title: Disease-causing mutations in exon 11 of the medium-chain acyl-CoA dehydrogenase gene.
abstract: Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial beta-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.
authors.affiliation: Center for Medical Molecular Biology, Arhus University Hospital, Denmark.
authors.forename: B S
authors.initials: BS
authors.lastname: Andresen
authors.forename: T G
authors.initials: TG
authors.lastname: Jensen
authors.forename: P
authors.initials: P
authors.lastname: Bross
authors.forename: I
authors.initials: I
authors.lastname: Knudsen
authors.forename: V
authors.initials: V
authors.lastname: Winter
authors.forename: S
authors.initials: S
authors.lastname: Kølvraa
authors.forename: L
authors.initials: L
authors.lastname: Bolund
authors.forename: J H
authors.initials: JH
authors.lastname: Ding
authors.forename: Y T
authors.initials: YT
authors.lastname: Chen
authors.forename: J L
authors.initials: JL
authors.lastname: Van Hove
mesh_terms: D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000032:analysis / Q000172:deficiency* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D005091:Exons / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase