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Research Notes: Short Chain Acyl-CoA Dehydrogenase DeficiencyJ Inherit Metab Dis. 2005. The 625G>A variant of the short-chain acyl-CoA dehydrogenase (SCAD) gene is considered to confer susceptibility for developing 'clinical SCAD deficiency' and appears to be common in the general population. To determine the frequency of the 625G>A variant in The Netherlands, we analysed 1036 screening cards of 5- to 8-day-old newborns and found 5.5% homozygous and 31.3% heterozygous for the 625G>A variant. An increased blood/plasma C4-carnitine concentration is considered to be one of the biochemical characteristics of SCAD deficiency. To explore the correlation of C4-carnitine levels with the 625G>A variant, we determined the C4-carnitine concentration, as well as the ratio of C4- to free carnitine, in blood spots from newborns, who were detected as homozygous, heterozygous or noncarriers for the gene variant. No significant differences were found between these groups. Our study demonstrates a high frequency of the 625G>A SCAD gene variant in the Dutch population, but no correlation to significantly increased C4-carnitine levels in blood spots taken between the 5th and 8th days of life. This latter observation might be the result of the relatively late timing of neonatal screening in our country, implying that fatty acid oxidation disorders may be missed at that stage. If the 625G>A variant is associated with clinical SCAD deficiency, the high frequency of the variant suggests a possible involvement of SCAD deficiency in the pathogenesis of common disorders, probably in relation to other genetic and/or environmental factors. However, homozygosity for the 625G>A variant might be only a biochemical phenomenon, representing a 'nondisease'. Pediatrics. 2003 Nov. OBJECTIVE: To understand the expanding clinical and biochemical spectrum of short-chain acyl-CoA dehydrogenase (SCAD) deficiency, the impact of which is not fully understood. STUDY DESIGN: We studied a family with SCAD deficiency and determined urinary ethylmalonic acid excretion, plasma C(4)-carnitine, SCAD enzyme activity in fibroblasts and lymphocytes, DNA mutations in the SCAD gene, and clinical expression. The index patient was born prematurely and had otherwise unexplained cholestasis and hepatomegaly during the first year of life. His mother developed a hemolysis-elevated liver enzymes-low platelets (HELLP) syndrome while pregnant with the index patient. RESULTS: Two siblings had a homozygous inactivating 1138C>T mutation, whereas the father was compound heterozygous for this mutation and the common 625G>A polymorphism. There was a good correlation between the type of SCAD mutation, the residual SCAD enzyme activity, and the levels of urinary ethylmalonic acid and plasma C(4)-carnitine in each of the eight family members. Retrospective acylcarnitine analysis of the index patient's Guthrie screening card confirmed the abnormal increase of C(4)-carnitine, suggestive of SCAD deficiency. None of the family members had hypotonia, developmental delay, or episodes of ketotic hypoglycemia. CONCLUSION: Homozygosity for an inactivating SCAD mutation does not necessarily result in disease. The previously held opinion that SCAD deficiency is always a serious disorder may have been influenced by a clinical bias. Homozygosity for an inactivating 1138C>T SCAD mutation was assessed by neonatal screening of blood spot acylcarnitines. SCAD deficiency may be associated with maternal HELLP syndrome. Pediatr Res. 2003 Aug. Tandem mass spectrometry was adopted for newborn screening by North Carolina in April 1999. Since then, three infants with short-chain acyl-CoA dehydrogenase (SCAD) and one with isobutyryl-CoA dehydrogenase deficiency were detected on the basis of elevated butyrylcarnitine/isobutyrylcarnitine (C4-carnitine) concentrations in newborn blood spots analyzed by tandem mass spectrometry. For three SCAD-deficient infants, biochemical evaluation included a plasma acylcarnitine profile with markedly elevated C4-carnitine, urine organic acid analysis with markedly elevated ethylmalonic and 2-methylsuccinic acids, and markedly elevated [U-13C]butyrylcarnitine concentrations in medium from fibroblasts incubated with [U-13C]palmitic acid and excess l-carnitine, consistent with classic SCAD deficiency. Two of three infants diagnosed with classic SCAD deficiency remained asymptomatic; however, the third infant presented with seizures and a cerebral infarct at 10 wk of age. All three infants had putatively inactivating mutations in both alleles of the SCAD gene. The highly elevated plasma C4-carnitine levels in the three infants detected by newborn screening tandem mass spectrometry differentiated them from infants and children who were homozygous or compound heterozygous for one of two SCAD gene susceptibility variations; for the latter group the C4-carnitine levels were normal. Isobutyryl-CoA dehydrogenase deficiency in a fourth infant was confirmed after isolated elevation of C4-carnitine in the acylcarnitine profile. Mol Genet Metab. 2003 Apr. Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a clinically heterogeneous disorder. The clinical phenotype varies from fatal metabolic decompensation in early life to subtle adult onset, some patients remain asymptomatic. Two mutations (511C>T; 625G>A) have been described in exons 5 and 6 of the SCAD gene. Although they alter the structural and catalytic properties of the SCAD protein, these variants are not true disease-causing mutations but confer disease susceptibility. Previous studies found these gene variants to be common in Europeans. We aimed to establish the frequency of these variants in the US population and to determine whether the presence of these variants correlates with elevated butyrylcarnitine (C(4)-acylcarnitine) concentrations in newborn blood spots. Based on the analysis of 694 samples, we found that the allele frequency of the 625G>A variant was significantly higher (22%) than that of the 511C>T variant (3%). These gene variants were detected in either homozygous or compound heterozygous form in 7% of the study population. Additionally, the frequency of the 625G>A allele in the Hispanic population (30%) was significantly higher than that of the African-American (9%) and Asian (13%) subpopulations. A previously unreported variant, IVS 5 (-10) C>T, was identified in three African-American newborns (0.3%). The C(4)-acylcarnitine concentration in blood spots was significantly higher in subjects homozygous for the 625A variant when compared to those homozygous for the wild type (p<0.0001). However, none of the observed genotypes was associated with a concentration of C(4)-acylcarnitine that would be consistent with a biochemical diagnosis of SCAD deficiency. Hum Mol Genet. 1998 Apr. 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. Introduction The importance of fatty acid [beta]-oxidation to sustain mitochondrial energy metabolism during periods of fasting is underscored by the potentially fatal manifestations of known inborn errors in this pathway (1-3). In particular, acyl-CoA dehydrogenase deficiencies constitute an important group of disorders which have been the focus of extensive investigations at the biochemical and molecular levels over the last decade. Despite this effort, short-chain acyl-CoA dehydrogenase (SCAD) deficiency still remains a poorly defined entity, since <10 verified patients with this disorder have been described (4-10), and only one case has been characterized previously at the molecular level (11). In sharp contrast to this limited number of patients with established SCAD deficiency, elevation of urinary ethylmalonic acid (EMA), a characteristic biochemical finding of diminished SCAD activity, is detected frequently in patients undergoing a metabolic work-up for a clinical suspicion of a metabolic disorder (12-14). Because urinary EMA elevation most likely reflects a cellular accumulation of butyryl-CoA (15), which is secondary to reduced SCAD catalytic activity, these patients are correctly considered as possibly having SCAD deficiency. In a previous study (16), we analysed the coding region of the SCAD gene in two patients with SCAD deficiency, whose diagnosis was based on the findings of elevated EMA excretion in urine and low SCAD activity in cultured skin fibroblasts (6). Surprisingly, the main result of the study was not the identification of new severe disease-causing mutations, but the discovery of a polymorphic variation in the SCAD gene, 625G/A. This variation results in a glycine/serine amino acid polymorphism at position 185 in the mature SCAD protein (G/S209 in the pre-SCAD protein) (16,17). In expression studies, the G185S variant enzyme, encoded by the 625A variant allele, was catalytically active, and therefore not a disease-causing mutation in a conventional sense, but the protein was more thermolabile than the wild-type enzyme. In addition, the 625A variant allele was found in homozygous form in 60% of 135 patients with elevated EMA excretion, analysed because of a suspicion of a metabolic disorder, compared with 7% of individuals in the general population (12). Taken together, these data suggest that the 625A variant allele is a susceptibility allele of the SCAD gene, which causes elevation of EMA in urine and, in combination with other genetic and/or environmental factors, may lead to a functional impairment of the enzyme's catalytic activity that, in some cases, is sufficiently severe to justify a diagnosis of SCAD deficiency in vitro. Isolated ethylmalonic aciduria may thus be a biochemical phenotype that can be subdivided into at least two groups. One group is comprised of patients with conventional SCAD deficiency caused by disruptive mutations in the SCAD gene. The second group consists of patients with a polygenic/multifactorial condition caused by the presence of a number of susceptibility alleles, located either in the SCAD locus, as is the 625A variant allele, or elsewhere. This distinction implies that patients with elevated EMA excretion, who are not homozygous for the 625A susceptibility allele, are the most likely candidates for carrying severe disease-causing mutations in the SCAD gene or, alternatively, other susceptibility polymorphisms. In the present study, we report the identification of three severe disease-causing SCAD gene mutations (274G -> T, 529T -> C and 1147C -> T) and one new disease-associated susceptibility mutation (511C -> T) in two patients, one of whom was homozygous and the other heterozygous for the common 625G allele. We have determined the frequencies of the mutations in normal control individuals and in a population of patients with elevation of EMA excretion in urine, and we have investigated the mechanism by which the mutations effect the protein function. [...] ...the present study has substantiated the view that ethylmalonic aciduria is a complex condition, with potential clinical consequences. In many patients, the condition is multifactorial, with the variant 625A allele emerging as the principal predisposing factor. Other susceptibility alleles, such as the 511T allele, and the presence of severe disease-causing mutations, such as 274G -> T, 529T -> C and 1147C -> T, may contribute together with as yet unknown cellular or genetic factors. Such factors include the protein quality control machinery or factors affecting the oxidative phosphorylation pathway, since an association between respiratory chain disorders and elevation of EMA excretion has been reported (26-28). In a small minority of cases with elevated EMA excretion, the disease is classically monogenic and caused by severe mutations such as 274G -> T and 529T -> C in patient 2 or 136C -> T and 319C -> T in the first case with SCAD deficiency to be characterized at the molecular level (11). These two groups, the multifactorial and the monogenic, are, however, not mutually exclusive, since in patients belonging to the monogenic group there may also be additional factors which modify the effect of the mutations, as observed for the variant R147W SCAD enzyme in patient 1 of this study. In addition to obvious diagnostic benefits, a comprehensive elucidation of the aetiology of ethylmalonic aciduria may also help us to understand better the processes which are important for the biogenesis, processing and degradation of aberrant mitochondrial proteins. Materials and methods Patient 1 This patient has been reported elsewhere (10): briefly, this Spanish girl presented at 3 months of age with coughing, vomiting and poor feeding. Bronchopneumonia was diagnosed and she recovered in 2 weeks. At 5 months of age, she was readmitted with hypotonia and a low level of consciousness. The urinary organic acid profile showed an increased level of EMA (126 mmol/mol creatinine; controls: <18 mmol/mol creatinine). At 4 years of age, her growth and development were normal. The range of EMA excretion during follow-up was 36-94 mmol/mol creatinine. Specific SCAD activity in cultured fibroblasts, determined by the ETF reduction assay (29), was reduced to 25% of controls, the results being confirmed in two independent laboratories. Patient 2 This African-American male presented with a shaking-staring spell on the first day of life (6,8). EMA excretion was strikingly abnormal (3900 mmol/mol creatinine), and remained elevated when tested during asymptomatic periods (range: 16-175 mmol/mol creatinine). Specific SCAD activity in fibroblasts showed severe deficiency, with <10% of control activity. Patients with elevated EMA excretion Blood spots for DNA analysis were collected prospectively in Germany, Denmark, the Czech Republic, Spain and the USA from patients with elevated EMA excretion (18-1185 mmol/mol creatinine). Typically, these patients were referred to a metabolic centre in order to investigate a possible inborn error of metabolism. Many of these patients showed neuromuscular signs such as hypotonia, convulsions and developmental delay, others showed symptoms suggestive of a possible fatty acid oxidation disorder, such as episodes of hypoglycaemia and lethargy. In about half of the cases, the abnormal EMA excretion was documented in more than one urine specimen. In these patients, there was no apparent correlation between the nature and severity of the symptoms and the level of EMA excretion. The samples used in the present study were blood spots from 133 of the 135 patients analysed previously for the SCAD 625G/A polymorphism (12). |