Clinical and molecular investigations of Japanese cases of glutaric acidemia type 2.

Glutaric acidemia type 2 (GA2) is an autosomal recessive disorder resulting from a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH) that manifests from most severe neonatal to late-onset forms. However, the genetic defect responsible for the disease and clinical severity is not well-characterized. In order to understand the relationship between the phenotype and genetic defect, we investigated the clinical and molecular features of 15 Japanese patients, including 4 previously reported cases. Three patients had the neonatal form and 8 patients had the late-onset form, 1 of whom presented an extremely mild phenotype. Immunoblot analysis showed that either ETFalpha, ETFbeta, or ETFDH was significantly reduced or absent in all patients. However, no specific enzyme deficiency predominated, and there were no associations with the clinical severity. Genetic analyses identified 15 mutations including non-sense, missense, splice site mutations, and small deletions, in ETFA, ETFB and ETFDH genes. Although almost all mutations were unique to Japanese patients and no common mutations were found, some of them appeared to be associated with a specific phenotype. Our results suggest that clinical and mutational spectrums of Japanese GA2 patients are heterogeneous and that genetic diagnoses may help to predict a prognosis and provide more accurate diagnostic information for patients and families with GA2.

Urinary organic metabolite screening of children with influenza-associated encephalopathy for inborn errors of metabolism using GC/MS.

BACKGROUND: Influenza-associated encephalopathy (IAE) occurs in childhood often with a serious clinical course and fatal outcomes. We screened children with IAE using GC/MS to determine whether they have metabolic disorders such as organic acidemias. METHODS: Urine samples from 70 Japanese children with IAE were analyzed between 2001 and 2005 using GC/MS with solvent extraction and direct drying methods. RESULTS: Apparent metabolic disorders in 6 of 70 tested children included vitamin B12-responsive methylmalonic acidemia (MMA; n=1), fructose-1,6-diphosphatase (FDPase) deficiency (n=1) and non-ketotic dicarboxylic aciduria (n=4) suggesting disorders of fatty acid oxidation. One child had an FDPase deficiency, for which glycerol infusion was contraindicated. Valproic acid metabolites were detected in 10 children and urinary glycerol excretion was increased in 22 of them after glycerol treatment. DISCUSSION: Our results showed that inborn errors of metabolism (IEM), such as organic acidemias, are detectable among children with IAE. Patients with metabolic disorders such as an FDPase deficiency and MMA often have hypoglycemia, hyperammonemia and acute lactic acidemia. Importantly, a detection of FDPase deficiency in which glycerol infusion is contraindicated suggests careful selection of a treatment strategy for "acute encephalopathy". We detected valproic acid and its metabolites at a rate of 14%, which was considerably higher than that in a control population. Thus, convulsive disorders might be a risk factor associated with IAE.

ESI-MS/MS study of acylcarnitine profiles in urine from patients with organic acidemias and fatty acid oxidation disorders.

Acylcarnitines in urine from 45 patients with organic acidemias and fatty acid oxidation disorders were evaluated using ESI-MS/MS. The urinary acylcarnitine profiles in organic acidemias, SCAD deficiency and MCAD deficiency were compatible with blood acylcarnitine profiles, and abnormalities in urinary acylcarnitine profiles in these conditions were enhanced following carnitine loading. Urinary acylcarnitine profiles were not helpful for characterization of long-chain fatty acid disorders, but a combination of urine and blood acylcarnitine analysis was useful for differential diagnosis of carnitine deficit.

In vitro probe acylcarnitine profiling assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry predicts severity of patients with glutaric aciduria type 2.

Glutaric aciduria type 2 (multiple acyl-CoA dehydrogenase deficiency, MAD) is a multiple defect of mitochondrial acyl-CoA dehydrogenases due to a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase. The clinical spectrum are relatively wide from the neonatal onset, severe form (MAD-S) to the late-onset, milder form (MAD-M). In the present study, we determined whether the in vitro probe acylcarnitine assay using cultured fibroblasts and electrospray ionization tandem mass spectrometry (MS/MS) can evaluate their clinical severity or not. Incubation of cells from MAD-S patients with palmitic acid showed large increase in palmitoylcarnitine (C16), whereas the downstream acylcarnitines; C14, C12, C10 or C8 as well as C2, were extremely low. In contrast, accumulation of C16 was smaller while the amount of downstream metabolites was higher in fibroblasts from MAD-M compared to MAD-S. The ratio of C16/C14, C16/C12, or C16/C10, in the culture medium was significantly higher in MAD-S compared with that in MAD-M. Loading octanoic acid or myristic acid led to a significant elevation in C8 or C12, respectively in MAD-S, while their effects were less pronounced in MAD-M. In conclusion, it is possible to distinguish MAD-S and MAD-M by in vitro probe acylcarnitine profiling assay with various fatty acids as substrates. This strategy may be applicable for other metabolic disorders.