Diagnosis of glutathione synthetase deficiency in newborn screening.

Glutathione synthetase (GSS) deficiency is a rare disorder of glutathione metabolism with varying clinical severity. Patients may present with haemolytic anaemia alone or together with acidosis and central nervous system impairment. Diagnosis is made by clinical presentation and detection of elevated concentrations of 5-oxoproline in urine and low GSS activity in erythrocytes or cultured skin fibroblasts. Diagnosis can be confirmed by mutational analysis. Treatment consists of the correction of acidosis, blood transfusion, and supplementation with antioxidants. The most important determinants for outcome and survival in patients with GSS deficiency are early diagnosis and early initiation of treatment. The case of a newborn with GSS deficiency diagnosed by tandem mass spectrometry (MS/MS)-based newborn screening is described. After onset of clinical symptoms on the 2nd day of life, expanded newborn screening revealed normal results for all disorders included in the German screening programme; however, selective MS/MS screening revealed a >10-fold elevation of 5-oxoproline in dried blood, leading to the presumptive diagnosis of GSS deficiency by the 5th day of life. Diagnosis was later confirmed by detection of markedly reduced glutathione concentration in erythrocytes and mutational analysis of the GSS gene. Presently, GSS deficiency is not included in newborn screening programmes in Europe. As outcome depends significantly on early start of treatment, routine inclusion of this disorder in newborn screening panels should be considered.

Chronic non-spherocytic hemolytic anemia associated with severe neurological disease due to gamma-glutamylcysteine synthetase deficiency in a patient of Moroccan origin.

A previously undescribed mutation of hereditary gamma-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage.

Progressive retinal dystrophy in two sisters with glutathione synthetase (GS) deficiency.

We report the ophthalmological findings of two sisters with severe glutathione synthetase deficiency, an autosomal recessive inborn error of metabolism resulting in very low intracellular levels of the free-radical scavenger glutathione. The patients were investigated because of declining visual acuity. The most prominent finding was progressive retinal dystrophy with hyperpigmentations and maculopathy. Generally disturbed functioning of both the outer and inner layers of the retina resulted in attenuated or nearly abolished electroretinograms. These findings agree with a rod/cone type of retinal dystrophy, and we suggest that this is due to glutathione deficiency. Treatment with antioxidants such as vitamins E and C seems to prevent the progression of CNS damage. We speculate that it might also prevent retinal dystrophy in patients with glutathione synthetase deficiency. We suggest that patients with retinal dystrophy and additional neurological signs should be investigated for a defect in glutathione metabolism. Also, we recommend that patients with low levels of glutathione should be examined for retinal dystrophy. Our results suggest that a decreased capacity for scavenging reactive oxygen species and/or increased oxidative stress may cause retinal dystrophy. If this is the case, the redox state in the retina should be a potentially useful therapeutic target to prevent reduced visual function and blindness.

Persistent 5-oxoprolinuria with normal glutathione synthase and 5-oxoprolinase activities.

5-Oxoprolinuria is primarily associated with inborn errors of the gamma-glutamyl cycle. In addition, transient 5-oxoprolinuria has been reported to occur in a variety of conditions, such as prematurity and malnutrition, and during medication. We report an unusual case of permanent 5-oxoprolinuria. The patient presented 3 days after birth with acidosis, and metabolic screening revealed massive excretion of 5-oxoproline. Following recovery, growth and psychomotor development were normal, but 5-oxoprolinuria persisted. Primary defects in the gamma-glutamyl cycle were ruled out since glutathione synthase and 5-oxoprolinase activities were normal. All known secondary causes of 5-oxoprolinuria were also excluded, leaving the basis of the permanent 5-oxoprolinuria in this patient unresolved.

Glutathione synthetase deficiency associated with antenatal cerebral bleeding.

We present a newborn with glutathione synthetase deficiency and intracranial haemorrhages. Because the latter are rare in term newborns a possible relationship with glutathione synthetase deficiency will be discussed.

Oxidative stress in inborn errors of metabolism: lessons from glutathione deficiency.

Oxidative stress has been thought to be involved in the pathogenesis of several inborn errors of metabolism, such as mitochondrial enzyme deficiencies, G6PD deficiency, methylmalonic acidaemia and PKU. The possible role of oxidative stress in the pathogenesis of inborn errors of metabolism is discussed in the light of inborn errors in the metabolism of glutathione.

Glutathione synthetase deficiency: is gamma-glutamylcysteine accumulation a way to cope with oxidative stress in cells with insufficient levels of glutathione?

Glutathione (GSH) plays a major role in the cellular defence against oxidative stress and other vital cellular functions. It therefore seems inevitable that patients with severe depletion of GSH will not survive. However, at least some with glutathione synthetase (GS) deficiency do. This study was done to determine whether these patients have a mechanism to compensate for their GSH deficiency. Cell-free extracts of cultured fibroblasts from 9 patients with GS deficiency and 9 control subjects were analysed by HPLC for low-molecular-weight thiol compounds. The patients' cells contained 7.4 nmol of GSH per mg of protein (median; range 2.8-25.2) compared to 33.0 nmol in control fibroblasts (range 26.7-51.4) (p < 0.01). On the other hand, the patients' cells accumulated 18.1 nmol of gamma-glutamylcysteine (gamma-GC) per mg of protein (median; range 6.9-71.7), whereas the control cells contained 0.1 nmol (range 0.05-0.16) (p < 0.01). The cysteine concentrations in the patients' cells were 20.7 nmol/mg protein (median; range 9.4-52.9) compared to 8.9 nmol in control cells (range 3.0-12.4) (p < 0.01). Cultured fibroblasts from patients with GS deficiency have low levels of GSH, but instead accumulate gamma-GC. We suggest that gamma-GC, which contains both reactive groups of GSH (i.e. the sulphydryl and gamma-glutamyl groups), can compensate for GSH in the cellular defence against oxidative stress. Thus, gamma-GC may alleviate, but only partly prevent, serious consequences of insufficient GSH levels in affected patients. Since the sum of the levels of GSH and gamma-GC in GS-deficient cells (median 31.5 nmol/mg protein, range 16.2-79.0) was similar to the level of GSH alone in control cells (33.0 nmol/mg protein, range 26.7-51.4), we propose that the cultured fibroblasts may have a mechanism to regulate in a coordinated way the levels of GSH and gamma-GC; for instance, by both compounds acting as feedback inhibitors of gamma-GC synthetase.

Long-term clinical outcome in patients with glutathione synthetase deficiency.

OBJECTIVE: The objective was to determine the long-term clinical outcome and the effects of treatment of patients with glutathione synthetase (GS) deficiency (n = 28). METHODS: The diagnosis was based on demonstration of a marked decrease in GS activity in erythrocytes or cultured fibroblasts in all patients and was supported by finding a decrease in erythrocyte or fibroblast glutathione, presence of 5-oxoprolinuria, or both. The treatment varied but usually included correction of acidosis and supplementation with vitamins C and/or E. RESULTS: Sixteen patients were severely affected with neurologic symptoms such as seizures and psychomotor retardation; 7 had died at the time of the study. None of the severely affected patients had been treated with both vitamins C and E from the neonatal period. No significant difference was found in GS activity between patients with or without neurologic symptoms or in erythrocyte or fibroblast glutathione levels. Five patients had recurrent bacterial infections. CONCLUSION: On the basis of clinical symptoms, patients with GS deficiency can be classified into 3 phenotypes: mild, moderate, and severe. Our results indicate that early supplementation with vitamins C and E may improve the long-term clinical outcome.

A missense mutation in the heavy subunit of gamma-glutamylcysteine synthetase gene causes hemolytic anemia.

gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine. In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year-old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS- deficient grandson were identified as homozygous for a 473C-->T substitution, changing codon 158 from CCC for proline into CTC for leucine. Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation.

Patients with genetic defects in the gamma-glutamyl cycle.

In the gamma-glutamyl cycle, hereditary defects have been described in four of the six enzymes namely: gamma-GC synthetase; GSH synthetase; gamma-glutamyl transpeptidase and 5-oxoprolinase. Mutants are still to be found in gamma-glutamyl cyclotransferase and in the dipeptidase. Deficiency of GSH synthatase or gamma-GC synthetases results in low levels of GSH. In gamma-GC synthetase deficiency hemolytic anemia is the most prominent symptom, with or without hepatosplenomegaly. In generalized GSH synthetase deficiency 5-oxoproline is overproduced due to lack of feedback inhibition of gamma-GC synthetase. These patients have metabolic acidosis, 5-oxoprolinuria, hemolytic anemia and about 50% of them also have progressive neurological symptoms. Treatment includes acidosis correction, high doses of vitamin E and C and avoidance of drugs precipitating hemolytic crises in G6PD deficiency. Therapeutic trials with GSH analogues, N-acetylcysteine and GSH esters have been carried out. Glutathione synthetase deficiency restricted to erythrocytes results in hemolytic anemia but no 5-oxoprolinuria. gamma-Glutamyl transpeptidase deficiency is associated with GSH-emia and GSH-uria whereas 5-oxoprolinase deficiency is associated with 5-oxoprolinuria. In diagnostic work it must be emphasized that erythrocytes contain an incomplete gamma-glutamyl cycle; they lack both gamma-glutamyl transpeptidase and 5-oxoprolinase and these enzyme activities must therefore be analyzed in other types of cells such as leukocytes and fibroblasts. It is also important to investigate other patients with inherited defects in the gamma-glutamyl cycle to learn more about the biological role of GSH in man.

Missense mutations in the human glutathione synthetase gene result in severe metabolic acidosis, 5-oxoprolinuria, hemolytic anemia and neurological dysfunction.

Severe glutathione synthetase (GS) deficiency is a rare genetic disorder with neonatal onset. The enzymatic block of the gamma-glutamyl cycle leads to a generalized glutathione deficiency. Clinically affected patients present with severe metabolic acidosis, 5-oxoprolinuria, increased rate of hemolysis and defective function of the central nervous system. The disorder is inherited in an autosomal recessive mode and, until recently, the molecular basis has remained unknown. We have sequenced 18 GS alleles associated with enzyme deficiency and we detected missense mutations by direct sequencing of cDNAs and genomic DNA. In total, 13 different mutations were identified. Four patients were found to be compound heterozygotes and two individuals were apparently homozygous. Reduced enzymatic activities were demonstrated in recombinant protein expressed from cDNAs in four cases with different missense mutations. The results from biochemical analysis of patient specimens, supported by the properties of the expressed mutant proteins, indicate that a residual activity is present in affected individuals. Our results suggest that complete loss of function of both GS alleles is probably lethal. It is postulated that missense mutations will account for the phenotype in the majority of patients with severe GS deficiency.