Gss deficiency causes age-related fertility impairment via ROS-triggered ferroptosis in the testes of mice.

Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss-/- male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss-/- mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.

Gas Chromatography Mass Spectrometry Aided Diagnosis of Glutathione Synthetase Deficiency.

Glutathione synthetase (GSS) deficiency is a rare disorder, occurring with a frequency of less than 1 in 100,000 individuals worldwide. The clinical presentation may vary from mild to severe, and manifestations include hemolytic anemia, hyperbilirubinemia, metabolic acidosis, neurological problems, and sepsis. Herein, we present a case of a newborn boy with the most severe phenotype of GSS deficiency, diagnosed based on clinical features and increased urinary 5-oxoproline levels determined via gas chromatography mass spectrometry (GCMS) testing.

Glutathione synthetase deficiency: a novel mutation with femur agenesis.

Introduction: Glutathione synthetase (GSS) deficiency is an autosomal recessive disorder (frequency < 1/1,000,000) with different varyingly severe clinical manifestations that include metabolic acidosis, hemolytic anemia, hyperbilirubinemia, neurological disorders and sepsis. Case report: This infant was small for gestational age, had hemolytic anemia, metabolic acidosis, bilateral subependymal pseudocysts and increased echogenicity of the basal ganglia. GSS deficiency was confirmed by genetic analysis. The patient also had unilateral right femur agenesis. Conclusion: By using next generation sequencing analysis, we identified a novel homozygous variant c.800G > A, p.Arg267Gln in the GSS gene of this patient. Femur agenesis had not previously been associated with GSS.

Lesson of the month 1: A rare adverse reaction between flucloxacillin and paracetamol.

Flucloxacillin, a beta-lactam antibiotic, is a commonly prescribed antibiotic for the treatment of infections caused by staphylococci and streptococci, most notably Staphylococcus aureus Paracetamol is one of the most dispensed medications by NHS England and is used for the treatment of fever and pain.1 However most doctors are unaware that concurrent use of these drugs can cause a potentially fatal drug interaction due to pyroglutamic acidosis (PGA), also known as 5-oxoprolinaemia. PGA is a rare cause of raised anion gap metabolic acidosis due to disruption of the γ-glutamyl cycle. We report the case of a patient with multiple comorbidities who developed PGA due to coadministration of paracetamol and flucloxacillin.

Treatment of cystoid macular edema in homozygous twins with glutathione synthetase deficiency and retinal dystrophy.

Monozygotic twins with glutathione synthetase deficiency, progressive retinal dystrophy and cystoid macular edema were followed for foveal changes on optical coherence tomography under different treatment modalities. The purpose of the study is to show the effect of topical dorzolamide in conjunction with systemic acetazolamide in terms of decreasing macular edema in this specific disease. The results showed that systemic acetazolamide alone or in combination with topical dorzolamide decreased CME in both patients for a certain period of time. The result can be temporary sustained after treatment discontinuation. In conclusion, topical dorzolamide, in conjunction with systemic acetazolamide, could reduce cystoid macular edema in GSSD.

A case of severe glutathione synthetase deficiency with novel GSS mutations.

Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

A Rare Cause of Neonatal Hemolytic Anemia: Glutathione Synthetase Deficiency.

BACKGROUND: Isolated hemolysis or hemolytic anemia and 5-oxoprolinuria are 2 distinct medical conditions in the clinical spectrum associated with glutathione synthetase deficiency. CLINICAL OBSERVATION: A 1-day-old female baby presented with anemia and respiratory distress. Her hemoglobin level was 9.5 g/dL and the total serum bilirubin level was 5.6 mg/dL. Metabolic acidosis was detected in her blood gas analysis. Metabolic acidosis recurred despite treatment and further investigation was required. Her 5-oxoproline level was 3815 mmol/mol creatinine in urine organic acid analysis, and a homozygous mutation [p.R125H (c.374G>A)] was found in the glutathione synthetase gene. CONCLUSIONS: GSD has been observed in very few patients and is rarely considered in the differential diagnosis of hemolytic anemia in newborns.

[Acetaminophen induced 5-oxoproline acidosis: An uncommon case of high anion gap metabolic acidosis]. / Acidose à la 5-oxoproline induite par le paracétamol : une cause rare d'acidose métabolique à trou anionique augmenté.

The most common causes of high anion gap metabolic acidosis (HAGMA) are lactic acidosis, ketoacidosis, and intoxications. Nevertheless, clinicians can be faced with unexplained HAGMA, with a need to look for less common etiologies. We describe a case of 5-oxoproline (pyroglutamate) acidosis due to chronic acetaminophen ingestion at therapeutic dose in a 79-year-old inpatient. The pathophysiology of this condition is detailed, with abnormalities in the gamma-glutamyl cycle due to acetaminophen ingestion and severe chronic morbidities, resulting in glutathione and cysteine deficiency and then accumulation of 5-oxoproline. In HAGMA, when usual causes have been excluded, 5-oxoproline acidosis should be suspected in patients with chronic morbidities and acetaminophen ingestion. This diagnosis should be kept in mind because it generally resolves quickly with cessation of acetaminophen and administration of intravenous fluids.

A case of severe glutathione synthetase deficiency with novel GSS mutations

Glutathione synthetase deficiency (GSSD) is a rare inborn error of glutathione metabolism with autosomal recessive inheritance. The severe form of the disease is characterized by acute metabolic acidosis, usually present in the neonatal period with hemolytic anemia and progressive encephalopathy. A case of a male newborn infant who had severe metabolic acidosis with high anion gap, hemolytic anemia, and hyperbilirubinemia is reported. A high level of 5-oxoproline was detected in his urine and a diagnosis of generalized GSSD was made. DNA sequence analysis revealed the infant to be compound heterozygous with two mutations, c.738dupG in exon 8 of GSS gene resulting in p.S247fs and a repetitive sequence in exon 3 of GSS gene. Treatment after diagnosis of GSSD included supplementation with antioxidants and oral sodium hydrogen bicarbonate. However, he maintained a variable degree of metabolic acidosis and succumbed shortly after his parents requested discontinuation of therapy because of dismal prognosis and medical futility when he was 18 days old.

Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione.IMPORTANCEYersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.

Forging Ahead.

The approach to clinical conundrums by an expert clinician is revealed through the presentation of an actual patient's case in an approach typical of a morning report. Similarly to patient care, sequential pieces of information are provided to the clinician, who is unfamiliar with the case. The focus is on the thought processes of both the clinical team caring for the patient and the discussant. This icon represents the patient's case. Each paragraph that follows represents the discussant's thoughts.

Recurrent Isolated Neonatal Hemolytic Anemia: Think About Glutathione Synthetase Deficiency.

Hemolytic anemia (HA) of the newborn should be considered in cases of rapidly developing, severe, or persistent hyperbilirubinemia. Several causes of corpuscular hemolysis have been described, among which red blood cell enzyme defects are of particular concern. We report a rare case of red blood cell enzyme defect in a male infant, who presented during his first months of life with recurrent and isolated neonatal hemolysis. All main causes were ruled out. At 6.5 months of age, the patient presented with gastroenteritis requiring hospitalization; fortuitously, urine organic acid chromatography revealed a large peak of 5-oxoproline. Before the association between HA and 5-oxoprolinuria was noted, glutathione synthetase deficiency was suspected and confirmed by a low glutathione synthetase concentration and a collapse of glutathione synthetase activity in erythrocytes. Moreover, molecular diagnosis revealed 2 mutations in the glutathione synthetase gene: a previously reported missense mutation (c.[656A>G]; p.[Asp219Gly]) and a mutation not yet described in the binding site of the enzyme (c.[902T>C]; p.[Leu301Pro]). However, 15 days later, a control sample revealed no signs of 5-oxoprolinuria and the clinical history discovered administration of acetaminophen in the 48 hours before hospitalization. Thus, in this patient, acetaminophen exposure allowed the diagnosis of a mild form of glutathione synthetase deficiency, characterized by isolated HA. Early diagnosis is important because treatment with bicarbonate, vitamins C and E, and elimination of trigger factors are recommended to improve long-term outcomes. Glutathione synthetase deficiency should be screened for in cases of unexplained newborn HA.

Expanding the phenotype of hawkinsinuria: new insights from response to N-acetyl-L-cysteine.

Hawkinsinuria is a rare disorder of tyrosine metabolism that can manifest with metabolic acidosis and growth arrest around the time of weaning off breast milk, typically followed by spontaneous resolution of symptoms around 1 year of age. The urinary metabolites hawkinsin, quinolacetic acid, and pyroglutamic acid can aid in identifying this condition, although their relationship to the clinical manifestations is not known. Herein we describe clinical and laboratory findings in two fraternal twins with hawkinsinuria who presented with failure to thrive and metabolic acidosis. Close clinical follow-up and laboratory testing revealed previously unrecognized hypoglycemia, hypophosphatemia, combined hyperlipidemia, and anemia, along with the characteristic urinary metabolites, including massive pyroglutamic aciduria. Treatment with N-acetyl-L-cysteine (NAC) restored normal growth and normalized or improved most biochemical parameters. The dramatic response to NAC therapy supports the idea that glutathione depletion plays a key role in the pathogenesis of hawkinsinuria.

Unravelling 5-oxoprolinuria (pyroglutamic aciduria) due to bi-allelic OPLAH mutations: 20 new mutations in 14 families.

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the γ-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology.

Metabolic acidosis and 5-oxoprolinuria induced by flucloxacillin and acetaminophen: a case report.

BACKGROUND: Frequent causes of high anion gap metabolic acidosis are well known: ethanol, methanol, and ethylene glycol intoxication; hyperglycemia; lactic or D-lactic acidosis; and impaired renal function. There are other causes, less frequent but also important. This report illustrates a rare case of a patient with increased anion gap metabolic acidosis due to a deficit of the γ-glutamyl cycle that led to 5-oxoproline (acid pyroglutamic) accumulation. CASE PRESENTATION: An 82-year-old white woman was admitted to our intensive care unit because of septic shock caused by right knee methicillin-sensitive Staphylococcus aureus-induced arthritis. She was treated for 10 days with flucloxacillin and rifampicin and developed metabolic acidosis with high anion gap. Her test results for methanol, ethanol, ethylene glycol, and acetylsalicylic acid were negative. Her glycemia, lactate level, and renal function were normal. However, the result of a urinary assay for pyroglutamate was positive. We concluded that the patient had metabolic acidosis induced by accumulation of 5-oxoproline. We modified her antibiotic treatment, administered acetylcysteine, and her acidosis resolved. CONCLUSIONS: 5-Oxoprolinuria (pyroglutamic acid accumulation) is a rare, probably underdiagnosed cause of transient metabolic acidosis with increased anion gap.

Nineteen-year follow-up of a patient with severe glutathione synthetase deficiency.

Glutathione synthetase deficiency is a rare autosomal recessive disorder resulting in low levels of glutathione and an increased susceptibility to oxidative stress. Patients with glutathione synthetase deficiency typically present in the neonatal period with hemolytic anemia, metabolic acidosis and neurological impairment. Lifelong treatment with antioxidants has been recommended in an attempt to prevent morbidity and mortality associated with the disorder. Here, we present a 19-year-old female who was diagnosed with glutathione synthetase deficiency shortly after birth and who has been closely followed in our metabolic clinic. Despite an initial severe presentation, she has had normal intellectual development and few complications of her disorder with a treatment regimen that includes polycitra (citric acid, potassium citrate and sodium citrate), vitamin C, vitamin E and selenium.

Clinical findings and effect of sodium hydrogen carbonate in patients with glutathione synthetase deficiency.

BACKGROUND: Glutathione synthetase (GS) deficiency is a rare inborn error of glutathione (GSH) metabolism manifested by severe metabolic acidosis, hemolytic anemia, neurological problems and massive excretion of pyroglutamic acid (5-oxoproline) in the urine. The disorder has mild, moderate, and severe clinical variants. We aimed to report clinical and laboratory findings of four patients, effect of sodium hydrogen carbonate treatment and long-term follow up of three patients. METHODS: Urine organic acid analysis was performed with gas chromatography-mass spectrometry. Molecular genetic analysis was performed in three patients, mutation was found in two of them. Enzyme analysis was performed in one patient. Clinical and laboratory findings of four patients were evaluated. RESULTS: One patient died at 4 months old, one patient's growth and development are normal, two patients have developed intellectual disability and seizures in the long term follow up period. Three patients benefited from sodium hydrogen carbonate treatment. CONCLUSIONS: The clinical picture varies from patient to patient, so it is difficult to predict the prognosis and the effectiveness of treatment protocols. We reported long term follow up of four patients and demonstrated that sodium hydrogen carbonate is effective for treatment of chronic metabolic acidosis in GS deficieny.

Transient 5-oxoprolinuria: unusually high anion gap acidosis in an infant.

Transient 5-oxoprolinuria is a phenomenon that is well recognised in adults. We illustrate an unusual paediatric case of transient 5-oxoprolinuria presenting during an episode of severe sepsis with concomitant paracetamol use. The 15-month-old patient had an extremely high anion gap metabolic acidosis. Adequate resuscitation failed to correct the biochemical disturbance, and high levels of 5-oxoproline were identified. A combination of haemofiltration, replenishment of glutathione stores with N-acetylcysteine and cessation of paracetamol administration resulted in the resolution of the acidosis. Subsequent testing following treatment of the sepsis revealed no ongoing 5-oxoprolinuria. CONCLUSION: Transient 5-oxoprolinuria has been previously reported in the adult population during episodes of severe sepsis and various pharmaceutical interventions. This case illustrates that it is a phenomenon that should be considered in paediatric patients where a very high anion gap metabolic acidosis exists that cannot be explained by the biochemical indices. WHAT IS KNOWN: • 5-oxoprolinuria in the paediatric population is usually secondary to an inborn error of metabolism. • Transient 5-oxoprolinuria is well recognised in adults during episodes of severe glutathione depletion. WHAT IS NEW: • Transient 5-oxoprolinuria is a phenomenon rarely reported in the paediatric population. • It highlights the importance of investigating a high anion gap such that unusual diagnoses are not missed.

Neutrophil functional disorder in childhood.

Neutrophil functional disorders thought to be uncommon, yet important as a cause of morbidity and mortality in infants and children. During the first years of life, when the immune system is still not completely mature, when the viral infections are frequent and antibiotic overuse can damage and alter the immune response, the inadequate nutrition followed with iron deficient anemia and malnutrition can lead the child`s organism in state of immunodeficiency. Sometimes is difficult to distinguish at the beginning weather the cause of patient suffering from frequent infections is existing of primary immunodeficiency disorder or the cause of the immunodeficiency state is just from exogenous factors. Fortunately, primary immune deficiencies are rare diseases and only 6-7% of all of them, due to the neutrophilic functional disorders. Unfortunately, many exogenous and environmental factors have influence to the immune system, and the percentage of secondary caused neutrophilic functional disorders is much higher and should be considered when children are investigated for immunodeficiency. So, when to suspect neutrophil functional disorder? The hallmarks for diseases related to the neutrophilic functional disorders are discussed in this article.