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Prenatal diagnosis for non-ketotic hyperglycinaemia (NKH) was performed by enzymatic analysis of chorionic villus samples in 28 families and by DNA analysis in two families. In 26 families, enzymatic analysis of the glycine cleavage multi-enzyme system (GCS) yielded an unambiguous diagnosis; inconclusive results in two families were due to borderline GCS activity. We analysed a second chorionic sample in these two families. In one case, GCS activity was normal in the second specimen, and the baby did not have NKH. In the other case, we again found extremely low GCS activity in the second specimen, but a healthy baby was born. The cause of this false-positive result is unknown. Molecular analysis of NKH has identified two prevalent mutations to date; the S564I mutation in a gene encoding the P-protein, a component of the GCS, in a Finnish population, and the H42R mutation in a gene encoding the T-protein in the Israeli-Arab population. These prevalent mutations allow us to obtain unambiguous prenatal diagnoses in both Finnish and Israeli-Arab families. GCS activity in samples from a Finnish family demonstrated a good agreement with DNA analysis, but the fetus of the Israeli-Arab family had an upper limit activity of the affected range, suggesting an advantages for DNA analysis., (Copyright 1999 John Wiley & Sons, Ltd.)

Nonketotic hyperglycinemia (NKH) is caused by a mutation in the genes encoding the components of the glycine cleavage multi-enzyme system. More than 80% of the patients have defects in the gene encoding P-protein, whereas the rest of the patients have defects in the gene encoding T-protein. We have found a large Israeli-Arab kindred with NKH. At least 14 children were affected, and all the patients had seizures and respiratory failure within 2 days after birth. Enzymatic analysis revealed that T-protein activity was deficient in the liver specimen from one propositus. We screened this family for a mutation in the protein-coding region and exon/intron boundaries of T-protein gene by direct sequencing analysis. A missense mutation was found in exon 2; this resulted in an amino acid substitution from histidine to arginine at position 42 (H42R). Histidine 42 is conserved in human, bovine, chicken, pea, and Escherichia coli, suggesting that it has an important role in catalytic functions. Genotype analyses of 26 family members confirmed that the homozygous H42R mutation was completely associated with the onset of NKH. The availability of DNA testing facilitates the prenatal diagnosis of NKH and the identification of carriers, which is necessary for genetic counseling in the affected families.

Two novel mutations in the gene encoding T-protein, a component of the glycine cleavage system, were identified in a Japanese family with nonketotic hyperglycinemia. The proband had two affected sibs, and enzymatic analysis of the liver sample from the proband revealed the T-protein deficiency. The first mutation, 183delC, was found in exon 1. One of six cytidine residues (base position 183-188) was deleted. The deletion was located in a coding region of the mitochondrial leader peptide and was deduced to create a truncated peptide with 94 amino acids. The second mutation was a base substitution from G to C at position 955 in exon 7. The G955C substitution caused an amino acid change from aspartate to histidine at position 276 (D276H). Aspartic acid at position 276 is evolutionarily conserved among human, bovine, chicken, and pea genes, and replaced by glutamic acid in Escherichia coli, suggesting that the presence of an acidic amino acid at 276 may be crucial for the enzymatic function. No base change other than the 183delC and the G955C was observed in the sequencing analysis. Familial analysis revealed that the 183delC and the D276H mutations were inherited from the father and the mother, respectively. This is the first report of T-protein gene mutation in Oriental patients with nonketotic hyperglycinemia.

Nonketotic hyperglycinemia (NKH) is a metabolic disorder with autosomal recessive inheritance, causing severe, frequently lethal, neurological symptoms in the neonatal period. The metabolic lesion of NKH is in the glycine cleavage system (GCS), a complex enzyme system with four enzyme components; P-, T-, H-, and L-protein. The enzymatic analysis revealed that 86% of the patients with NKH are deficient of P-protein activity. The cDNA clones encoding all four components were isolated and their primary structures were determined. Several mutations have been identified in P- and T-protein genes: One missense mutation, S564I, in P-protein gene accounts for 70% of the mutant alleles in Finland where the incidence of NKH is unusually high. The immunochemical and in situ hybridization analyses revealed that the strong GCS expression was observed in rat hippocampus, olfactory bulbus, and cerebellum. The distribution resembled that of N-methyl-D-aspartic acid (NMDA) receptor which has binding site for glycine. It is, therefore, suggested that the neurological disturbance in NKH may be caused by excitoneurotoxicity through the NMDA receptor allosterically activated by high concentration of glycine. Based on the hypothesis the NMDA antagonists such as ketamine and dextromethorphan were administered to the patients. We treated three neonatal case with dextromethorphan and it ameliorated their findings on electroencephalogram and behavior in two out of three patients. Thus the GCS is suggested to play a role in regulation of glycine level around the NMDA receptor.

Tissue concentrations of D-serine and other chiral and non-chiral amino acids were measured post-mortem in the cerebral cortex of the neonatal or infantile individuals with (three cases) or without (seven cases) non-ketotic hyperglycinemia (NKH) using high performance liquid chromatography with fluorometric detection. In the cortical tissues of the NKH patients lacking activity of glycine cleavage system, there was a marked reduction and elevation of the contents of D-serine and glycine, respectively, compared to non-NKH controls. Systemic administration of an inhibitor of glycine cleavage system (GCS), cysteamine, mimicked the changes in the cortical concentrations of these amino acids in the 8-day-old rats. Augmentation of brain glycine levels by means of intraperitoneal injection of glycine itself resulted in an increase in cortical D-serine contents in the neonatal rats with normal activity of GCS. These findings provide the first evidence that GCS might be implicated in the biosynthesis or content regulation of endogenous D-serine in the mammalian brain.

Non-ketotic hyperglycinaemia (NKH) is a well-recognized metabolic cause of life-threatening illness in the neonate. The fundamental defect is in the glycine cleavage system, which consists of four protein components. Our study revealed that the majority of NKH patients had a specific defect in P-protein (glycine decarboxylase). The primary lesion of NKH at gene level was investigated, using cDNA encoding human glycine decarboxylase. A three-base deletion resulting in deletion of Phe756 was found in a Japanese patient with NKH. The majority of NKH patients in Finland, where there is a high incidence of NKH, were found to be due to a common mutation, a point mutation resulting in the amino acid substitution of Ile564 for Ser564. Prenatal diagnosis is feasible by determining the activity of the glycine cleavage system and is also possible by DNA analysis. Recent findings suggest that a high concentration of glycine in the brain may contribute to the pathophysiology of NKH by overactivating N-methyl-D-aspartate receptors allosterically, which may result in intracellular calcium accumulation, DNA fragmentation and neuronal death. These provide the possibility that early treatment with N-methyl-D-aspartate receptor antagonist may prevent brain damage in NKH.

Nonketotic hyperglycinemia (NKH) is an autosomal recessive metabolic disorder caused by the defects in the glycine cleavage system (GCS; EC 2.1.2.10), a multienzyme system that consists of four individual components. NKH is a rare disorder in many countries, but with a very high incidence in northern Finland. To understand the genetic background of this high incidence, we examined the GCS in a typical case of NKH at the molecular level. The activity of P protein, a component of the GCS, was not detected in the lymphoblasts of the patient, while P protein mRNA of a normal size and level was present in the cells. Structural analysis of P protein mRNA from the patient revealed a single nucleotide substitution from G to T in the protein coding region, which resulted in an amino acid alteration from Ser564 to Ile564. No P protein activity was detected when the mutant P protein with this amino acid substitution was expressed in COS 7 cells. The patient was homozygous for this mutation. Furthermore, this mutation was present in 70% (14 of 20) of P protein gene alleles in Finnish patients with NKH, whereas it was not found in 20 alleles of non-Finnish patients. The results suggest that this mutation is responsible for the high incidence of NKH in Finland.

Non-ketotic hyperglycinemia (NKH) is a well-recognized metabolic cause of life-threatening illness in the neonate. The fundamental defect is in the glycine cleavage enzyme (GCE), which consists of four protein components. Our study revealed that the majority of NKH patients had a specific defect in P-protein (glycine decarboxylase). The primary lesion of NKH in gene level was investigated, using cDNA encoding human glycine decarboxylase. A three-base deletion; resulting in deletion of Phe756 was found in a Japanese patient with NKH. In the majority of NKH patients in Finland, where there is a high incidence of NKH, it was found to be due to a common mutation--a point mutation resulting in amino acid alternation from Ser564 to Ile564. Prenatal diagnosis is possible by determining the activity of GCE and also by DNA analysis. Recent findings suggest that the high concentrations of glycine in the brain may contribute to the pathophysiology of NKH by overactivating NMDA receptors via an action at the associated glycine modulatory site. These provide a possibility that early treatment with NMDA receptor antagonist may prevent brain damage in NKH.

We developed a new enzymatic assay for the glycine cleavage system that used Epstein-Barr virus-transformed lymphoblasts instead of liver biopsy specimens. Patients with nonketotic hyperglycinemia from a deficiency of P-protein could be clearly distinguished from control subjects by activities in their lymphoblasts, suggesting the clinical usefulness of this method.

The localization of the glycine cleavage system was examined in the rat brain by immunohistochemistry using an antibody to P-protein (a constituent of the system). In all sites studied, the enzyme was confined to the astrocytes. The intensity of astrocyte staining varied in different brain regions, with the strongest staining being noted in the hippocampus, the cerebellar cortex, the Bergmann glia in the cerebellum and the Muller cells in the retina. The weakest staining was seen in the brainstem and spinal cord. P-protein was found to be located in the mitochondria by an ultrastructual study.

A multiple pregnancy of high rank may occur in a couple at risk for a Mendelian disorder. Prenatal diagnosis is hampered by the difficulty of (1) obtaining chorionic villi from each zygote and (2) unequivocally relating each sample to the corresponding embryo. The calculation of the genetic risk according to the number of zygotes led us to propose a diagnostic strategy based on embryo reduction, a technique initially designed to improve the perinatal outcome of multiple pregnancies with normal embryos. We report a case in which this approach allowed rational use of first-trimester chorionic villus sampling in a quintuplet pregnancy at risk for non-ketotic hyperglycinaemia, resulting in the selective birth of unaffected twins.

We examined ophthalmologically two siblings with nonketotic hyperglycinemia who had negligible activities of glycine cleavage system in the liver. Both had jerky nystagmus. The elder brother had a normal electroretinographic response, while the younger brother's response was subnormal. There may be a close association of subnormal electroretinographic response and hyperglycinemia, because glycine can act as an inhibitory neurotransmitter in the retina.

Glycine decarboxylase, a constituent of the glycine cleavage system, in patients with either nonketotic or ketotic hyperglycinemia (NKH and KH) was examined using an anti-chicken glycine decarboxylase antibody. Patients with NKH who have lesion in glycine decarboxylase are differentiated by its expressed level in the liver. One group is cases of the neonatal onset type who have neither activity of the enzyme nor protein reactive to the antibody. The other is a case of the late onset type who shows low but detectable activity of the enzyme and the desirable amount of the immunoreactive material. In the liver of a patient with KH not showing the appreciable activity of H-protein, ubiquitous amount of protein reactive to anti-H-protein IgG is detected and amount of glycine decarboxylase has also been lowered. It is suggested that several mechanisms may be involved in determining the expressed level of glycine decarboxylase in patients with hyperglycinemias.

The effects of ten metabolites of the branched-chain amino acids, CoA and cysteamine (beta-mercaptoethylamine), on the glycine cleavage system were investigated with the liver extracts. It was found that CoA derivatives including tiglyl CoA, isobutyryl CoA, succinyl CoA, methylmalonyl CoA, isovaleryl CoA, propionyl CoA and CoA itself and cysteamine significantly inhibited the glycine cleavage system of the liver extracts. Further studies on the glycine-14CO2 exchange catalyzed by p-protein and H-protein purified from chicken liver indicated that tiglyl CoA inhibited the activity of P-protein in an apparently competitive manner with respect to H-protein, and that cysteamine inhibited the activity of P-protein in two ways, by increasing the Km value for glycine and changing Vmax.

A 44-year-old woman had a fundus appearance similar to that of gyrate atrophy, a macular lesion, and excessive urinary excretion of proline, hydroxyproline, and glycine. The patient also had abnormal ciliary processes and patchy atrophy of the irides. To our knowledge, this is the first reported case of ocular manifestations associated with iminoglycinuria.

The clinical and metabolic data of 2 cases of methylmalonic acidaemia with propionic acidaemia are reported together with those of 3 other patients with nonketotic hyperglycinaemia. Liver enzymatic studies showed decreased activity in vitro of the glycine cleavage enzyme in one patient with methylmalonic acidaemia as well as in 2 unrelated patients with nonketotic hyperglycinaemia, while the activity of the serine hydroxymethylase enzyme was normal. Hyperammonaemia was substantiated in one patient with methylmalonic acidaemia and also in one child with nonketotic hyperglycinaemia. The activity of the enzymes of the urea cycle, determined in the liver of this nonketotic child, was normal except for a decrease of the carbamyl phosphate synthetase enzyme to 15% of normal.

Non-ketotic hyperglycinaemia was diagnosed in a girl at 3 weeks of age because of the typical clinical presentation, the elevated glycine concentration in urine, plasma and especially in cerebrospinal fluid and the normal profile of organic acids in urine. An EEG showed the typical burst suppression pattern. Therapeutic approaches with either pyridoxine (50 mg d-1) alone or in combination with N5-formyltetrahydrofolate (3 X 3 mg d-1) or with strychnine (0.3 mg per kg body weight) did not result in improvement. In postmortem liver and brain of the patient the overall activity of the glycine cleavage system was deficient; examination of the activity of the individual components of the glycine cleavage system in the tissues revealed that the activity of the T-protein was undetectable, whereas that of the other components and of lipoamide dehydrogenase was normal.

Non-ketotic hyperglycinaemia is an autosomal recessive disorder of glycine metabolism characterized by elevated concentrations of glycine in plasma, urine and cerebrospinal fluid. The fundamental defect was found to lie in the glycine cleavage system. It is of significance that the major pathway for the catabolism of glycine was elucidated through the studies of hyperglycinaemia. The present knowledge about non-ketotic hyperglycinaemia is described in clinical and biochemical aspects.

The molecular nature of the glycine cleavage system was investigated in 16 patients with nonketotic hyperglycinemia (NKH). The overall activity of the glycine cleavage system was found to be decreased in all of the liver and brain tissue studied. It was undetectable or extremely low in the neonatal type of NKH, whereas there was some residual activity in the infantile type of NKH. Thus the clinical phenotypes do seem to relate to the degree of the defect in the glycine cleavage system. In the neonatal type, a specific defect in P protein was found in 9 cases and a specific defect in T protein in 2 cases. In the infantile type, a partial defect in T protein was found in 2 cases. Differential diagnosis between NKH and ketotic hyperglycinemia is described. A feasibility of prenatal diagnosis of NKH by chorionic villus biopsy is provided.

Glycine cleavage activity was compared in the livers from three cases of ketotic hyperglycinemia (two cases of propionic acidemia and one case of methylmalonic acidemia) and three controls. In one case of propionic acidemia, glycine cleavage activity (5.2 nmole/mg protein/hr) was normal in the liver obtained at biopsy when the patient was well controlled by the treatment with low protein diet (0.8 g/kg/day) and the level of serum glycine was lowered to normal. In the two other cases of ketotic hyperglycinemia, glycine cleavage activity was significantly reduced in the liver obtained at autopsy when the patients died in the state of metabolic acidosis. Its activity in the liver of one case of propionic acidemia (0.7 nmole/mg protein/hr) was 6-26% of that in controls (2.7-10.8 nmole/mg protein/hr), and 2-7% in the case of methylmalonic acidemia (0.2 nmole/mg protein/hr). Analysing of the individual components of the glycine cleavage system, a marked decrease in the activity of H-protein was revealed in the livers of the both patients; it (0.2 nmole/mg protein/hr) was only 3-4% of that in controls (4.9-6.3 nmole/mg Protein/hr). These findings suggest that the reduction of the glycine cleavage system in the liver of ketotic hyperglycinemia occurs secondarily as speculated previously and is caused mainly by a decrease of H-protein activity.

The glycine cleavage system was investigated in the livers and brains of two patients with typical nonketotic hyperglycinemia who died in the neonatal period. The overall activity of the glycine cleavage system was found to be extremely low in both the liver and brain of each patient. In one patient, the disturbance of the glycine cleavage system was due to absence of activity of the P-protein. Immunochemical analysis indicated that this resulted from an absence of the enzyme protein. In the other patient, the activity of the T-protein was undetectable in the brain and was extremely low in the liver. Clinically classic nonketotic hyperglycinemia resulted from molecular defects in two different protein components of the glycine cleavage system.

Sisters with a mild variant of non-ketotic hyperglycinaemia resulting from a defect in the T-protein of the glycine cleavage system had different clinical outcomes. The older sister was ascertained at 6 months of age because of mental retardation. She received only brief treatment with sodium benzoate from 11-15 months and at 15 years of age is profoundly retarded and has epilepsy. The younger sister was diagnosed 36 h after birth, was treated with strychnine, sodium benzoate and arginine from the neonatal period and at 27 months of age is only moderately retarded and free of seizures. The possible role of strychnine in the improved outcome is discussed.

Serum bile acids and their conjugates were analysed in 20 breast-fed infants with prolonged jaundice. The mean total bile acid levels in serum were increased in the breast-fed infants with jaundice, as compared with those in either breast- or bottle-fed infants without jaundice. However, there were no significant differences between the groups. All the breast-fed infants examined, regardless of association with jaundice, had a bile acid pattern dominated by taurine conjugates (the ratio of glycine- to taurine-conjugated bile acid, G/T ratio, less than 1.00). In contrast, the bottle-fed infants without jaundice had a pattern dominated by glycine conjugates (G/T ratio, more than 1.00). Among the breast-fed infants with jaundice, the mean G/T ratio in those who had serum bilirubin levels over 10 mg/100 ml was significantly lower than that in those who had serum bilirubin levels of less than 10 mg/100 ml. The altered bile acid metabolism might be associated with the pathology of breast milk jaundice.

We diagnosed a 22-year-old man with psychomotor retardation, rare seizures, hyperglycinemia, and hyperglycinuria as an atypical variant of nonketotic hyperglycinemia (NKH). Despite this clinical phenotype and a CSF/plasma ratio confirming a mild variant, measurement of hepatic glycine cleavage activity and the P-protein component indicated the more severe neonatal variant.

The molecular nature of the glycine cleavage system was investigated in eight patients with typical (neonatal) and two patients with atypical (late onset) nonketotic hyperglycinemia (NKH). The overall activity of the glycine cleavage system was found to be decreased in all of the liver and brain tissue studied, but it was undetectable or extremely low in typical NKH, whereas there was some residual activity in atypical NKH. Six patients with typical NKH had a specific defect in the P protein, and one a defect in the T protein; the activity of the T protein was defective in one patient with atypical NKH.