adolescents with the good to intermediate attenuated form of non-ketotic hyperglycinemia (NKH) and to explore associations between the behavioral phenotype and age, sex, plasma glycine levels and drug treatment. METHOD: Parents of children with attenuated NKH completed questionnaires assessing maladaptive behavior, adaptive behavior, social communication, speech/language development and motor development in addition to demographic and medical questions. RESULTS AND INTERPRETATION: Twelve children, age 6 to 21y, functioned at mild to severe intellectual disability levels. Their speech/language development was in line with their developmental quotient. Relative to their intellectual functioning, their motor development and communication were weaker in comparison to their general development. Their adaptive behavior, however, appeared a relative strength. There was no evidence for autism spectrum disorder occurring more frequently than expected, rather social skills, except for communication, were rated as a relative strength. Maladaptive behaviors with ADHD-like characteristics were present in more than two thirds of children. Maladaptive behaviors were significantly related to female sex and to taking dextromethorphan, but no significant relation between plasma glycine levels and behavior was found. Future studies will need to evaluate causality in the observed relation between dextromethorphan use and maladaptive behaviors. Clinicians should reconsider the benefit of dextromethorphan when presented with disruptive behaviors in children with attenuated NKH.

Glycine disrupts myelin, glutamatergic neurotransmission, and redox homeostasis in a neonatal model for non ketotic hyperglycinemia.

Non ketotic hyperglycinemia (NKH) is an inborn error of glycine metabolism caused by mutations in the genes encoding glycine cleavage system proteins. Classic NKH has a neonatal onset, and patients present with severe neurodegeneration. Although glycine accumulation has been implicated in NKH pathophysiology, the exact mechanisms underlying the neurological damage and white matter alterations remain unclear. We investigated the effects of glycine in the brain of neonatal rats and MO3.13 oligodendroglial cells. Glycine decreased myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) in the corpus callosum and striatum of rats on post-natal day (PND) 15. Glycine also reduced neuroglycan 2 (NG2) and N-methyl-d-aspartate receptor subunit 1 (NR1) in the cerebral cortex and striatum on PND15. Moreover, glycine reduced striatal glutamate aspartate transporter 1 (GLAST) content and neuronal nucleus (NeuN), and increased glial fibrillary acidic protein (GFAP) on PND15. Glycine also increased DCFH oxidation and malondialdehyde levels and decreased GSH concentrations in the cerebral cortex and striatum on PND6, but not on PND15. Glycine further reduced viability but did not alter DCFH oxidation and GSH levels in MO3.13 cells after 48- and 72-h incubation. These data indicate that impairment of myelin structure and glutamatergic system and induction of oxidative stress are involved in the neuropathophysiology of NKH.

GLRX5-associated [Fe-S] cluster biogenesis disorder: further characterisation of the neurological phenotype and long-term outcome.

BACKGROUND: Identification and characterisation of monogenic causes of complex neurological phenotypes are important for genetic counselling and prognostication. Bi-allelic pathogenic variants in the gene encoding GLRX5, a protein involved in the early steps of Fe-S cluster biogenesis, are rare and cause two distinct phenotypes: isolated sideroblastic anemia and a neurological phenotype with variant non-ketotic hyperglycinemia. In this study, we analysed the evolution of clinical and MRI findings and long-term outcome of patients with GLRX5 mutations. METHODS: Four patients from three Australian families of Lebanese descent were identified. All patients presented in childhood and were followed up into adult life through multiple clinical assessments. All were prescribed sodium benzoate. RESULTS: All patients (all females, age range 18-56 years) showed a complex neurological phenotype characterised by varying combinations of spastic paraparesis, length-dependent motor/sensory-motor axonal polyneuropathy, and psychiatric disturbances with variable intellectual disability. All had non-ketotic hyperglycinemia and a homozygous pathogenic c.151_153delAAG (p.K51del) change in GLRX5. Motor disability gradually progressed reaching moderate disability during adolescence and moderately severe disability during adult life. The major MRI finding was the upper cervical spinal cord signal changes with contrast enhancement noted in all and additional leukoencephalopathy in one. On follow up MRI, the white matter lesions diminished on a subsequent scan and then remained static over time. The spinal cord showed gliotic changes. Two patients have previously demonstrated low pyruvate dehydrogenase complex deficiency but none had plasma lactate elevation, nor biochemical evidence of branch-chain keto-dehydrogenase deficiency. Glycine levels reduced in patients that tolerated sodium benzoate, possibly stabilising clinical manifestations. CONCLUSIONS: This report demonstrates that the p.K51del GLRX5 variant causes a distinct and predictable neurological phenotype. The clinical assessments spanning from childhood to adult life enable physicians to infer the natural history of GLRX5 related neurological disorder. There may be widespread metabolic consequences, and optimal management is unknown.

Regulation of glycine metabolism by the glycine cleavage system and conjugation pathway in mouse models of non-ketotic hyperglycinemia.

Glycine abundance is modulated in a tissue-specific manner by use in biosynthetic reactions, catabolism by the glycine cleavage system (GCS), and excretion via glycine conjugation. Dysregulation of glycine metabolism is associated with multiple disorders including epilepsy, developmental delay, and birth defects. Mutation of the GCS component glycine decarboxylase (GLDC) in non-ketotic hyperglycinemia (NKH) causes accumulation of glycine in body fluids, but there is a gap in our knowledge regarding the effects on glycine metabolism in tissues. Here, we analysed mice carrying mutations in Gldc that result in severe or mild elevations of plasma glycine and model NKH. Liver of Gldc-deficient mice accumulated glycine and numerous glycine derivatives, including multiple acylglycines, indicating increased flux through reactions mediated by enzymes including glycine-N-acyltransferase and arginine: glycine amidinotransferase. Levels of dysregulated metabolites increased with age and were normalised by liver-specific rescue of Gldc expression. Brain tissue exhibited increased abundance of glycine, as well as derivatives including guanidinoacetate, which may itself be epileptogenic. Elevation of brain tissue glycine occurred even in the presence of only mildly elevated plasma glycine in mice carrying a missense allele of Gldc. Treatment with benzoate enhanced hepatic glycine conjugation thereby lowering plasma and tissue glycine. Moreover, administration of a glycine conjugation pathway intermediate, cinnamate, similarly achieved normalisation of liver glycine derivatives and circulating glycine. Although exogenous benzoate and cinnamate impact glycine levels via activity of glycine-N-acyltransferase, that is not expressed in brain, they are sufficient to lower levels of glycine and derivatives in brain tissue of treated Gldc-deficient mice.

Características electroclínicas en pacientes menores de 18 años con errores innatos del metabolismo (EIM) y epilepsia / Electro clinical characteristics in pediatric patients with inborn errors of metabolism (IEM) and epilepsy

INTRODUCCIÓN: Los errores innatos del metabolismo (EIM) son un grupo de enfermedades de origen genético, que entre el 40 % y el 60 % pueden manifestar crisis convulsivas. OBJETIVO: En este estudio se establecieron las características clínicas y electroencefalográficas en una muestra de 20 niños con diagnóstico de EIM y epilepsia. MATERIALES Y METODOS: La metodología utilizada fue un estudio descriptivo de series de casos retrospectivo. RESULTADOS: El 65 % de los pacientes de la muestra eran niños, el EIM de moléculas pequeñas fue el más frecuente (70 %). En cuanto a las variables clínicas, 90 % tenían encefalopatía, 75 % epilepsia refractaria y 55 °% crisis generalizadas. En electroencefalografía (EEG), 90 % de los pacientes tenían ritmo de fondo anormal, 80 % grafoelementos del sueño mal estructurados, 36 % de los afectados por EIM de moléculas pequeñas tenían patrón EEG multifocal y 100 % de los pacientes con déficit de producción de energía tuvieron patrón EEG focal. CONCLUSION: El tipo de EIM más frecuente en el estudio fue de moléculas pequeñas, con grados variables de encefalopatía y epilepsia refractaria. La anormalidad electroencefalográfica más frecuente fue el ritmo de fondo anormal debido a grafoelementos de sueño mal estructurados, en tanto que el patrón eléctrico fue dependiente de la edad y el tipo de EIM.

SUMMARY INTRODUCTION: Inborn errors of metabolism (IEM) are a group of diseases of genetic origin and they may manifest with seizures at some point of their evolution such as 40 to 60 percent of cases. SUBJECT: In this study, the clinical and electroencephalographic characteristics were established in a sample of 20 children diagnosed with IEM and epilepsy. METHODS: The methodology was a descriptive way of retrospective case series. RESULTS: The group was constituted 65 % by males. The EIM of small molecules was the most frequent (70 %). Regarding the clinical variables, 90 % had encephalopathy, 75 % refractory epilepsy and 55 % generalized epilepsy. About the electroencephalographic facts, 90 % had an abnormal basal activity, 80 % poorly structured sleep elements. The most frequent electroencephalographic pattern in small molecules disease's patients was multifocal (36 %) but in deficit of energy production's patients was focal (100 %). CONCLUSION: The type of IEM that predominated in this study was small molecules, with varying degrees of encephalopathy and refractory epilepsy. The most frequent electroencephalographic variable was abnormal background rhythm, with poorly structured sleep graphoelements. The electroencephalographic pattern depends on the age and type of IEM.

Bezafibrate Prevents Glycine-Induced Increase of Antioxidant Enzyme Activities in Rat Striatum.

Non-ketotic hyperglycinemia (NKH) is a severe neurological disorder caused by defects in glycine (GLY) catabolism and characterized by a high cerebrospinal fluid/plasma GLY ratio. Treatment is often ineffective and limited to the control of symptoms and detoxification of GLY. In the present work, we investigated the in vivo effects of GLY intracerebroventricular administration on oxidative stress parameters in rat striatum, cerebral cortex, and hippocampus. In vitro effects of GLY were also evaluated in striatum. The effects of bezafibrate (BEZ), a potential neuroprotective agent, on the possible alterations caused by GLY administration were further evaluated. Our in vivo results showed that GLY increased the activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) in striatum. Furthermore, GLY decreased the concentrations of total glutathione and reduced glutathione (GSH), as well as GSH/oxidized glutathione ratio in vivo in hippocampus. In vitro data also showed that GLY induced lipid peroxidation and decreased GSH in striatum. Regarding the effects of BEZ, we found that GLY-induced increase of GPx, SOD, and GR activities was attenuated or prevented by this compound. However, BEZ did not alter GLY-induced decrease of GSH in hippocampus. We hypothesize that GLY-induced increase of the activities of antioxidant enzymes in striatum occurs as a mechanism to avoid accumulation of reactive oxygen species and consequent oxidative damage. Furthermore, since BEZ prevented GLY-induced alterations, it might be considered as an adjuvant therapy for NKH.

The case of pyridoxine dependent epilepsy misdiagnosed as non-ketotic hyperglycinemia.

Gazeteci-Tekin H, Demir M, Aktan G, Tekgül H, Gökben S. The case of pyridoxine dependent epilepsy misdiagnosed as non-ketotic hyperglycinemia. Turk J Pediatr 2019; 61: 599-603. Pyridoxine-dependent epilepsy (PDE) is a rare but an important condition, since early diagnosis and treatment result in normal or near normal psychomotor development. It is caused by mutations in the Antiquitin (ALDH7A1) gene. Different clinical findings may appear in the deficiency of pyridoxine, which is the cofactor of many enzymes. A wide variety of clinical and laboratory findings can cause confusion during diagnosis. We present a male with neonatal convulsions; structural brain anomaly, hyperglycinemia in CSF/plasma, with ALDH7A1 Compound heterozygote mutation.

A novel compound heterozygous variant identified in GLDC gene in a Chinese family with non-ketotic hyperglycinemia.

BACKGROUND: Non-ketotic hyperglycinemia (NKH) is a rare, devastating autosomal recessive disorder of glycine metabolism with a very poor prognosis. Currently, few studies have reported genetic profiling of Chinese NKH patients. This study aimed to identify the genetic mutations in a Chinese family with NKH. METHODS: A Chinese family of Han ethnicity, with three siblings with NKH was studied. Sanger sequencing and multiplex ligation-dependent probe amplification combined with SYBR green real-time quantitative PCR was used to identify potential mutations in the GLDC, AMT and GCSH genes. The potential pathogenicity of the identified missense mutation was analyzed using SIFT, PolyPhen-2, PROVEAN and MutationTaster software. RESULTS: All patients exhibited severe and progressive clinical symptoms, including lethargy, hypotonia and seizures, and had greatly elevated glycine levels in their plasma and CSF. Molecular genetic analysis identified compound heterozygous variants in the GLDC gene in these three siblings, including a novel missense variant c.2680A > G (p.Thr894Ala) in exon 23 and a heterozygous deletion of exon 3, which were inherited respectively from their parents. In silico analysis, using several different types of bioinformatic software, predicted that the novel variant c.2680A > G in the GLDC gene was pathogenic. Moreover, the deletion of exon 3 was identified for the first time in a Chinese population. CONCLUSIONS: A novel missense variant and a previously reported deletion in GLDC gene were identified. The two variants of GLDC gene identified probably underlie the pathogenesis of non-ketotic hyperglycinemia in this family, and also enrich the mutational spectrum of GLDC gene.

Partitioning of One-Carbon Units in Folate and Methionine Metabolism Is Essential for Neural Tube Closure.

Abnormal folate one-carbon metabolism (FOCM) is implicated in neural tube defects (NTDs), severe malformations of the nervous system. MTHFR mediates unidirectional transfer of methyl groups from the folate cycle to the methionine cycle and, therefore, represents a key nexus in partitioning one-carbon units between FOCM functional outputs. Methionine cycle inhibitors prevent neural tube closure in mouse embryos. Similarly, the inability to use glycine as a one-carbon donor to the folate cycle causes NTDs in glycine decarboxylase (Gldc)-deficient embryos. However, analysis of Mthfr-null mouse embryos shows that neither S-adenosylmethionine abundance nor neural tube closure depend on one-carbon units derived from embryonic or maternal folate cycles. Mthfr deletion or methionine treatment prevents NTDs in Gldc-null embryos by retention of one-carbon units within the folate cycle. Overall, neural tube closure depends on the activity of both the methionine and folate cycles, but transfer of one-carbon units between the cycles is not necessary.

Clinical heterogeneity of glycine encephalopathy in three Palestinian siblings: A novel mutation in the glycine decarboxylase (GLDC) gene.

INTRODUCTION: Glycine encephalopathy (GE), also known as non-ketotic hyperglycinemia (NKH), is a rare inborn error of glycine metabolism caused by a defect in glycine cleavage system, a multi-enzyme complex located in mitochondrial membrane. This defect results in elevated glycine concentration in plasma and cerebrospinal fluid (CSF). Clinical manifestations vary from severe lethargy, hypoactivity and apneic episodes in the neonatal form, mild or moderate psychomotor delay and seizures in the infantile form, and abnormal behaviors, ataxia and choreoathetoid movements in late onset form. More than 50 GLDC mutations were found, reflecting large heterogeneity of the gene. METHODS: We describe the clinical, biochemical and molecular characteristics of three Palestinian siblings who have distinct clinical phenotypes. Molecular study was performed utilizing standard Polymerase Chain Reaction (PCR) amplification then direct DNA sequencing for the affected family members. RESULTS: Their phenotypes included severe symptoms in neonatal period, infantile onset of seizure and psychomotor delay and a mild late-onset form with speech delay at age 20months. All siblings were homozygous for a novel mutation Y164H in exon 4 of GLDC gene. The described novel homozygous variant in our study is predicted deleterious and pathogenic. CONCLUSIONS: This article further expands the genetic spectrum of glycine encephalopathy and adds an evidence of the clinical heterogeneity of glycine encephalopathy even in siblings with identical mutation.

Caracterización clínica, bioquímica e imagenológica en una cohorte de pacientes diagnosticados con hiperglicinemia no cetósica clásica: estudio ambispectivo 2000-2014, Medellín, Colombia / Clinical, biochemical and imaging characterization in a cohort of patients diagnosed with classical nonketotic hyperglycinemia: ambispective study 2000-2014, Medellin-Colombia

Introducción: La hiperglicinemia no cetósica (HGNC) es un error innato del metabolismo del grupo de las aminoacidopatías, de carácter autosómico recesivo, causado por un defecto en el sistema de clivaje de la glicina. Es una entidad rara y no se conoce su incidencia en Colombia. Objetivo: Describir características clínicas, bioquímica e imagenológicas en una cohorte de pacientes diagnosticados con hiperglicinemia no cetósica clásica Materiales y métodos: Estudio de tipo descriptivo, ambispectivo, en el periodo enero 2000-2014, en varios centros de Medellín. Resultados: Se incluyeron 20 pacientes que cumplían criterios de inclusión, de los 35 pacientes que cumplían con el criterio de búsqueda, en su mayoría de sexo femenino y con un Apgar adecuado al nacer. El promedio de inicio de los síntomas fue de 2,6 días; somnolencia, hipoactividad, apnea, convulsiones y singulto fueron los principales síntomas, y las convulsiones de tipo focal las más frecuentes. La relación glicina LCR/plasma en promedio fue 0,42. El patrón estallido-supresión en el electroencefalograma y la ausencia o retraso en la mielinización de la sustancia blanca supratentorial en la resonancia magnética fueron hallazgos comunes. Conclusión: La HGNC es frecuente en nuestro medio, por lo cual es necesario que se disponga de pruebas bioquímicas y moleculares necesarias para diagnóstico oportuno, manejo integral y asesoría genética.

Introduction: Nonketotic Hyperglycinemia is an inborn error of metabolism in a group of aminoacidopathies, autosomal recessive, caused by a defect in the system of the glycine cleavage. It is rare, and the incidence is unknown in Colombia. Objective:To describe clinical, biochemical and imaging characteristics in a cohort of patients diagnosed with classical nonketotic hyperglycinemia. Materials and methods: This is a descriptive-ambispective study during the period January 2000 - 2014 in some centers of Medellin. Results: There were 35 patients who met the search criteria and finally 20 patients who met inclusion criteria. We found in this cohort more girls than boys, and most of them with a good APGAR. The average onset of symptoms was 2.6 days, with drowsiness, hypoactivity, apnea, seizures and singultus the main symptoms. The focal seizures were the most frequent type. The average value of CSF glycine to plasma glycine ratio was 0.42. The burst suppression pattern in the EEG and the absence or delayed myelination in the supratentorial white matter on MRI were common findings. All patients received dextromethorphan as part of their treatment and the vast majority of sodium benzoate. Conclusion: HGNC is common in our environment. It´s necessary to have available biochemical and molecular evidence for timely diagnosis, comprehensive management and genetic counseling.

The alanine-serine-cysteine-1 (Asc-1) transporter controls glycine levels in the brain and is required for glycinergic inhibitory transmission.

Asc-1 (SLC7A10) is an amino acid transporter whose deletion causes neurological abnormalities and early postnatal death in mice. Using metabolomics and behavioral and electrophysiological methods, we demonstrate that Asc-1 knockout mice display a marked decrease in glycine levels in the brain and spinal cord along with impairment of glycinergic inhibitory transmission, and a hyperekplexia-like phenotype that is rescued by replenishing brain glycine. Asc-1 works as a glycine and L-serine transporter, and its transport activity is required for the subsequent conversion of L-serine into glycine in vivo. Asc-1 is a novel regulator of glycine metabolism and a candidate for hyperekplexia disorders.

Concurrent non-ketotic hyperglycinemia and propionic acidemia in an eight year old boy.

This is the first reported case of a patient with both non-ketotic hyperglycinemia and propionic acidemia. At 2 years of age, the patient was diagnosed with non-ketotic hyperglycinemia by elevated glycine levels and mutations in the GLDC gene (paternal allele: c.1576_1577insC delT and c.1580delGinsCAA; p.S527Tfs*13, and maternal allele: c.1819G>A; p.G607S). At 8 years of age after having been placed on ketogenic diet, he became lethargic and had severe metabolic acidosis with ketonuria. Urine organic acid analysis and plasma acylcarnitine profile were consistent with propionic acidemia. He was found to have an apparently homozygous mutation in the PCCB gene: c.49C>A; p.Leu17Met. The patient was also treated with natural protein restriction, carnitine, biotin, and thiamine and had subjective and biochemical improvement.