The Role of Excitotoxicity, Oxidative Stress and Bioenergetics Disruption in the Neuropathology of Nonketotic Hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is an inherited disorder of amino acid metabolism biochemically characterized by the accumulation of glycine (Gly) predominantly in the brain. Affected patients usually manifest with neurological symptoms including hypotonia, seizures, epilepsy, lethargy, and coma, the pathophysiology of which is still not completely understood. Treatment is limited and based on lowering Gly levels aiming to reduce overstimulation of N-methyl-D-aspartate (NMDA) receptors. Mounting in vitro and in vivo animal and human evidence have recently suggested that excitotoxicity, oxidative stress, and bioenergetics disruption induced by Gly are relevant mechanisms involved in the neuropathology of NKH. This brief review gives emphasis to the deleterious effects of Gly in the brain of patients and animal models of NKH that may offer perspectives for the development of novel adjuvant treatments for this disorder.

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.

Non-ketotic hyperglycinaemia masquerading as a hypotonic-hyporesponsive episode following vaccination in an infant.

Non-ketotic hyperglycinaemia (NKH) is an inborn error of glycine metabolism with autosomal recessive inheritance. A female infant presented to our emergency department with intractable seizures, lethargy and hypotonia, 2 weeks after her routine vaccination. Detailed infective and metabolic workup revealed normal blood sugar, ketone, lactate ammonia, and a high level of glycine in serum and cerebrospinal fluid suggesting NKH. Diagnosis of NKH was further confirmed on genetic analysis for AMT gene mutation. The child showed clinical improvement with oral sodium benzoate. Here, we report the inheritance, pathophysiology, diagnostic approach, genetic confirmation, management and prognosis of a child with 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.

Homozygosity for disease-causing variants in AMT and GLDC in a patient with severe nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is a relatively well-characterized inborn error of metabolism that results in a combination of lethargy, hypotonia, seizures, developmental arrest, and, in severe cases, death early in life. Three genes encoding components of the glycine cleavage enzyme system-GLDC, AMT, and GCSH-are independently associated with NKH. We report on a patient with severe NKH in whom the homozygous pathogenic variant in AMT (NM_000481.3):c.602_603del (p.Lys201Thrfs*75) and the homozygous likely pathogenic variant in GLDC(NM_000170.2):c.2852C>A (p.Ser951Tyr) were both identified. Our patient demonstrates a novel combination of two homozygous disease-causing variants impacting the glycine cleavage pathway at two different components, and elicits management- and genetic counseling-related challenges for the family.

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Non-Ketotic Hyperglycinemia: A Rare Presentation with Neurological and Skeletal Abnormalities.

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Teaching NeuroImage: Glutaredoxin-5-Associated Variant Nonketotic Hyperglycinemia.

A 5-year-old boy presented with subacute motor regression since age 2.5 years. Examination revealed spasticity of bilateral lower extremities, generalized dystonia, and pseudobulbar palsy. Investigations revealed raised plasma lactate (2.5 mmol/L, normal range 0.8-1.5 mmol/L) and no evidence of sideroblastic anemia. Neuroimaging showed cavitating leukoencephalopathy with involvement of long tracts (corticospinal, spinothalamic tracts) and dorsolateral columns of cervicothoracic cord (Figures 1 and 2). A next-generation sequencing test identified a novel homozygous missense variant (c.171C > A, p.Phe57Leu) in exon 1 of the Glutaredoxin-5 (GLRX5) gene.

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.

Pathogenic variants in GCSH encoding the moonlighting H-protein cause combined nonketotic hyperglycinemia and lipoate deficiency.

Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here, we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modeling, expression analysis in GCSH knockdown COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency, whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options.

Ketogenic diet as a glycine lowering therapy in nonketotic hyperglycinemia and impact on brain glycine levels.

BACKGROUND: Nonketotic hyperglycinemia (NKH) is a severe neurometabolic disorder characterized by increased glycine levels. Current glycine reduction therapy uses high doses of sodium benzoate. The ketogenic diet (KD) may represent an alternative method of glycine reduction. AIM: We aimed to assess clinical and biochemical effects of two glycine reduction strategies: high dose benzoate versus KD with low dose benzoate. METHODS: Six infants with NKH were first treated with high dose benzoate therapy to achieve target plasma glycine levels, and then switched to KD with low dose benzoate. They were evaluated as clinically indicated by physical examination, electroencephalogram, plasma and cerebral spinal fluid amino acid levels. Brain glycine levels were monitored by magnetic resonance spectroscopy (MRS). RESULTS: Average plasma glycine levels were significantly lower with KD compared to benzoate monotherapy by on average 28%. Two infants underwent comparative assessments of brain glycine levels via serial MRS. A 30% reduction of brain glycine levels was observed in the basal ganglia and a 50% reduction in the white matter, which remained elevated above normal, and was equivalent between the KD and high dose benzoate therapies. CSF analysis obtained while participants remained on the KD showed a decrease in glycine, serine and threonine levels, reflecting their gluconeogenetic usage. Clinically, half the patients had seizure reduction on KD, otherwise the clinical impact was variable. CONCLUSION: KD is an effective glycine reduction method in NKH, and may provide a more consistent reduction in plasma glycine levels than high-dose benzoate therapy. Both high-dose benzoate therapy and KD equally reduced but did not normalize brain glycine levels even in the setting of low-normal plasma glycine.

Integrative Approach to Predict Severity in Nonketotic Hyperglycinemia.

OBJECTIVE: Glycine encephalopathy, also known as nonketotic hyperglycinemia (NKH), is an inherited neurometabolic disorder with variable clinical course and severity, ranging from infantile epileptic encephalopathy to psychiatric disorders. A precise phenotypic characterization and an evaluation of predictive approaches are needed. METHODS: Longitudinal clinical and biochemical data of 25 individuals with NKH from the patient registry of the International Working Group on Neurotransmitter Related Disorders were studied with in silico analyses, pathogenicity scores, and molecular modeling of GLDC and AMT variants. RESULTS: Symptom onset (p < 0.01) and diagnosis occur earlier in life in severe NKH (p < 0.01). Presenting symptoms affect the age at diagnosis. Psychiatric problems occur predominantly in attenuated NKH. Onset age ≥ 3 months (66% specificity, 100% sensitivity, area under the curve [AUC] = 0.87) and cerebrospinal fluid (CSF)/plasma glycine ratio ≤ 0.09 (57% specificity, 100% sensitivity, AUC = 0.88) are sensitive indicators for attenuated NKH, whereas CSF glycine concentration ≥ 116.5µmol/l (100% specificity, 93% sensitivity, AUC = 0.97) and CSF/plasma glycine ratio ≥ 0.15 (100% specificity, 64% sensitivity, AUC = 0.88) are specific for severe forms. A ratio threshold of 0.128 discriminates the overlapping range. We present 10 new GLDC variants. Two mild variants resulted in attenuated, whereas 2 severe variants or 1 mild and 1 severe variant led to severe phenotype. Based on clinical, biochemical, and genetic parameters, we propose a severity prediction model. INTERPRETATION: This study widens the phenotypic spectrum of attenuated NKH and expands the number of pathogenic variants. The multiparametric approach provides a promising tool to predict disease severity, helping to improve clinical management strategies. ANN NEUROL 2022;92:292-303.

Cerebrospinal fluid amino acids glycine, serine, and threonine in nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is caused by deficient glycine cleavage enzyme activity and characterized by elevated brain glycine. Metabolism of glycine is connected enzymatically to serine through serine hydroxymethyltransferase and shares transporters with serine and threonine. We aimed to evaluate changes in serine and threonine in NKH patients, and relate this to clinical outcome severity. Age-related reference values were developed for cerebrospinal fluid (CSF) serine and threonine from 274 controls, and in a cross-sectional study compared to 61 genetically proven NKH patients, categorized according to outcome. CSF d-serine and l-serine levels were stereoselectively determined in seven NKH patients and compared to 29 age-matched controls. In addition to elevated CSF glycine, NKH patients had significantly decreased levels of CSF serine and increased levels of CSF threonine, even after age-adjustment. The CSF serine/threonine ratio discriminated between NKH patients and controls. The CSF glycine/serine aided in discrimination between severe and attenuated neonates with NKH. Over all ages, the CSF glycine, serine and threonine had moderate to fair correlation with outcome classes. After age-adjustment, only the CSF glycine level provided good discrimination between outcome classes. In untreated patients, d-serine was more reduced than l-serine, with a decreased d/l-serine ratio, indicating a specific impact on d-serine metabolism. We conclude that in NKH the elevation of glycine is accompanied by changes in l-serine, d-serine and threonine, likely reflecting a perturbation of the serine shuttle and metabolism, and of one-carbon metabolism. This provides additional guidance on diagnosis and prognosis, and opens new therapeutic avenues to be explored.

Manejo anestésico y perioperatorio en paciente pediátrico con hiperglicinemia no cetósica / Anesthetic and perioperative management in children with non-ketotic hyperglycinemiawith non-ketotic hyperglycinemia

Presentación del caso: se reporta un paciente pediátrico con diagnóstico de hiperglicinemia no cetósica (HNC), enfermedad neurometabólica poco frecuente ocasionada por una deficiencia en el sistema de segmentación de la glicina, codificada por los genes GLDC, GCSH, AMT y GCSL que conduce a niveles elevados de glicina en la sinapsis generando un efecto agonista prolongado en los receptores N-metil-D-aspartato (NMDA). Discusión y conclusiones: se asocia con hipotonía, convulsiones y trastornos de la deglución, los cuales dependerán de la edad de presentación. Se revisa la literatura actual para el abordaje perioperatorio.

Case presentation: we report a child with a diagnosis of non-ketotic hyperglycinemia (NKGH), a rare neurometabolic disease caused by a defect in the glycine cleavage system, encoded by the GLDC, GCSH, AMT and GCSL genes resulting in elevated synaptic glycine levels generating a prolonged agonist effect on N-methyl-D-aspartate (NMDA) receptors. Discussion and conclusions: it is associated with hypotonia, seizures and swallowing disorders, which will depend on the age at presentation. A literature review was conducted to tailor perioperative approach.

Biallelic start loss variant, c.1A > G in GCSH is associated with variant nonketotic hyperglycinemia.

The glycine cleavage system H protein (GCSH) is an integral part of the glycine cleavage system with its additional involvement in the synthesis and transport of lipoic acid. We hypothesize that pathogenic variants in GCSH can cause variant nonketotic hyperglycinemia (NKH), a heterogeneous group of disorders with findings resembling a combination of severe NKH (elevated levels of glycine in plasma and CSF, progressive lethargy, seizures, severe hypotonia, no developmental progress, early death) and mitochondriopathies (lactic acidosis, leukoencephalopathy and Leigh-like lesions on MRI). We herein report three individuals from two unrelated Indian families with clinical, biochemical, and radiological findings of variant NKH, harboring a biallelic start loss variant, c.1A > G in GCSH.

Genotypic and phenotypic features in Turkish patients with classic nonketotic hyperglycinemia.

Nonketotic hyperglycinemia is an autosomal recessive inborn error of glycine metabolism, characterized by deficient activity of the glycine cleavage enzyme system. Classic nonketotic hyperglycinemia is caused by mutations or genomic changes in genes that encode the protein components of the glycine cleavage enzyme system. We aimed to investigate clinical, biochemical, radiological findings and molecular genetic data in ten Turkish patients with classic nonketotic hyperglycinemia. Ten Turkish patients who were diagnosed with classic nonketotic hyperglycinemia in a single center from 2013 to 2019 were included in this study. Their clinical, radiological, electrophysiological and laboratory data were collected retrospectively. Sixty percent of the patients were in neonatal group, while 40 % of the patients were infantile. There were no late-onset patients. 90 % of the patients had the severe form. All patients had developmental delay and seizures. Mortality ratio was 30 % in all groups and 50 % in the neonatal group, while no mortality was seen in infantile group. Median (range) values of cerebrospinal fluid (CSF) glycine levels, plasma glycine levels and CSF/plasma glycine ratios were 148 (15-320) µmol/L, 896 (87-1910) µmol/L, 0.17 (0.09-0.21) respectively. Diffuse hypomyelination and corpus callosum anomaly were the most common cranial MRI findings and multifocal epileptic activity and burst supression pattern were the most common electroencephalographic findings. Six patients had variants in GLDC gene and four in AMT gene; five novel variants including AMT gene deletion were detected. Prognosis was poor and treatment was not effective, especially in the severe form. Classic nonketotic hyperglycinemia causes high morbidity and mortality. Neonatal-onset disease was more common and severe than infantile-onset disease. The ratio of AMT gene variants might be higher in Turkey than other countries. AMT gene deletion also plays a role in the etiology of classic nonketotic hyperglycinemia.

Elevated preoptic brain activity in zebrafish glial glycine transporter mutants is linked to lethargy-like behaviors and delayed emergence from anesthesia.

Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 - / -) mutant model. We compared locomotor behaviors; dose-response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1-/- mutants and their siblings. To identify differentially active brain regions in glyt1-/- mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1-/- mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose-response curves, glyt1-/- mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1-/- mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1-/- mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1-/- mutants.