Structural organization and regulatory regions of the human medium-chain acyl-CoA dehydrogenase gene

Abstract

Medium-chain acyl-CoA dehydrogenase (MCAD) is a highly regulated mitochondrial flavo-enzyme that catalyzes the initial reaction in fatty acid beta-oxidation. Deficiency of MCAD is a common inherited defect in energy metabolism. We have previously shown that the mRNA encoding MCAD in an MCAD-deficient child is homozygous for the point mutation A985 to G [Kelly et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9236-9420]. To define the molecular basis of MCAD deficiency and as an initial step in the study of the regulation of MCAD gene expression, we determined the structure and organization of the human MCAD gene. The gene is comprised of 12 exons which span 44 kb of DNA. Comparison of the MCAD gene to MCAD mRNAs from the MCAD-deficient child revealed that missplicing was common, resulting in a variety of exon deletions and intron insertions. The MCAD gene promoter region is extremely GC-rich and lacks prototypical TATA and CAAT boxes. Several regions upstream of the promoter are homologous with mitochondrial enhancers purportedly involved in coordinate expression of nuclear genes encoding mitochondrial proteins. Transfection of chimeric plasmid constructs with 299 bp of upstream sequence into HepG2 cells revealed high-level transcriptional activity. We conclude that the precursor MCAD mRNA is misspliced to a high degree and complexity in association with the G985 mutation and the MCAD gene contains a strong promoter which shares some structural features with other "housekeeping" genes encoding mitochondrial proteins.

Medium Chain Acyl-CoA Dehydrogenase Deficiency in Pennsylvania: Neonatal Screening Shows High Incidence and Unexpected Mutation Frequencies

“…The common A985G mutation was detected by restriction fragment digestion of PCR product as described by Yokota et al (12), with the exception that the reverse primer used for the amplification was as follows (5'-CCTCCCAAGCTGCTCTCTGG-3'). Oligonucleotide primer pairs covering each exon from the exon/intron boundries were designed (13) to amplify all the MCAD exons. Primers to amplify exon 5 were as follows.…”

A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

“…Construction of MCADCAT (-361/ + 189) (with reference to the transcription start site as + 1) has been described (65). pTKCAT is a pUC-based plasmid constructed by replacing the herpes simplex virus tkl gene/ chloramphenicol acetyltransferase (CAT) gene hybrid of pUT KAT3 (45) with a Kozak consensus sequence upstream of the CAT open reading frame, followed by the simian virus 40 early region splice and polyadenylation signals.…”

“…Accordingly, MCAD is a pivotal enzyme in cellular fatty acid oxidative metabolism. This fact is underscored by the severe consequences resulting from inherited MCAD deficiency, including hypoglycemia and sudden death (15,51).As an initial step in determining the molecular mechanisms involved in transcriptional regulation of nuclear genes involved in mitochondrial metabolic pathways, we have begun to delineate cis-acting regulatory elements in the MCAD gene promoter (10,46,65). In this pursuit, we identified a retinoic acid (RA) response element (RARE) that confers retinoid-mediated activation of the MCAD gene promoter (46).…”

“…As an initial step in determining the molecular mechanisms involved in transcriptional regulation of nuclear genes involved in mitochondrial metabolic pathways, we have begun to delineate cis-acting regulatory elements in the MCAD gene promoter (10,46,65). In this pursuit, we identified a retinoic acid (RA) response element (RARE) that confers retinoid-mediated activation of the MCAD gene promoter (46).…”

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

“…54 ACADM gene consists of 12 exons that encode 421 amino acids. 55 The use of NBS for early detection of MCAD deficiency has revealed a more varied mutational and biochemical spectrum of MCAD deficiency than the studies done on clinically ascertained populations. 56 More than 100 different mutations of all types -missense, nonsense, splicing, and small insertions/deletions -are now known in the ACADM gene and are distributed to all the exons .…”