Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.     

Identification of a common mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common recessively inherited metabolic diseases in man. We have studied fibroblast cultures obtained from three patients with MCAD deficiency by sequencing the entire coding region of MCAD mRNA. A single A to G nucleotide replacement which resulted in lysine329-to-glutamic acid329 substitution of the MCAD protein was identified in all cultures. Furthermore, this point mutation was present in 91% (31 of 34) of mutant MCAD alleles, indicating that the majority of cases with MCAD deficiency are caused by this type of mutation.     

Prevalence of carriers of the most common medium-chain acyl-CoA dehydrogenase (MCAD) deficiency mutation (G985A) in The Netherlands

The G985A mutation represents about 90% of all medium-chain acyl-CoA dehydrogenase (MCAD) allele mutations that cause the clinical symptoms of MCAD deficiency. The prevalence of carriers varies between different European populations, with high frequencies in the northwestern part of Europe. To determine the prevalence of MCAD carriers with the G985A mutation in The Netherlands, we collected 6195 Guthrie cards of newborns. Mutation detection was performed with the polymerase chain reaction (PCR), in which a NcoI restriction site was created in the presence of a G985A mutation in the PCR product, followed by NcoI digestion, and gel electrophoresis. We detected a G985A carrier frequency of 1 in 59 (95% CI 1/50–1/73) in The Netherlands. The total prevalence of carriers was estimated to be 1 in 55 (95% CI 1/46– 1/68), based on a relative G985A frequency of 94% in The Netherlands. Received: 18 December 1995 / Revised: 14 February 1996     

The most common mutation causing medium-chain acyl-CoA dehydrogenase deficiency is strongly associated with a particular haplotype in the region of the gene

Summary RFLP haplotypes in the region containing the medium-chain acyl-CoA dehydrogenase (MCAD) gene on chromosome 1 have been determined in patients with MCAD deficiency. The RFLPs were detected after digestion of patient DNA with the enzymes BanII, PstI and TaqI and with an MCAD cDNA-clone as a probe. Of 32 disease-causing alleles studied, 31 possesed the previously publised AG point-mutation at position 985 of the cDNA. This mutation has been shown to result in inactivity of the MCAD enzyme. In at least 30 of the 31 alleles carrying this G985 mutation a specific RFLP haplotype was present. In contrast, the same haplotype was present in only 23% of normal alleles (P3.4×10^-18). These findings are consistent with the existence of a pronounced founder effect, possibly combined with biological and/or sampling selection.     

A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the β-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine²⁸ and glutamate⁸⁶, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.     

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated cDNA, containing the entire coding region, was placed between the SV40 early promotor and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species.     

Immunoreactive enzyme protein in medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of mitochondrial fatty acid oxidation. To determine if immunoreactive enzyme protein is present in patients with MCAD deficiency, we studied cultured skin fibroblasts from patients with the 985 point mutation, present in about 85% of cases, and cell lines from patients in which the point mutation is not present or only involves one allele. Immunoblotting studies, using a polyclonal antibody to the purified protein, showed an absence of immunoreactive protein in mitochondrial fractions prepared from fibroblasts from MCAD-deficient patients. To determine whether MCAD protein accumulated in the cytosol because of impaired transport into the mitochondria, we immunoprecipitated MCAD protein from the fibroblast homogenate. MCAD protein was detected in the immunoprecipitates from controls, but not in those from the MCAD-deficient patients. These results suggest that either the MCAD protein is not synthesised or, if produced, it is rapidly degraded.     

Molecular survey of a prevalent mutation, 985A-to-G transition, and identification of five infrequent mutations in the medium-chain Acyl-CoA dehydrogenase (MCAD) gene in 55 patients with MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty-acid oxidation that is characterized by fasting intolerance and recurrent episodes of hypoglycemic coma which can be fatal. Its incidence is one of the highest among genetic metabolic disorders. Using a modified PCR and NcoI digestion method, we have surveyed 46 additional, unrelated MCAD-deficient patients for a prevalent mutation, an 985A-to-G transition (985A----G), that we previously identified in nine MCAD-deficient patients. Among the total of 55 studied, 44 were homozygous and 10 were heterozygous for the 985G allele, whereas one did not carry this mutant allele, indicating that the prevalence of the 985G allele is 89.1%. Furthermore, we identified five other types of mutation: one each in three of the compound heterozygotes and two in the single non-985G patient. An RFLP study of 12 985G-homozygotes showed that all 24 alleles fell into a single haplotype. A questionnaire regarding the ethnic and national origin of their patients was sent to all referring investigators. All 41 patients for whom this information was provided were Caucasians. Of 29 patients whose country of origin was specified, 19 and five were from the British Isles and Germany, respectively. These data suggest that 985A----G may have occurred in a single person in an ancient Germanic tribe.     

Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs.

The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta-thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+-thalassemia in some populations.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

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 β-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 programed 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.     

Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase with 3-thiaoctanoyl-CoA

The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.     

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.     

Binding of the human "electron transferring flavoprotein" (ETF) to the medium chain acyl-CoA dehydrogenase (MCAD) involves an arginine and histidine residue

The interaction between the "electron transferring flavoprotein" (ETF) and medium chain acyl-CoA dehydrogenase (MCAD) enables successful flavin to flavin electron transfer, crucial for the beta-oxidation of fatty acids. The exact biochemical determinants for ETF binding to MCAD are unknown. Here we show that binding of human ETF, to MCAD, was inhibited by 2,3-butanedione and diethylpyrocarbonate (DEPC) and reversed by incubation with free arginine and hydroxylamine respectively. Spectral analyses of native ETF vs modified ETF suggested that flavin binding was not affected and that the loss of ETF activity with MCAD involved modification of one ETF arginine residue and one ETF histidine residue respectively. MCAD and octanoyl-CoA protected ETF against inactivation by both 2,3-butanedione and DEPC indicating that the arginine and histidine residues are present in or around the MCAD binding site. Comparison of exposed arginine and histidine residues among different ETF species, however, indicates that arginine residues are highly conserved but that histidine residues are not. These results lead us to conclude that this single arginine residue is essential for the binding of ETF to MCAD, but that the single histidine residue, although involved, is not.     

Evidence that the ancestral haplotype in Australian hemochromatosis patients may be associated with a common mutation in the gene.

Hemochromatosis (HC) is a common inherited disorder of iron metabolism for which neither the gene nor biochemical defect have yet been identified. The aim of this study was to look for clinical evidence that the predominant ancestral haplotype in Australian patients is associated with a common mutation in the gene. We compared indices of iron metabolism and storage in three groups of HC patients categorized according to the presence of the ancestral haplotype (i.e., patients with two copies, one copy, and no copies of the ancestral haplotype). We also examined iron indices in two groups of HC heterozygotes (those with the ancestral haplotype and those without) and in age-matched controls. These analyses indicate that (i) HC patients with two copies of the ancestral haplotype show significantly more severe expression of the disorder than those with one copy or those without, (ii) HC heterozygotes have partial clinical expression, which may be influenced by the presence of the ancestral haplotype in females but not in males, and (iii) the high population frequency of the HC gene may be the result of the selective advantage conferred by protecting heterozygotes against iron deficiency.     

Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed no activity either with or without chaperonin, but in this case a strong MCAD protein band was seen in the western blots throughout. The proteins were also purified, and the enzyme function and thermostability investigated. The K364R protein showed only moderate kinetic impairment, whereas the R256T protein was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though clinically asymptomatic thus far, both mutations have a severe impact on the biochemical phenotype of the protein. K364R, like several previously described MCAD mutant proteins, appears to be defective in folding. R256T, by contrast, is a well-folded protein that is nevertheless devoid of catalytic activity. How the mutations specifically affect the catalytic activity and the folding is further discussed.