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.

Large scale analyses of genotype-phenotype relationships of glycine decarboxylase mutations and neurological disease severity.

Monogenetic diseases provide unique opportunity for studying complex, clinical states that underlie neurological severity. Loss of glycine decarboxylase (GLDC) can severely impact neurological development as seen in non-ketotic hyperglycinemia (NKH). NKH is a neuro-metabolic disorder lacking quantitative predictors of disease states. It is characterized by elevation of glycine, seizures and failure to thrive, but glycine reduction often fails to confer neurological benefit, suggesting need for alternate tools to distinguish severe from attenuated disease. A major challenge has been that there are 255 unique disease-causing missense mutations in GLDC, of which 206 remain entirely uncharacterized. Here we report a Multiparametric Mutation Score (MMS) developed by combining in silico predictions of stability, evolutionary conservation and protein interaction models and suitable to assess 251 of 255 mutations. In addition, we created a quantitative scale of clinical disease severity comprising of four major disease domains (seizure, cognitive failure, muscular and motor control and brain-malformation) to comprehensively score patient symptoms identified in 131 clinical reports published over the last 15 years. The resulting patient Clinical Outcomes Scores (COS) were used to optimize the MMS for biological and clinical relevance and yield a patient Weighted Multiparametric Mutation Score (WMMS) that separates severe from attenuated neurological disease (p = 1.2 e-5). Our study provides understanding for developing quantitative tools to predict clinical severity of neurological disease and a clinical scale that advances monitoring disease progression needed to evaluate new treatments for NKH.

Nonketotic Hyperglycinemia in Tunisia. Report upon a Series of 69 Patients.

AIM: The aim of the study is to report on epidemiological, clinical, and biochemical characteristics of nonketotic hyperglycinemia (NKH) in Tunisia. METHODS: Patients diagnosed with NKH in Laboratory of Biochemistry at Rabta hospital (Tunis, Tunisia) between 1999 and 2018 were included. Plasma and cerebrospinal fluid (CSF) free amino acids were assessed by ion exchange chromatography. Diagnosis was based on family history, patient's clinical presentation and course, and increased CSF to plasma glycine ratio. RESULTS: During 20 years, 69 patients were diagnosed with NKH, with 25 patients originating from Kairouan region. Estimated incidences were 1:55,641 in Tunisia and 1:9,684 in Kairouan. Consanguinity was found for 73.9% of the patients and 42% of the families have history of infantile death due to a disease of similar clinical course than the propositus. Clinical symptoms initiated within the first week of life in 75% of the patients and within the first 3 months in 95.7% ones. The phenotype was severe in 76.8% of the patients. Main symptoms were hypotonia, feeding difficulties, coma, apnea, and seizures. Most patients died within few days to months following diagnosis. CSF to plasma glycine ratio was increased in all patients. CSF and plasma glycine levels were negatively correlated with age of disease onset and severity. CONCLUSION: NKH is quite frequent in Tunisia. Kairouan region has the highest NKH incidence rate, worldwide. However, due to lack of confirmatory enzymatic and genetic tests, NKH diagnosis was based on first-line biochemical tests. Characterization of causal mutations is needed for accurate diagnosis and prenatal diagnosis of this devastating life-threatening disease.

Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine.

Glycine is a major neurotransmitter that activates inhibitory glycine receptors and is a co-agonist for excitatory glutamatergic N-methyl-D-aspartate (NMDA) receptors. Two transporters, GLYT1 and GLYT2, regulate extracellular glycine concentrations within the CNS. Dysregulation of the extracellular glycine has been associated with hyperekplexia and nonketotic hyperglycinemia. Here, we report four individuals from two families who presented at birth with facial dysmorphism, encephalopathy, arthrogryposis, hypotonia progressing to hypertonicity with startle-like clonus, and respiratory failure. Only one individual survived the respiratory failure and was weaned off ventilation but has significant global developmental delay. Mildly elevated cerebrospinal fluid (CSF) glycine and normal serum glycine were observed in two individuals. In both families, we identified truncating mutations in SLC6A9, encoding GLYT1. We demonstrate that pharmacologic or genetic abolishment of GlyT1 activity in mice leads to mildly elevated glycine in the CSF but not in blood. Additionally, previously reported slc6a9-null mice and zebrafish mutants also display phenotypes consistent with the affected individuals we examined. Our data suggest that truncating SLC6A9 mutations lead to a distinct human neurological syndrome hallmarked by mildly elevated CSF glycine and normal serum glycine.

Inherited metabolic disorders presenting as hypoxic ischaemic encephalopathy: A case series of patients presenting at a tertiary care hospital in Pakistan.

In spite of the efforts and interventions by the Government of Pakistan and The World Health Organization, the neonatal mortality in Pakistan has declined by only 0.9% as compared to the global average decline of 2.1% between 2000 and 2010. This has resulted in failure to achieve the global Millennium Development Goal 4. Hypoxic-ischaemic encephalopathy, still birth, sepsis, pneumonia, diarrhoea and birth defects are commonly attributed as leading causes of neonatal mortality in Pakistan. Inherited metabolic disorders often present at the time of birth or the first few days of life. The clinical presentation of the inherited metabolic disorders including hypotonia, seizure and lactic acidosis overlap with clinical features of hypoxic-ischaemic encephalopathy and sepsis. Thus, these disorders are often either missed or wrongly diagnosed as hypoxicischaemic encephalopathy or sepsis unless the physicians actively investigate for the underlying inherited metabolic disorders. We present 4 neonates who had received the diagnosis of hypoxic-ischaemic encephalopathy and eventually were diagnosed to have various inherited metabolic disorders. Neonates with sepsis and hypoxic-ischaemic encephalopathy-like clinical presentation should be evaluated for inherited metabolic disorders.

The genetic basis of classic nonketotic hyperglycinemia due to mutations in GLDC and AMT.

PURPOSE: The study's purpose was to delineate the genetic mutations that cause classic nonketotic hyperglycinemia (NKH). METHODS: Genetic results, parental phase, ethnic origin, and gender data were collected from subjects suspected to have classic NKH. Mutations were compared with those in the existing literature and to the population frequency from the Exome Aggregation Consortium (ExAC) database. RESULTS: In 578 families, genetic analyses identified 410 unique mutations, including 246 novel mutations. 80% of subjects had mutations in GLDC. Missense mutations were noted in 52% of all GLDC alleles, most private. Missense mutations were 1.5 times as likely to be pathogenic in the carboxy terminal of GLDC than in the amino-terminal part. Intragenic copy-number variations (CNVs) in GLDC were noted in 140 subjects, with biallelic CNVs present in 39 subjects. The position and frequency of the breakpoint for CNVs correlated with intron size and presence of Alu elements. Missense mutations, most often recurring, were the most common type of disease-causing mutation in AMT. Sequencing and CNV analysis identified biallelic pathogenic mutations in 98% of subjects. Based on genotype, 15% of subjects had an attenuated phenotype. The frequency of NKH is estimated at 1:76,000. CONCLUSION: The 484 unique mutations now known in classic NKH provide a valuable overview for the development of genotype-based therapies.Genet Med 19 1, 104-111.

d-Glyceric aciduria does not cause nonketotic hyperglycinemia: A historic co-occurrence.

Historically, d-glyceric aciduria was thought to cause an uncharacterized blockage to the glycine cleavage enzyme system (GCS) causing nonketotic hyperglycinemia (NKH) as a secondary phenomenon. This inference was reached based on the clinical and biochemical results from the first d-glyceric aciduria patient reported in 1974. Along with elevated glyceric acid excretion, this patient exhibited severe neurological symptoms of myoclonic epilepsy and absent development, and had elevated glycine levels and decreased glycine cleavage system enzyme activity. Mutations in the GLYCTK gene (encoding d-glycerate kinase) causing glyceric aciduria were previously noted. Since glycine changes were not observed in almost all of the subsequently reported cases of d-glyceric aciduria, this theory of NKH as a secondary syndrome of d-glyceric aciduria was revisited in this work. We showed that this historic patient harbored a homozygous missense mutation in AMT c.350C>T, p.Ser117Leu, and enzymatic assay of the expressed mutation confirmed the pathogeneity of the p.Ser117Leu mutation. We conclude that the original d-glyceric aciduria patient also had classic NKH and that this co-occurrence of two inborn errors of metabolism explains the original presentation. We conclude that no evidence remains that d-glyceric aciduria would cause NKH.

[MR spectroscopy in metabolic disorders of the brain]. / MR-Spektroskopie bei Stoffwechselerkrankungen des Gehirns.

CLINICAL ISSUE: Metabolic disorders of the brain often present a particular challenge for the neuroradiologist, since the disorders are rare, changes on conventional MR are often non-specific and there are numerous differential diagnoses for the white substance lesions. STANDARD RADIOLOGICAL METHODS: As a complementary method to conventional brain MRI, MR spectroscopy may help to reduce the scope of the differential diagnosis. Entities with specific MR spectroscopy patterns are Canavan disease, maple syrup urine disease, nonketotic hyperglycinemia and creatine deficiency.

[Clinical and molecular genetic study of nonketotic hyperglycinemia in a Chinese family].

Nonketotic hyperglycinemia (NKH) is a rare, inborn error of metabolism. In this case report, a Chinese male infant was diagnosed with NKH caused by GLDC gene mutation. The clinical characteristics and genetic diagnosis were reported. The infant presented with an onset of early metabolic encephalopathy and Ohtahara syndrome. Both blood and urinary levels of metabolites were in the normal range. Brain MRI images indicated a poor development of corpus callosum, and a burst suppression pattern was found in the EEG. Results of target gene sequencing technology combined with multiplex ligation-dependent probe amplification (MLPA) indicated a heterozygous missense mutation of c.1786 C>T (p.R596X) in maternal exon 15 and a loss of heterozygosity of 4-15 exon gross deletions in paternal GLDC gene. These definite pathogenic mutations confirmed the diagnosis of NKH. The infant's clinical condition was not improved after treatment with adreno-cortico-tropic-hormone, topiramate and dextromethorphan, and he finally died at 4 months of age. Patients with NKH often exhibit complicated clinical phenotypes and are lack of specific symptoms. NKH could be diagnosed by metabolic screening and molecular genetic analysis.

Neurodevelopmental Outcome and Treatment Efficacy of Benzoate and Dextromethorphan in Siblings with Attenuated Nonketotic Hyperglycinemia.

OBJECTIVE: To evaluate the impact of sodium benzoate and dextromethorphan treatment on patients with the attenuated form of nonketotic hyperglycinemia. STUDY DESIGN: Families were recruited with 2 siblings both affected with attenuated nonketotic hyperglycinemia. Genetic mutations were expressed to identify residual activity. The outcome on developmental progress and seizures was compared between the first child diagnosed and treated late with the second child diagnosed at birth and treated aggressively from the newborn period using dextromethorphan and benzoate at dosing sufficient to normalize plasma glycine levels. Both siblings were evaluated with similar standardized neurodevelopmental measures. RESULTS: In each sibling set, the second sibling treated from the neonatal period achieved earlier and more developmental milestones, and had a higher developmental quotient. In 3 of the 4 sibling pairs, the younger sibling had no seizures whereas the first child had a seizure disorder. The adaptive behavior subdomains of socialization and daily living skills improved more than motor skills and communication. CONCLUSIONS: Early treatment with dextromethorphan and sodium benzoate sufficient to normalize plasma glycine levels is effective at improving outcome if used in children with attenuated disease with mutations providing residual activity and when started from the neonatal period.

Biochemical and molecular predictors for prognosis in nonketotic hyperglycinemia.

OBJECTIVE: Nonketotic hyperglycinemia is a neurometabolic disorder characterized by intellectual disability, seizures, and spasticity. Patients with attenuated nonketotic hyperglycinemia make variable developmental progress. Predictive factors have not been systematically assessed. METHODS: We reviewed 124 patients stratified by developmental outcome for biochemical and molecular predictive factors. Missense mutations were expressed to quantify residual activity using a new assay. RESULTS: Patients with severe nonketotic hyperglycinemia required multiple anticonvulsants, whereas patients with developmental quotient (DQ) > 30 did not require anticonvulsants. Brain malformations occurred mainly in patients with severe nonketotic hyperglycinemia (71%) but rarely in patients with attenuated nonketotic hyperglycinemia (7.5%). Neonatal presentation did not correlate with outcome, but age at onset ≥ 4 months was associated with attenuated nonketotic hyperglycinemia. Cerebrospinal fluid (CSF) glycine levels and CSF:plasma glycine ratio correlated inversely with DQ; CSF glycine > 230 µM indicated severe outcome and CSF:plasma glycine ratio ≤ 0.08 predicted attenuated outcome. The glycine index correlated strongly with outcome. Molecular analysis identified 99% of mutant alleles, including 96 novel mutations. Mutations near the active cleft of the P-protein maintained stable protein levels. Presence of 1 mutation with residual activity was necessary but not sufficient for attenuated outcome; 2 such mutations conferred best outcome. Divergent outcomes for the same genotype indicate a contribution of other genetic or nongenetic factors. INTERPRETATION: Accurate prediction of outcome is possible in most patients. A combination of 4 factors available neonatally predicted 78% of severe and 49% of attenuated patients, and a score based on mutation severity predicted outcome with 70% sensitivity and 97% specificity.

Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5.

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.

Diagnosis of glycine encephalopathy in a pediatric patient by detection of a GLDC mutation during initial next generation DNA sequencing.

Early diagnosis for metabolic encephalopathy caused by inborn errors of metabolism is very important for the initiation of early treatment and also for prevention of sequela. Metabolic encephalopathy in the form of seizures can result from many inborn errors of metabolism and considering the large number of disorders causing metabolic encephalopathy, enzyme assays or conventional molecular tests are expensive and take considerably long period of time which results in delayed treatment. In our center we have used next generation DNA sequencing technology as an initial diagnostic test to look for about 700 disorders at the same time for the etiologic diagnosis of a 4-month-old female infant suffering from intractable seizures. The patient was found to have glycine encephalopathy resulting from a previously defined mutation in the GLDC gene. The diagnostic result was obtained much sooner than other conventional investigations. Up to our knowledge, this would be the first case with glycine encephalopathy in the literature who was approached by this novel panel method initially. Although currently, classical evaluation methods such as physical examination, biochemical and conventional molecular investigations are still accepted as the gold standards to clarify the etiology of the metabolic encephalopathy it is obvious that next generation sequence analysis will play a very significant role in the future.

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

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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.

Neonatal nonketotic hyperglycinemia: a case study and review of management for the advanced practice nurse.

Nonketotic hyperglycinemia (NKH) is an autosomal recessive inborn error of glycine metabolism. In this article, I will present the case of baby girl S. who presented to the emergency room on Day 4 of life with severe lethargy, seizures, and respiratory depression requiring mechanical ventilation. A diagnosis of NKH was made secondary to elevated plasma and cerebrospinal fluid glycine concentrations. I will review the pathophysiology of NKH, methods of diagnosis, and the differential diagnosis. There are a variety of different pharmacologic and alternative therapies for NKH. Despite these treatments, the prognosis for infants with NKH is poor, with severe neurologic impairment, intractable seizures, and death common before 5 years of age. I will address the role of the advanced practice nurse in caring for an infant with NKH including clinical, educational, and research implications.

Nonketotic hyperglycinemia presenting as pulmonary hypertensive vascular disease and fatal pulmonary edema in response to pulmonary vasodilator therapy.

The association of pulmonary hypertensive vascular disease with nonketotic hyperglycinemia is rare. We describe 5 infants diagnosed with nonketotic hyperglycinemia, in whom pulmonary hypertensive vascular disease was the main presenting feature, and who developed severe pulmonary edema in response to pulmonary vasodilators.

Prediction of long-term outcome in glycine encephalopathy: a clinical survey.

OBJECTIVE: Glycine encephalopathy (GE) is a rare autosomal recessive inborn error of glycine degradation resulting in severe encephalopathy with ensuing poor outcome. Attenuated variants with a significantly better outcome have been reported. Early prediction of long-term outcome is not yet possible. METHODS: We compared the clinical and biochemical features of 45 children, each with a different course of the disease, to help determine predictors of long-term outcome. RESULTS: The most common presenting symptoms were hypotonia, seizures, and coma. In this study, 85% of the patients presented within the first week of life, and 15% presented after the neonatal period up to the age of 12 months. Developmental progress was made by 19% of those children presenting during the neonatal period and by 50% of those presenting in infancy. Initial CSF and plasma glycine concentrations were not useful in differentiating severe and attenuated outcome. A severe outcome was significantly associated with early onset of spasticity, frequent hiccupping, EEG burst-suppression or hypsarrhythmia patterns, microcephaly, and congenital or cerebral malformations, e.g. corpus callosum hypoplasia. An attenuated outcome was significantly associated with hyperactivity and choreiform movement disorders. We describe a severity score which facilitates the prediction of the outcome in patients with GE. CONCLUSION: Prediction of the outcome of GE may be facilitated by recognizing selected clinical parameters and early neuroimaging findings.

Diagnostic tools of metabolic and structural brain disturbances in neonatal non-ketotic hyperglycinemia.

Non-ketotic hyperglycinemia (NKH) is a rare autosomal recessive disorder of glycine metabolism. We report a newborn case of NKH and discuss the effects of this rare disease on brain metabolism and structure together with amplitude-integrated electroencephalography, cranial magnetic resonance and magnetic resonance spectroscopy findings which are very rarely reported together so far.