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

Abstract

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-mitochon drial 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. Con sequently, 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 sub _titution from adenine (A) to guanine (G) at position ~85 in the MCAD cDNA as the only consistent base-var iation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mu tated 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 geno mic DNA containing G 985 . The same assay consistently revealed A 985 in genomic DNA from 26 control individu als. 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 in ability to form active tetrameric MCAD. All the experi ments are consistent with the contention that the G 985 mutation, resulting in a lysine to glutamate shift at posi tion 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the Offprint requests to: N. Gregersen.

Effects of Two Mutations Detected in Medium Chain Acyl-CoA Dehydrogenase (MCAD)-deficient Patients on Folding, Oligomer Assembly, and Stability of MCAD Enzyme

“…Plasmids-The previously used plasmid pBMCK2- (8), which carries a gene encoding the mature part of human MCAD preceded by an artificial initiator methionine under control of the lac promoter, was modified in order to allow straightforward subcloning ofPCR amplified MCAD cDNA derived from patient material. For this purpose pB-MCK2-was modified in aseries of steps involving standard PCR and cloning techniques in the following way.…”

“…Besides aseries of rare point mutations, short insertions, and deletions (5-7), one prevalent point mutation (8)(9)(10)(11) is present in more than 90% ofthe alleles in patients with MCAD deficiency (5). This mutation is an A~G transition at position 985 of the cDNA sequence (G985), leading to replacement of lysine 304 in the mature protein with glutamic acid (K304E).…”

Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation

“…MCAD deficiency is an autosomal length specific enzymes which catalyze the first step in recessive genetic disease which, in the majority of cases mitochondrial l3-oxidation of straight-chain fatty acids (approx. 90% of the alleles, [4]), is due to a point mutation at position 985 of the cDNA sequence [5][6][7][8] causing a lysine to glutamic acid exchange at position correctly [9]. We have shown that, unlike the wild formation of insoluble aggregates [5,10].…”

“…90% of the alleles, [4]), is due to a point mutation at position 985 of the cDNA sequence [5][6][7][8] causing a lysine to glutamic acid exchange at position correctly [9]. We have shown that, unlike the wild formation of insoluble aggregates [5,10]. Upon overex pression in COS-7 cells, the K304E mutant has been demonstrated to be unstable and only very small amounts of tetrameric mutant MCAD could be detected [11].…”

Screening for medium-chain acyl CoA dehydrogenase deficiency: current perspectives

“…Approximately, 80% of these patients were homozygous for this mutation, and 18% were heterozygous for c.985A.G. 7,[58][59][60][61][62][63] In order to explore the founder effect hypothesis, Leal et al in 2014 combined genotyping and screening data from 43 publications reporting the frequency of c.985A.G spanning over 10 million individuals, and they found significant variation in the frequency of the mutation across regions supporting a reported founder effect. 58 The proportion Prior to NBS, the incidence of MCAD deficiency was approximately in the range 1 in 30,000 to 1 in 135,000.…”