Short-chain acyl-CoA Research Articles

A Rare Case of Short-Chain Acyl-COA Dehydrogenase Deficiency: The Apparent Rarity of the Disorder Results in Under Diagnosis

Short-chain acyl-CoA dehydrogenase (ACAD) deficiency is an extremely rare inherited mitochondrial disorder of fat metabolism. This belongs to a group of diseases known as fatty acid oxidation disorders. Screening programmes have provided evidence that all the fatty acid oxidation disorders combined are among the most common inborn errors of metabolism. Mitochondrial beta oxidation of fatty acids is an essential energy producing pathway. It is a particularly important pathway during prolonged periods of starvation and during periods of reduced caloric intake due to gastrointestinal illness or increased energy expenditure during febrile illness. The most common presentation is an acute episode of life threatening coma and hypoglycemia induced by a period of fasting due to defective hepatic ketogenesis. Here, the case of a 4 month old female patient who had seizures since the third day of her birth and persistent hypoglycemia is described. She was born to parents of second degree consanguinity after 10years of infertility treatment. There was history of delayed cry after birth. Metabolic screening for TSH, galactosemia, 17-OHP, G6PD, cystic fibrosis, biotinidase were normal. Tandem mass spectrometric (TMS) screening for blood amino acids, organic acids, fatty acids showed elevated butyryl carnitine (C4) as 3.40 μmol/L (normal<2.00 μmol/L), hexanoyl carnitine (C6) as 0.92 μmol/L (normal<0.72 μmol/L), C4/C3 as 2.93 μmol/L (normal<1.18 μmol/L). The child was started immediately on carnitor syrup (carnitine) 1/2ml twice daily. Limitation of fasting stress and dietary fat was advised. Baby responded well by gaining weight and seizures were controlled. Until now, less than 25 patients have been reported worldwide. The limited number of patients diagnosed until now is due to the rarity of the disorder resulting in under diagnosis.

Exacerbation of vulnerability of short-chain acyl-CoA dehydrogenase deficient (SCADD) fibroblasts to oxidative stress in vitro by risk factors mimicking an infectious crisis in patients and response to rescue with normothermia and glucose

Exacerbation of vulnerability of short-chain acyl-CoA dehydrogenase deficient (SCADD) fibroblasts to oxidative stress in vitro by risk factors mimicking an infectious crisis in patients and response to rescue with normothermia and glucose

Vulnerability to oxidative stress may contribute to pathophysiology of short-chain acyl-CoA dehydrogenase deficient (SCADD) patients and is independent of the genotypic severity

Vulnerability to oxidative stress may contribute to pathophysiology of short-chain acyl-CoA dehydrogenase deficient (SCADD) patients and is independent of the genotypic severity

Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC‐1α by PRMT1 and SIRT1

Cardiomyopathies occur by mechanisms that involve inherited and acquired metabolic disorders. Both folate and vitamin B12 deficiencies are associated with left ventricular dysfunction, but mechanisms that underlie these associations are not known. However, folate and vitamin B12 are methyl donors needed for the synthesis of S-adenosylmethionine, the substrate required for the activation by methylation of regulators of energy metabolism. We investigated the consequences of a diet lacking methyl donors in the myocardium of weaning rats from dams subjected to deficiency during gestation and lactation. Positron emission tomography (PET), microscope and metabolic examinations evidenced a myocardium hypertrophy, with cardiomyocyte enlargement, disturbed mitochondrial alignment, lipid droplets, decreased respiratory activity of complexes I and II and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio. The increased concentrations of triglycerides and acylcarnitines were consistent with a deficit in fatty acid oxidation. These changes were explained by imbalanced acetylation/methylation of PGC-1α, through decreased expression of SIRT1 and PRMT1 and decreased S-adenosylmethionine:S-adenosylhomocysteine ratio, and by decreased expression of PPARα and ERRα. The main changes of the myocardium proteomic study were observed for proteins regulated by PGC-1α, PPARs and ERRα. These proteins, namely trifunctional enzyme subunit α-complex, short chain acylCoA dehydrogenase, acylCoA thioesterase 2, fatty acid binding protein-3, NADH dehydrogenase (ubiquinone) flavoprotein 2, NADH dehydrogenase (ubiquinone) 1α-subunit 10 and Hspd1 protein, are involved in fatty acid oxidation and mitochondrial respiration. In conclusion, the methyl donor deficiency produces detrimental effects on fatty acid oxidation and energy metabolism of myocardium through imbalanced methylation/acetylation of PGC-1α and decreased expression of PPARα and ERRα. These data are of pathogenetic relevance to perinatal cardiomyopathies.

Reversible High Affinity Inhibition of Phosphofructokinase-1 by Acyl-CoA: A MECHANISM INTEGRATING GLYCOLYTIC FLUX WITH LIPID METABOLISM

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki∼1 μM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.

Vulnerability to Oxidative Stress In Vitro in Pathophysiology of Mitochondrial Short-Chain Acyl-CoA Dehydrogenase Deficiency: Response to Antioxidants

ObjectiveTo elucidate the pathophysiology of SCAD deficient patients who have a unique neurological phenotype, among fatty acid oxidation disorders, with early developmental delay, CNS malformations, intractable seizures, myopathy and clinical signs suggesting oxidative stress.MethodsWe studied skin fibroblast cultures from patients homozygous for ACADS common variant c.625G>A (n = 10), compound heterozygous for c.625G>A/c.319C>T (n = 3) or homozygous for pathogenic c.319C>T (n = 2) and c.1138C>T (n = 2) mutations compared to fibroblasts from patients with carnitine palmitoyltransferase 2 (CPT2) (n = 5), mitochondrial trifunctional protein (MTP)/long-chain L-3-hydroxyacyl-CoA dehydrogenase (LCHAD) (n = 7), and medium-chain acyl-CoA dehydrogenase (MCAD) deficiencies (n = 4) and normal controls (n = 9). All were exposed to 50 µM menadione at 37°C. Additonal conditions included exposure to 39°C and/or hypoglycemia. Time to 100% cell death was confirmed with trypan blue dye exclusion. Experiments were repeated with antioxidants (Vitamins C and E or N-acetylcysteine), Bezafibrate or glucose and temperature rescue.ResultsThe most significant risk factor for vulnerability to menadione-induced oxidative stress was the presence of a FAO defect. SCADD fibroblasts were the most vulnerable compared to other FAO disorders and controls, and were similarly affected, independent of genotype. Cell death was exacerbated by hyperthermia and/or hypoglycemia. Hyperthermia was a more significant independent risk factor than hypoglycemia. Rescue significantly prolonged survival. Incubation with antioxidants and Bezafibrate significantly increased viability of SCADD fibroblasts.InterpretationVulnerability to oxidative stress likely contributes to neurotoxicity of SCADD regardless of ACADS genotype and is significantly exacerbated by hyperthermia. We recommend rigorous temperature control in SCADD patients during acute illness. Antioxidants and Bezafibrate may also prove instrumental in their management.

Multi‐factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions

Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi-modular polyketide synthases utilizing short-chain acyl-CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl-CoA's. As a result, metabolic engineering of these pathways is necessary for high-level production of heterologous polyketides. In this study, we over-express three different pathways for provision of the two substrates (propionyl-CoA and (2S)-methylmalonyl-CoA) utilized for the biosynthesis of 6-deoxyerythronolide B (6-dEB; the macrolactone precursor of erythromycin): (1) a propionate → propionyl-CoA → (2S)-methylmalonyl-CoA pathway, (2) a methylmalonate → methylmalonyl-CoA → propionyl-CoA pathway, and (3) a succinate → succinyl-CoA → (2R)-methylmalonyl-CoA → (2S)-methylmalonyl-CoA → propionyl-CoA pathway. The current study revealed that propionate is a necessary component for greater than 5 mg L(-1) titers. Deletion of the propionyl-CoA:succinate CoA transferase (ygfH) or over-expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L(-1), while the combination of the two improved titer to over 130 mg L(-1). The addition of exogenous methylmalonate could also improve titer to over 100 mg L(-1). Expression of a Streptomyces coelicolor A3(2) methylmalonyl-CoA epimerase, in conjunction with over-expression of Escherichia coli's native methylmalonyl-CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6-dEB core.

High Prevalence of Short-Chain Acyl-CoA Dehydrogenase Deficiency in the Netherlands, but No Association with Epilepsy of Unknown Origin in Childhood

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of metabolism, most frequently associated with developmental delay and/or epilepsy. Most SCADD patients carry common SCAD-encoding gene ( ACADS) variants or these variants in combination with a rare ACADS mutation, in the Netherlands predominantly the c.1058C>T. Epilepsy in childhood often remains unexplained and patients with epilepsy related to SCADD may remain undiagnosed because studies for SCADD are often not performed. To test this hypothesis and to further estimate the extent of the Dutch SCADD population, we performed a study on blood spot samples in 131 paediatric patients with epilepsy and 909 anonymous newborns and investigated the presence of the 2 common ACADS variants and the rare c.1058C>T mutation. Overall, the 2 common ACADS variants and the rare c.1058C>T mutation were detected in either homozygous or compound heterozygous forms in 9.2% of the epilepsy and 7.5% of the reference group. A birth prevalence of SCADD with a mutation/variant genotype in the Netherlands as high as >1:1,000 was calculated. This is in contrast with the low number of patients diagnosed clinically and supports the hypothesis that SCADD is clinically irrelevant. Furthermore our study does not support an association between SCADD and epilepsy.

Multiple sources of metabolic disturbance inETHE1‐related ethylmalonic encephalopathy

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

Evidence in Colombia of 625G&gt;A polymorphism in the short chain acyl-CoA dehydrogenase gene, a variation which could cause glutaric aciduria in our populations

Introduction: Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid b-oxidation. The SCAD gene is located on chromosome 12q22 and is approximately 13 kb long with 10 exons and 1236 nucleotides of coding sequence. Hereditary SCAD deficiency has been reported and only a few cases of this disorder have been described. Objective: The present study was conducted to determine the possible presence of the 625G>A variation in the short-chain acyl-CoA dehydrogenase gene in Caldas (Colombia), given that variations 625G>A and 511C>T are present in 14% of some studied populations; thereby sometimes causing its deficiency. Methods: This is a descriptive study; blood samples from three-hundred adult volunteers were tested for 625G>A polymorphism, analysing the polymerase chain reaction amplified cDNA, using a single-stranded conformation polymorphism assay. The results were confirmed by direct bidirectional cycle sequencing using DNA from the positive persons. Results: The polymorphism was identified and confirmed in four healthy persons. Conclusion: This is evidence of the presence of 625G>A polymorphism in the short-chain acyl-CoA dehydrogenase gene in Colombia, meaning that some people in our populations can be at risk of suffering SCAD deficiency and its main complication: the ethylmalonic aciduria.

Brown adipose tissue function in short-chain acyl-CoA dehydrogenase deficient mice

Brown adipose tissue is a highly specialized organ that uses mitochondrial fatty acid oxidation to fuel non-shivering thermogenesis. In mice, mutations in the acyl-CoA dehydrogenase family of fatty acid oxidation genes are associated with sensitivity to cold. Brown adipose tissue function has not previously been characterized in these knockout strains. Short-chain acyl-CoA dehydrogenase (SCAD) deficient mice were found to have increased brown adipose tissue mass as well as modest cardiac hypertrophy. Uncoupling protein-1 was reduced by 70% in brown adipose tissue and this was not due to a change in mitochondrial number, nor was it due to decreased signal transduction through protein kinase A which is known to be a major regulator of uncoupling protein-1 expression. PKA activity and in vitro lipolysis were normal in brown adipose tissue, although in white adipose tissue a modest increase in basal lipolysis was seen in SCAD−/− mice. Finally, an in vivo norepinephrine challenge of brown adipose tissue thermogenesis revealed normal heat production in SCAD−/− mice. These results suggest that reduced brown adipose tissue function is not the major factor causing cold sensitivity in acyl-CoA dehydrogenase knockout strains. We speculate that other mechanisms such as shivering capacity, cardiac function, and reduced hepatic glycogen stores are involved.

Crystal Structures and Mutational Analyses of Acyl-CoA Carboxylase β Subunit of Streptomyces coelicolor,

The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.

The Multifunctional Protein in Peroxisomal β-Oxidation: STRUCTURE AND SUBSTRATE SPECIFICITY OF THE ARABIDOPSIS THALIANA PROTEIN MFP2

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal beta-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.

Redesign of Cosubstrate Specificity and Identification of Important Residues for Substrate Binding to hChAT

In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.

Nationwide survey of extended newborn screening by tandem mass spectrometry in Taiwan

In Taiwan, during the period March 2000 to June 2009, 1,495,132 neonates were screened for phenylketonuria (PKU) and homocystinuria (HCU), and 1,321,123 neonates were screened for maple syrup urine disease (MSUD), methylmalonic academia (MMA), medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, isovaleric academia (IVA), and glutaric aciduria type 1 (GA-1) using tandem mass spectrometry (MS/MS). In a pilot study, 592,717 neonates were screened for citrullinemia, 3-methylcrotonyl-CoA carboxylase deficiency (3-MCC) and other fatty acid oxidation defects in the MS/MS newborn screening. A total of 170 newborns and four mothers were confirmed to have inborn errors of metabolism. The overall incidence was approximately 1/5,882 (1/6,219 without mothers). The most common inborn errors were defects of phenylalanine metabolism [five classic PKU, 20 mild PKU, 40 mild hyperphenylalaninemia (HPA), and 13 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency]. MSUD was the second most common amino acidopathy and, significantly, most MSUD patients (10/13) belonged to the Austronesian aboriginal tribes of southern Taiwan. The most frequently detected among organic acid disorders was 3-MCC deficiency (14 newborns and four mothers). GA-1 and MMA were the second most common organic acid disorders (13 and 13 newborns, respectively). In fatty acid disorders, five carnitine transport defect (CTD), five short-chain acyl-CoA dehydrogenase deficiency (SCAD), and two medium-chain acyl-CoA dehydrogenase (MCAD) deficiency were confirmed. This is the largest case of MS/MS newborn screening in an East-Asian population to date. We hereby report the incidences and outcomes of metabolic inborn error diseases found in our nationwide MS/MS newborn screening program.

Comparative studies of skeletal muscle proteome and transcriptome profilings between pig breeds

Two genetically different pig breeds, the Korean native pig (KNP) and the Western meat-producing Landrace, show breed-specific traits in stress responsiveness (stress hormone levels), growth performance (live weight), and meat quality (intramuscular fat content). We analyzed expression levels within the proteome and transcriptome of the longissimus muscles of both breeds using two-dimensional electrophoresis (2-DE) and microarray analysis. We constructed a porcine proteome database focused mainly on mitochondrial proteins. In total, 101 proteins were identified, of which approximately 60% were metabolic enzymes and mitochondrial proteins. We screened several proteins and genes related to stress and metabolism in skeletal muscles using comparative analysis. In particular, three stress-related genes (heat shock protein beta-1, stress-70 protein, and heat shock 70 kDa protein) were more highly expressed in the Landrace than in the KNP breed. Six metabolism-related genes (peroxisome proliferative activated receptor alpha, short-chain acyl-CoA dehydrogenase, succinate dehydrogenase, NADH-ubiquinone oxidoreductase, glycerol-3-phosphate dehydrogenase, and sterol regulatory element binding protein-1c), all of which are involved in energy and lipid metabolism, were more highly expressed at the protein or mRNA level in the KNP breed. These data may reflect the breed dependence of traits such as stress responsiveness, growth performance, and meat quality.

Screening and sequence analysis of the hemolysin gene of Fusobacterium necrophorum

Fusobacterium necrophorum is the main pathogen that causes numerous necrobacilloses. Hemolysin is one of the major virulence factors involved in fusobacterial infections. In order to investigate the genetic basis of hemolytic activity and the regulation mechanism of the hemolysin expression, a genomic library was constructed from F. necrophorum DNA by ligating DNA fragments generated by partial HindIII digestion with pUC18 vector. The screening of the genomic library with polymerase chain reaction, DNA sequencing and sequence assembly led to a 7.45 kb sequence which includes the putative hly gene and upstream sequence. Clustered putative genes encoding short chain acyl-CoA dehydrogenase (Scad) and electron transfer flavoprotein (Etf) α and β subunits locate upstream of hly. A 535 bp non-coding sequence, possibly with some cis-regulatory elements involved in the regulation of the hemolysin expression in F. necrophorum, locates between etf-β and hly. The nucleotide sequence of the hly gene indicates it encodes hemolysin. It is the first characterized hemolysin coding gene in F. necrophorum.

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the beta-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programmed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.

Newborn screening for disorders of fatty‐acid oxidation: experience and recommendations from an expert meeting

Experience with new-born screening (NBS) for disorders of fatty-acid oxidation (FAOD) is now becoming available from an increasing number of programs worldwide. The spectrum of FAOD differs widely between ethnic groups. Incidence calculations from reports from Australia, Germany, and the USA of a total of 5,256,999 newborns give a combined incidence of all FAOD of approximately 1:9,300. However, it appears to be much lower in Asians. Consequently, a significant prevalence and evidence for a clear benefit of NBS is proven for medium-chain acyl-CoA dehydrogenase deficiency (MCAD) only in countries with a high percentage of Caucasians, with very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD) and long-chain 3-hydroxy acyl-CoA dehydrogenase deficiency (LCHAD) being additional candidates. The long-term benefit for many disorders has still to be evaluated and will require international collaboration, especially for the rarest disorders. Short-chain acyl-CoA dehydrogenase deficiency (SCAD) [as well as Systemic carnitine transporter deficiency (CTD) and dienoyl-CoA reductase deficiency (DE-RED)] are conditions of uncertain clinical significance, but most FAOD have a spectrum of clinical presentations (healthy-death). Confirmatory diagnostic procedures should be agreed upon to ensure international comparability of results and evidence-based modifications. The case of short-chain acyl-CoA dehydrogenase deficiency (SCAD) deficiency shows that even inclusion of conditions without a clearly known natural course may prove useful with respect to gain of knowledge and consecutive exclusion of a biochemical abnormality without clinical significance, although this line of argument implies the existence of structured follow-up programs and bears ethical controversies. As a final conclusion, the accumulated evidence suggests all FAOD should to be included into tandem mass spectrometry (MS/MS)-based NBS programs provided sufficient laboratory performance is guaranteed.

Expression and Purification of Sunflower Cotyledon Acetoacetyl CoA Thiolase Mutants

Sunflower (Helianthus annuus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of Thiolase II from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and this construct was transformed into BL‐21 E. coli that were induced for production of the Thiolase II with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble Thiolase II was purified to apparent homogeneity using Ni‐NTA agarose affinity chromatography as indicated by a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.