[Show abstract][Hide abstract] ABSTRACT: We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild-type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.
Human Molecular Genetics 04/1998; 7(4):619-27. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.
Human Molecular Genetics 06/1997; 6(5):695-707. · 6.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inherited enzyme defects in mitochondrial fatty acid oxidation (FAO) are associated with acute metabolic crisis and sudden death. Necropsy findings may be subtle, yielding no diagnosis and precluding genetic counseling. Preliminary identification of an FAO disorder requires the use of sophisticated tools (e.g., GC/MS) and specific body fluids, and the diagnosis rests on molecular analysis or enzyme assay. At present, confirmation of long-chain or short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency relies on measurement of enzyme activity. Here, we report our examination of the effect of storage temperature (25, 4, -20, and -70 degrees C) and the postmortem interval on enzyme activities in rat and human liver. Enzyme activity decreases 50% in 30 h in samples stored at 25 degrees C, whereas 55 h at 4 degrees C is required to reach this value; freezing minimizes this loss. Regardless of rate of degradation, however, the short-chain to long-chain activity ratio remains constant--which should make it possible to differentiate postmortem degradation from enzyme deficiency.
[Show abstract][Hide abstract] ABSTRACT: The similarity of the hepatic pathology in acute fatty liver of pregnancy (AFLP) to that seen in children with inherited disorders of intramitochondrial fatty acid oxidation (FAO) suggests that there may be a genetic basis for some cases of AFLP.
The purpose of this study was to examine patients with AFLP and their offspring to determine if there were women with AFLP who were heterozygous for the FAO defect, long chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency.
We evaluated 12 women previously diagnosed with AFLP. Provocative fasting studies and skin biopsies for examination of their cultured skin fibroblasts were performed to search for a generalized defect in FAO both in vivo and in vitro. Cultured skin fibroblasts from AFLP patients, their children, and their husbands were also examined specifically for LCHAD activity.
Of 12 women with a previous episode of AFLP, eight had reduced LCHAD activity consistent with being heterozygous for LCHAD deficiency. The eight heterozygotes had a total of nine pregnancies complicated by AFLP. In seven of those nine pregnancies, the women developed severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. Of the nine offspring delivered from these pregnancies, four were confirmed to be affected with homozygous LCHAD deficiency. Three other deceased infants were presumed to be LCHAD-deficient based on clinical findings, postmortem examination, and confirmed heterozygote parents. The remaining two infants delivered after pregnancies complicated by AFLP had LCHAD activity in the heterozygous range and are healthy at 18 and 24 months of age. Consistent with the known autosomal recessive nature of this defect, five tested husbands of LCHAD heterozygous women with a history of AFLP and affected infants also showed reduced LCHAD activity.
These studies indicate that a significant subgroup of women with AFLP are heterozygous for LCHAD deficiency and that careful observation of their offspring for signs of this disorder is warranted. Severe preeclampsia appears to increase the risk of AFLP in LCHAD heterozygous women.
The American Journal of Gastroenterology 12/1996; 91(11):2293-300. · 9.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acute fatty liver of pregnancy (AFLP) is a devastating late gestational complication with many similarities to the inherited disorders of mitochondrial fatty acid oxidation. We report the molecular defects in a woman with AFLP and her infant who subsequently was diagnosed with trifunctional protein (TFP) deficiency. We used single-stranded conformation variance and DNA sequence analyses of the human TFP alpha-subunit gene, which encodes the long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity, to demonstrate a C to T mutation (C1678T) in exon 16 present on one allele in the mother and the affected infant. This creates a premature termination codon (R524Stop) in the LCHAD domain. Using reverse transcriptase-PCR amplification of the alpha-subunit mRNA from cultured fibroblasts, we demonstrated that transcripts containing this R524Stop mutation are present at very low levels, presumably because of rapid mRNA degradation. The affected infant also had the common E474Q mutation (nucleotide G1528C) on the second allele. Thus, he is a compound heterozygote. The father and two normal siblings are heterozygous for this E474Q mutation. This initial delineation of the R524Stop mutation provides evidence of the heterogeneity of genetic defects responsible for TFP deficiency and AFLP.
Pediatric Research 10/1996; 40(3):393-8. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endocardial fibroelastosis (EFE) has previously been shown to be associated with tissue carnitine deficiency, although the basis for the carnitine deficiency has not been documented. A patient with the classical features of EFE and marked deficiency of carnitine in heart muscle, skeletal muscle, and liver is presented in this report. Cultured skin fibroblasts from both parents demonstrated levels of carnitine uptake at 50% of the normal rate. This is consistent with heterozygosity for the plasma membrane carnitine transporter defect, indicating likely homozygosity for this recently recognized inborn error in the index patient.
[Show abstract][Hide abstract] ABSTRACT: beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 220.127.116.11] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.
Proceedings of the National Academy of Sciences 11/1995; 92(23):10496-500. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Negative chemical ionization (NCI) mass spectrometry was used to quantify the acyl-CoA intermediates present in human fibroblasts growing in media containing the long-chain fatty acid, palmitate. The acyl-CoA intermediates were detected as the N-acyl pentafluorobenzyl glycinates. In fibroblasts from normal individuals only saturated acyl-CoA esters were detected, supporting the concept that the acyl-CoA dehydrogenase reaction is the rate-limiting step of intramitochondrial fatty acid oxidation. In patients with inherited enzymatic defects of intramitochondrial long-chain fatty acid oxidation, there was not a significant increase in the amount of long-chain acyl-CoA compounds, with palmitoyl-CoA amounts similar to those found in controls. However, there was a sharp decrease in the relative amount of lauroyl-CoA and a resultant sixfold elevation in the palmitoyl-CoA:lauroyl-CoA ratio. In contrast, fibroblasts with a defect involving the transport of fatty acids across the mitochondrial membrane, carnitine palmitoyl transferase 1 deficiency, had a fourfold increase in palmitoyl-CoA. Our results suggest that acyl-CoA esters in biological tissues are readily detectable using NCI mass spectrometry. This approach is significantly more sensitive than previous methods for the detection of these important metabolic intermediates, and may prove useful in the study of fatty acid oxidation in both normal and enzyme-deficient tissues.
Biochemical and Molecular Medicine 07/1995; 55(1):15-21.
[Show abstract][Hide abstract] ABSTRACT: We identified two additional patients with short-chain acyl-coenzyme A (CoA), further characterized the clinical and biochemical features of this defect, and compared it with other fatty acid oxidation defects.
We have measured the in vitro short-chain acyl-coenzyme A dehydrogenase (SCAD) activity in six affected persons with the electron-transfer flavoprotein-linked assay in the presence and absence of anti-medium-chain acyl-CoA dehydrogenase antibody. Urine organic acids, acylglycines, acylcarnitines, and radiolabeled substrate catabolism by skin fibroblasts were also examined.
All patients had some neurologic abnormalities, including hypotonia, hypertonia, or seizures. None of the patients had episodes of hypoglycemia; in the only patient tested, fasting ketogenesis was not impaired. Four patients were initially seen in the neonatal period, two with profound metabolic acidosis and two with mild acidemia; the other two cases were recognized in infancy. Enzymatic analysis of cultured skin fibroblasts demonstrated approximately 10% activity of SCAD when compared with control fibroblasts. Gas chromatography and mass spectrometry of urine revealed that ethylmalonic acid was present in all samples but not always at elevated concentrations; methylsuccinic acid and butyrylglycine were sporadically elevated. n-Butyrylcarnitine was often found in urine and plasma. Radiolabeled substrate metabolism was reduced to 40% to 60% of control values.
Because affected persons do not consistently excrete characteristic metabolites, the diagnosis of this enzymatic deficiency is difficult. It is necessary to collect and analyze several urine and plasma specimens when the diagnosis is being considered in patients with neurologic abnormalities suggestive of this disorder.
Journal of Pediatrics 07/1995; 126(6):910-5. · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial long chain fatty acid beta-oxidation provides the major source of energy in the heart. Deficiencies of human beta-oxidation enzymes produce sudden, unexplained death in childhood, acute hepatic encephalopathy, skeletal myopathy, or cardiomyopathy. Long chain 3-hydroxyacyl-CoA dehydrogenase [LCHAD; long-chain-(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 18.104.22.168] catalyzes the third step in beta-oxidation, and this activity is present on the C-terminal portion of the alpha subunit of mitochondrial trifunctional protein. We used single-stranded conformation variance analysis of the exons of the human LCHAD (alpha subunit) gene to determine the molecular basis of LCHAD deficiency in three families with children presenting with sudden unexplained death or hypoglycemia and abnormal liver enzymes (Reye-like syndrome). In all families, the mothers had acute fatty liver and associated sever complications during pregnancies with the affected infants. The analysis in two affected children revealed a G to C mutation at position 1528 (G1528C) of the alpha subunit of the trifunctional protein on both alleles. This is in the LCHAD domain and substitutes glutamine for glutamic acid at position 474 of mature alpha subunit. The third child had this G1528C mutation on one allele and a different mutation (C1132T) creating a premature termination codon (residue 342) on the second allele. Our results demonstrate that mutations in the LCHAD domain of the trifunctional protein alpha subunit in affected offspring are associated with maternal acute fatty liver of pregnancy. This is the initial delineation of the molecular basis of isolated LCHAD deficiency.
Proceedings of the National Academy of Sciences 02/1995; 92(3):841-5. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A neonate with signs of neurologic dysfunction was noted to have elevated blood lactic acid levels. Organic acid analysis revealed transient elevations in ethylmalonate, methylsuccinate, butyrylglycine, and butyrylcarnitine. Enzyme assay in cultured skin fibroblasts confirmed short-chain acyl coenzyme. A dehydrogenase deficiency. The intermittent nature of the characteristic metabolic markers for this deficiency make diagnosis difficult. The apparent rarity of the disorder may be the result of underdiagnosis.
Journal of Pediatrics 02/1995; 126(1):69-71. · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic diseases of mitochondrial fatty acid oxidation have recently emerged as important disorders to consider in the differential diagnosis of hypoglycemia, cardiomyopathy, or skeletal muscle weakness in infants and children. A total of 16 different defects have been identified over the past decade that involve almost all of the possible enzyme steps in the pathway. One of these disorders, medium-chain acyl-coenzyme A dehydrogenase deficiency has a frequency as high as 1 in 10,000 births and is the single most common genetic defect of intermediary metabolism. The disorders are frequently mistaken for Reye syndrome or sudden infant death syndrome. Improved methods have simplified the diagnosis of some of the fatty acid oxidation defects. However, recognition of these disorders remains challenging. Rapid advances have continued to be made over the past year in defining clinical phenotypes, diagnostic methods, and therapeutic strategies. Familiarity with this new group of disorders is becoming increasingly important for general pediatricians as well as subspecialists in metabolism, endocrinology, gastroenterology, cardiology, neurology, and genetics.
Current Opinion in Pediatrics 09/1994; 6(4):476-81. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of acute fatty liver of pregnancy is unknown, but similarities in the clinical presentation and the histological appearance of the liver with those found in children with metabolic defects in the intramitochondrial beta-oxidation pathway of the liver suggest that a disturbance in hepatic fatty acid oxidation may play a role. We report a woman with acute fatty liver of pregnancy who gave birth to a seemingly normal full-term infant who was seen at 4 mo of age with hypoglycemia, coma and profound hepatic steatosis. The infant had a defect in fatty acid oxidation, long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency, and the mother proved to be heterozygous for this metabolic condition. We hypothesize that the interaction of an affected fetus with a female heterozygous for this defect in fatty acid oxidation in the late third trimester accounts for some cases of acute fatty liver of pregnancy.
[Show abstract][Hide abstract] ABSTRACT: We describe four Italian male infants with a novel clinical phenotype characterized by orthostatic acrocyanosis, relapsing petechiae, chronic diarrhea, progressive pyramidal signs, mental retardation, and brain magnetic resonance imaging abnormalities. The first symptoms appeared after the termination of breast-feeding and introduction of formula feeding. Marked persistent 2-ethylmalonic aciduria was associated with abnormal excretion of C4-C5(n-butyryl-, isobutyryl-, isovaleryl-, and 2-methylbutyryl-)acylglycines and acylcarnitines and with intermittent lactic acidosis. Short- and branched-chain plasma acylcarnitine levels were also elevated. 2-Ethylmalonic aciduria is generally regarded as being indicative of a defect in fatty acid oxidation. Extensive studies of cultured fibroblasts failed to reveal such a defect. The observation of intermittent urinary excretion of 2-ethylhydracrylic acid pointed to involvement of the isoleucine R pathway in ethylmalonate biosynthesis. This hypothesis was tentatively corroborated by the biochemical responses to an oral isoleucine challenge in two patients. However, fibroblast studies showed normal oxidation rates of (14C)isoleucine (ul), indicating that this is not a defect of isoleucine oxidation expressed in skin fibroblasts. In one of two patients tested, cytochrome c oxidase activity was partially reduced (45%) in cultured fibroblasts. This unique clinical and biochemical phenotype identifies a new metabolic encephalopathy of yet undetermined cause.
Journal of Pediatrics 02/1994; 124(1):79-86. · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a frequent and sometimes fatal inherited metabolic disorder of fatty acid beta-oxidation. A eukaryotic expression system was used to study naturally occurring mutations in MCAD. The 1263 nucleotide coding region of human MCAD cDNA was inserted downstream of the SV40 early promoter for high-level expression in Chinese hamster ovary cells. Both normal MCAD cDNA and a mutant MCAD cDNA containing the common, disease-causing A to G transition at position 985 (A985G), which alters a lysine to a glutamic acid (K304E), were inserted into expression vectors. Transient transfection of Chinese hamster ovary cells was performed with the expression constructs. The steady state level of expressed normal MCAD protein antigen was substantially higher (5-fold) than the expressed mutant protein. The MCAD enzymatic activity in protein extracts from cells containing the expressed normal MCAD cDNA was also much higher (6-fold) than the activity in cells expressing the mutant MCAD. Therefore, these data confirm that the common K304E mutation causes MCAD deficiency primarily by decreased protein stability rather than reduction of catalytic activity and, in fact, demonstrate that the K304E mutant protein has a similar sp act against octanoyl CoA substrate as the normal protein.
Pediatric Research 12/1993; 34(5):694-7. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 28-month-old Turkish girl presented with recurrent bronchopneumonia and severe muscular hypotonia. Urinary excretion of ethylmalonic acid was persistently elevated, methylsuccinate appearing only in stress situations. Studies in cultured fibroblasts showed a deficiency of short-chain acyl-CoA dehydrogenase.
European Journal of Pediatrics 12/1993; 152(11):922-4. · 1.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reduced plasma and tissue concentrations of carnitine, a cofactor required for fatty acid oxidation, are present in patients with inherited disorders of mitochondrial acyl-CoA oxidation that are associated with accumulations of acylcarnitines. To determine whether the secondary carnitine deficiency in these patients is due to excessive urinary loss of acylcarnitines, the development of carnitine deficiency was examined in patients with four different acyl-CoA oxidation disorders, including medium-chain and long-chain fatty acyl-CoA dehydrogenase deficiencies, isovaleric acidemia, and propionic acidemia. After a 3-mo period of treatment with oral carnitine to raise plasma total carnitine concentrations to or above normal, patients were started on a carnitine-free diet and the changes in plasma total and free carnitine levels and urinary total and free carnitine excretion were followed for 5 d. Patients with all four disorders showed a return of plasma carnitine levels and urinary carnitine excretion to baseline within 2 to 4 d. The rapidity of these changes could not be explained solely by excessive acylcarnitine wasting. Continued excretion of free carnitine in all patients indicated the additional presence of an impairment in renal transport of free carnitine. Consistent with this interpretation, estimates of renal thresholds for free carnitine gave values that were less than that for a control child in all four disorders and ranged as low as one half those reported in normal individuals. These results suggest that secondary carnitine deficiency in the acyl-CoA oxidation disorders is due to indirect as well as direct effects of accumulated acylcarnitines.(ABSTRACT TRUNCATED AT 250 WORDS)
Pediatric Research 08/1993; 34(1):89-97. · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Screening for medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency by urinary 3-phenylpropionylglycine may not be reliable in early infancy because young infants are not colonized with adult-type colonic flora. In this study we delineated the microbes that produce 3-phenylpropionic acid, the precursor of 3-phenylpropionylglycine. We found that the use of some antibiotics may alter gut flora, thereby confounding this method of screening for MCAD deficiency.
Journal of Pediatrics 02/1993; 122(1):100-3. · 3.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An infant with glycogen storage disease and prolonged malnourishment showed a urinary organic acid profile during an episode of fasting hypoglycaemia with inappropriate hypoketotic dicarboxylic aciduria that was indistinguishable from that reported in long-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency. Although there was a striking elevation of urinary 3-hydroxydecanedioic acid, the ratios between hydroxydicarboxylic acids were consistent with values reported to be indicate of medium-chain acyl-CoA dehydrogenase deficiency. We suspect that the fasting 3-hydroxydicarboxylic aciduria was attributable to secondarily impaired enzyme activities, the consequence of malnutrition, early infancy, and/or glycogen storage disease. Caution is advised in the interpretation of urinary organic acid patterns that indicate a 3-hydroxydicarboxylic aciduria, as well as an inappropriate hypoketotic dicarboxylic aciduria, as they may represent non-specific findings.
[Show abstract][Hide abstract] ABSTRACT: Medium chain acyl-coenzyme A dehydrogenase deficiency (MCADD) was the first metabolic disorder found to be associated with sudden infant death syndrome. This review covers recent advances in the biochemical and molecular understanding of MCADD.
New Jersey medicine: the journal of the Medical Society of New Jersey 10/1992; 89(9):675-8.