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ABSTRACT: Multiple acyl-CoA dehydrogenase deficiency (MADD, OMIM 231680) or glutaric aciduria type II (GAII) is an inherited autosomal recessive disease affecting fatty acid, amino acid and choline metabolism, due to mutations in one of three genes namely, electron transfer flavoprotein alpha-subunit, ETFA (OMIM 608053), electron transfer flavoprotein beta-subunit, ETFB (OMIM 130410) and electron transfer flavoprotein dehydrogenase, ETFDH (OMIM 231675). Some MADD patients are responsive to riboflavin treatment with an excellent prognosis. Recently, mutations in ETFDH were found to be responsible for all riboflavin-responsive MADD patients. In this study, we present the clinical features and molecular studies of 2 Chinese families with riboflavin-responsive MADD.
Genomic DNA was extracted from peripheral blood samples or skin fibroblast cultures from the patients and normal controls. The thirteen exons of ETFDH were amplified by PCR. PCR products were sequenced in both forward and reverse directions. To rule out mutations in other genes, phenotype segregation was studied in the families by microsatellite markers in the proximity of the 3 genes, ETFA, ETFB and ETFDH.
Four novel mutations in ETFDH were detected in the 2 families. In family 1, a frame shift mutation, c.1355delG which introduced a premature-termination codon (PTC), I454X in exon 11 of ETFDH was found. Another mutation was a c.250G>A transition in exon 3 of ETFDH, A84T. In family 2, two novel missense mutations were identified, P137S, in exon 4 and G467R in exon 11. No carrier of these four mutations was identified from about 150 alleles of healthy Chinese control subjects.
Four novel mutations (3 missenses and 1 deletion) in ETFDH were found in Chinese families that presented with riboflavin-responsive MADD, which further expands the list of mutations found in patients with riboflavin-responsive MADD. Furthermore, we illustrated the utility of phenotype-genotype segregation in MADD families to prioritize genes for sequencing or to rule out the presence of disease causing mutation in other genes in MADD and other diseases caused by multiple genes.
Clinica chimica acta; international journal of clinical chemistry 03/2009; 404(2):95-9. · 2.54 Impact Factor
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ABSTRACT: Two separate and complementary assays, total mitochondrial fatty acid beta-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by measurement of deuterated water enrichment and to combine it with the conventional acylcarnitine profiling method into a single tracer incubation experiment.
Skin fibroblasts were incubated in a medium containing universal deuterium-labeled palmitate ((2)H(31)-palmitate) and l-carnitine without glucose supplementation for 96 h. The culture medium was assayed for deuterated water enrichment using isotope ratio mass spectrometry (IRMS) and acylcarnitine profiling by electrospray-ionization tandem mass spectrometry (ESI/MS/MS).
The medians of (2)H(2)O enrichment after 96 h of incubation of (2)H(31)-palmitate of the control, other inherited metabolic diseases and FAOD cell lines were 109.9, 102 and 23.1 ppm/mg protein/96 h, respectively. All fibroblasts with FAOD except carnitine uptake defective, multiple acyl-CoA dehydrogenase and short-chain 3-hydroxyacyl-CoA dehydrogenase deficient cells were well separated from the control (<60% control median, p<0.05) and could be identified by IRMS assay. Accumulations of disease-specific acylcarnitines due to blockage in the carnitine cycle and FAO spiral were also demonstrated by acylcarnitine profiling.
This novel functional assay is less time consuming and relatively simple by comparison to other published methods and can be used to investigate patients suspected to have FAO defects.
Clinica Chimica Acta 08/2007; 382(1-2):25-30. · 2.54 Impact Factor
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ABSTRACT: We tested the application of bioinformatic algorithms in studying the metabolomic profiles of neonatal urine samples with clinical evidence of severe asphyxia at birth and subsequent neurodevelopmental handicap.
The clinical outcomes of 256 newborns that required direct admission to neonatal intensive care unit for respiratory support or did not require direct admission were studied. Urinary metabolite profiles were measured by high throughput mass spectrometry and analyzed by bioinformatic methods.
We found a positive relationship between suppressed biochemical networks involved in macromolecular synthesis and birth asphyxia associated with significant neonatal oxidative stress and morbidity. The metabolomic discriminators between good neonatal outcome and poor neonatal outcome were established using hierarchical clustering analysis. Concentrations of eight urinary organic acids in distinct biochemical pathways were elevated and significantly associated with the prognosis of neurodevelopmental handicap with high sensitivity and specificity: ethylmalonate, 3-hydroxy-3-methylglutarate, 2-hydroxy-glutarate and 2-oxo-glutarate were associated with good neonatal outcome, whereas glutarate, methylmalonate, 3-hydroxy-butyrate and orotate were associated with poor outcome.
The data demonstrated the potential application of bioinformatics methods in this metabolomic study and proved its clinical relevance.
Clinical Biochemistry 04/2006; 39(3):203-9. · 2.08 Impact Factor
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ABSTRACT: BackgroundMultiple acyl-CoA dehydrogenase deficiency (MADD, OMIM 231680) or glutaric aciduria type II (GAII) is an inherited autosomal recessive disease affecting fatty acid, amino acid and choline metabolism, due to mutations in one of three genes namely, electron transfer flavoprotein alpha-subunit, ETFA (OMIM 608053), electron transfer flavoprotein β-subunit, ETFB (OMIM 130410) and electron transfer flavoprotein dehydrogenase, ETFDH (OMIM 231675). Some MADD patients are responsive to riboflavin treatment with an excellent prognosis. Recently, mutations in ETFDH were found to be responsible for all riboflavin-responsive MADD patients. In this study, we present the clinical features and molecular studies of 2 Chinese families with riboflavin-responsive MADD.MethodsGenomic DNA was extracted from peripheral blood samples or skin fibroblast cultures from the patients and normal controls. The thirteen exons of ETFDH were amplified by PCR. PCR products were sequenced in both forward and reverse directions. To rule out mutations in other genes, phenotype segregation was studied in the families by microsatellite markers in the proximity of the 3 genes, ETFA, ETFB and ETFDH.ResultsFour novel mutations in ETFDH were detected in the 2 families. In family 1, a frame shift mutation, c.1355delG which introduced a premature-termination codon (PTC), I454X in exon 11 of ETFDH was found. Another mutation was a c.250G>A transition in exon 3 of ETFDH, A84T. In family 2, two novel missense mutations were identified, P137S, in exon 4 and G467R in exon 11. No carrier of these four mutations was identified from about 150 alleles of healthy Chinese control subjects.ConclusionsFour novel mutations (3 missenses and 1 deletion) in ETFDH were found in Chinese families that presented with riboflavin-responsive MADD, which further expands the list of mutations found in patients with riboflavin-responsive MADD. Furthermore, we illustrated the utility of phenotype–genotype segregation in MADD families to prioritize genes for sequencing or to rule out the presence of disease causing mutation in other genes in MADD and other diseases caused by multiple genes.
Clinica Chimica Acta.
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[show abstract]
[hide abstract]
ABSTRACT: BackgroundTwo separate and complementary assays, total mitochondrial fatty acid β-oxidation (FAO) flux rate and acylcarnitine profiling, have been used to establish a definitive diagnosis of FAO defects (FAOD) in cultured cells. We developed a novel functional assay for total FAO rate assay by measurement of deuterated water enrichment and to combine it with the conventional acylcarnitine profiling method into a single tracer incubation experiment.MethodsSkin fibroblasts were incubated in a medium containing universal deuterium-labeled palmitate (2H31-palmitate) and l-carnitine without glucose supplementation for 96 h. The culture medium was assayed for deuterated water enrichment using isotope ratio mass spectrometry (IRMS) and acylcarnitine profiling by electrospray-ionization tandem mass spectrometry (ESI/MS/MS).ResultsThe medians of 2H2O enrichment after 96 h of incubation of 2H31-palmitate of the control, other inherited metabolic diseases and FAOD cell lines were 109.9, 102 and 23.1 ppm/mg protein/96 h, respectively. All fibroblasts with FAOD except carnitine uptake defective, multiple acyl-CoA dehydrogenase and short-chain 3-hydroxyacyl-CoA dehydrogenase deficient cells were well separated from the control (< 60% control median, p < 0.05) and could be identified by IRMS assay. Accumulations of disease-specific acylcarnitines due to blockage in the carnitine cycle and FAO spiral were also demonstrated by acylcarnitine profiling.ConclusionsThis novel functional assay is less time consuming and relatively simple by comparison to other published methods and can be used to investigate patients suspected to have FAO defects.
Clinica Chimica Acta.
