d-Glycerate kinase deficiency in a neuropediatric patient.

d-Glyceric aciduria (DGA) due to d-glycerate kinase deficiency (DGKD) is a rare autosomal-recessive inborn error of metabolism that is usually linked to the metabolism of fructose and serine. We describe a Moroccan patient with DGKD whose metabolic defect has been characterized by metabolite studies, sequencing of genomic DNA and by studies on the RNA level. Since birth the index patient presented with severe muscular hypotonia, joint hypermobility and tremor. Enantioselective analysis showed elevated d-glyceric acid in the urine of the patient, but not in that of his parents. DNA analysis revealed homozygosity in the GLYCTK gene for c.517G>T [p.(Val173Leu)], the first mutation reported for exon 3 of this gene, as well as for the c.530-4A>G polymorphism. RNA studies suggest that none of these sequence variants affects splicing. The mother was heterozygous for both sequence variants, the father heterozygous for the first one and homozygous for the polymorphism, which further supports that c.517G>T is the functionally relevant nucleotide change. The conservation of GLYCTK throughout evolution suggests an important biological role of this enzyme, although it is not known yet how mutations are linked to clinical features. Future studies should investigate the molecular defect in a more general way and search for additional roles of GLYCTK beyond its established role in catabolism of serine and fructose.

Acylpeptide hydrolase (APEH) sequence variants with potential impact on the metabolism of the antiepileptic drug valproic acid.

Acylpeptide hydrolase (APEH) is a serine protease involved in the recycling of amino acids from acylated peptides. Beyond that, APEH participates in the metabolism of the antiepileptic drug valproic acid (2-propylpentanoic acid; VPA) by catalyzing the hydrolysis of the VPA metabolite valproylglucuronide (VPA-G) to its aglycon. It has been shown that the inhibition of APEH by carbapenem antibiotics decreases therapeutic VPA levels by enhancing the urinary elimination of VPA in form of VPA-G. As various sequence variants of the APEH gene (which encodes the APEH protein) are listed in databases, but have not been functionally characterized yet, we assume, that some APEH sequence variants may have pharmacogenetic relevance due to their impaired cleavage of VPA-G. APEH sequence variants predicted to affect enzyme activity were selected from databases, and overexpressed in HEK293 cells (stable transfection), a cell line derived from human embryonic kidney cells. APEH activity in cell homogenates was determined spectrophotometrically by monitoring the hydrolysis of the synthetic substrate N-acetyl-L-alanine-nitroanilide. APEH enzyme activity and protein expression of the sequence variants were compared with those of APEH with the reference sequence. Three out of five tested missense sequence variants resulted in a considerable decrease of enzyme activity assessed with the standard substrate N-acetyl-L-alanine-nitroanilide, suggesting an effect on pharmacokinetics of VPA. Our work underlines the need to consider the APEH genotype in investigations of altered VPA metabolism.

Further evidence that d-glycerate kinase (GK) deficiency is a benign disorder.

d-Glyceric aciduria is caused by deficiency in d-glycerate kinase (GK) due to recessive mutations in the GLYCTK gene. GK catalyzes the conversion of d-glycerate to 2-phosphoglycerate which is an intermediary reaction in the catabolism of serine and fructose. Deficiency of GK leads to accumulation of d-glycerate, which may be detected in urine organic acid analysis. Debate exists as to whether this is a benign or disease-causing disorder as the reported phenotypes vary significantly. We report two siblings from a consanguineous Pakistani family. The index case is a 5year old boy with severe autism and global developmental delay. His urine organic acid analysis showed markedly increased excretion of glycerate, determined as d-form by enantioselective gas chromatography. There was no oxalic aciduria. His younger sister (3years old) is asymptomatic and developmentally normal (already bilingual). Her urine showed similar amounts of d-glycerate. Both children are homozygous for the novel mutation c.767C>G in exon 5 of the GLYCTK gene, predicted to affect the enzyme by replacing the evolutionarily conserved Proline with Arginine (P256R). Both parents are heterozygous carriers. These cases support the view that d-glycerate kinase deficiency is a benign disorder. Long term follow-up studies with a greater number of individuals may be required for further confirmation.

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.

Heterozygous Monocarboxylate Transporter 1 (MCT1, SLC16A1) Deficiency as a Cause of Recurrent Ketoacidosis.

We describe two half-siblings with monocarboxylate transporter 1 (MCT1, SLC16A1) deficiency, a defect on ketone body utilization, that has only recently been identified (van Hasselt et al., N Engl J Med, 371:1900-1907, 2014) as a cause for recurrent ketoacidoses. Our index patient is a boy with non-consanguineous parents who had presented acutely with impaired consciousness and severe metabolic ketoacidosis following a 3-day history of gastroenteritis at age 5 years. A 12.5-year-old half-brother who shared the proband's mother also had a previous history of recurrent ketoacidoses. Results of mutation and enzyme activity analyses in proband samples advocated against methylacetoacetyl-coenzyme A thiolase ("beta-ketothiolase") and succinyl-coenzyme A: 3-oxoacyl coenzyme A transferase (SCOT) deficiencies. A single heterozygous c.982C>T transition in the SLC16A1 gene resulting in a stop mutation (p.Arg328Ter) was detected in both boys. It was shared by their healthy mother and by the proband's half-sister, but was absent in the proband's father. MCT1 deficiency may be more prevalent than is apparent, as clinical manifestations can occur both in individuals with bi- and monoallelic mutations. It may be an important differential diagnosis in recurrent ketoacidosis with or without hypoglycemia, particularly in the absence of any specific metabolic profiles in blood and urine. Early diagnosis may enable improved disease management. Careful identification of potential triggers of metabolic decompensations in individuals even with single heterozygous mutations in the SLC16A1 gene is indicated.