Skip to main content
PMC home page
American Journal of Human Genetics logoLink to American Journal of Human Genetics
. 1996 Jan;58(1):97–106.

Mutation analysis of very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency: identification and characterization of mutant VLCAD cDNAs from four patients.

M Souri 1, T Aoyama 1, K Orii 1, S Yamaguchi 1, T Hashimoto 1
PMCID: PMC1914938  PMID: 8554073

Abstract

Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is a newly identified disease. A 105-bp deletion in the VLCAD cDNA in two patients has been reported, and detailed molecular characterization of this disease has remained to be done. We report here five mutations identified in four patients: a 135-bp deletion encompassing bases 343-477, a C-1837-to-T transition (R613W), 3-bp deletions at the nucleotide positions 388-390 (E130del) and 895-897 (K299del), and an A-1144-to-C transversion (K382Q). Sequencing of genomic DNA amplified by PCR revealed a 135-bp deletion caused by exon skipping due to a 1-bp deletion in a 3' splice site of an intron. In cDNA expression experiments using Chinese hamster ovary (CHO) cells, we found that each of the mRNAs derived from E130del and K299del clones were unstable and that translation products from R613W, E130del, K299del, and K382Q clones were labile. Each of R613W, E130del, K299del, and K382Q proteins expressed in CHO cells appeared abnormal in dimer assembly, as shown in gel-filtration analysis. VLCAD activity was not detected in mutants' transfectants. Thus, we verified that all five mutations identified in these four patients were disease-causing alterations.

Full text

PDF
97

Images in this article

100Figure 1
on p.100
101Figure 3
on p.101
102Figure 4
on p.102
102Figure 5
on p.102
103Figure 6
on p.103
103Figure 7
on p.103

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akli S., Chelly J., Lacorte J. M., Poenaru L., Kahn A. Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments. Genomics. 1991 Sep;11(1):124–134. doi: 10.1016/0888-7543(91)90109-r. [DOI] [PubMed] [Google Scholar]
  2. Aoyama T., Souri M., Ueno I., Kamijo T., Yamaguchi S., Rhead W. J., Tanaka K., Hashimoto T. Cloning of human very-long-chain acyl-coenzyme A dehydrogenase and molecular characterization of its deficiency in two patients. Am J Hum Genet. 1995 Aug;57(2):273–283. [PMC free article] [PubMed] [Google Scholar]
  3. Aoyama T., Souri M., Ushikubo S., Kamijo T., Yamaguchi S., Kelley R. I., Rhead W. J., Uetake K., Tanaka K., Hashimoto T. Purification of human very-long-chain acyl-coenzyme A dehydrogenase and characterization of its deficiency in seven patients. J Clin Invest. 1995 Jun;95(6):2465–2473. doi: 10.1172/JCI117947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aoyama T., Uchida Y., Kelley R. I., Marble M., Hofman K., Tonsgard J. H., Rhead W. J., Hashimoto T. A novel disease with deficiency of mitochondrial very-long-chain acyl-CoA dehydrogenase. Biochem Biophys Res Commun. 1993 Mar 31;191(3):1369–1372. doi: 10.1006/bbrc.1993.1368. [DOI] [PubMed] [Google Scholar]
  5. Aoyama T., Ueno I., Kamijo T., Hashimoto T. Rat very-long-chain acyl-CoA dehydrogenase, a novel mitochondrial acyl-CoA dehydrogenase gene product, is a rate-limiting enzyme in long-chain fatty acid beta-oxidation system. cDNA and deduced amino acid sequence and distinct specificities of the cDNA-expressed protein. J Biol Chem. 1994 Jul 22;269(29):19088–19094. [PubMed] [Google Scholar]
  6. Bertrand C., Largillière C., Zabot M. T., Mathieu M., Vianey-Saban C. Very long chain acyl-CoA dehydrogenase deficiency: identification of a new inborn error of mitochondrial fatty acid oxidation in fibroblasts. Biochim Biophys Acta. 1993 Jan 22;1180(3):327–329. doi: 10.1016/0925-4439(93)90058-9. [DOI] [PubMed] [Google Scholar]
  7. Chelly J., Gilgenkrantz H., Lambert M., Hamard G., Chafey P., Récan D., Katz P., de la Chapelle A., Koenig M., Ginjaar I. B. Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker muscular dystrophies. Cell. 1990 Dec 21;63(6):1239–1248. doi: 10.1016/0092-8674(90)90419-f. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Coates P. M., Indo Y., Young D., Hale D. E., Tanaka K. Immunochemical characterization of variant medium-chain acyl-CoA dehydrogenase in fibroblasts from patients with medium-chain acyl-CoA dehydrogenase deficiency. Pediatr Res. 1992 Jan;31(1):34–38. doi: 10.1203/00006450-199201000-00006. [DOI] [PubMed] [Google Scholar]
  10. Daar I. O., Maquat L. E. Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol Cell Biol. 1988 Feb;8(2):802–813. doi: 10.1128/mcb.8.2.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibbs R. A., Nguyen P. N., McBride L. J., Koepf S. M., Caskey C. T. Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1919–1923. doi: 10.1073/pnas.86.6.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  13. Hale D. E., Stanley C. A., Coates P. M. The long-chain acyl-CoA dehydrogenase deficiency. Prog Clin Biol Res. 1990;321:303–311. [PubMed] [Google Scholar]
  14. Indo Y., Coates P. M., Hale D. E., Tanaka K. Immunochemical characterization of variant long-chain acyl-CoA dehydrogenase in cultured fibroblasts from nine patients with long-chain acyl-CoA dehydrogenase deficiency. Pediatr Res. 1991 Sep;30(3):211–215. doi: 10.1203/00006450-199109000-00001. [DOI] [PubMed] [Google Scholar]
  15. Inglehearn C. F., Bashir R., Lester D. H., Jay M., Bird A. C., Bhattacharya S. S. A 3-bp deletion in the rhodopsin gene in a family with autosomal dominant retinitis pigmentosa. Am J Hum Genet. 1991 Jan;48(1):26–30. [PMC free article] [PubMed] [Google Scholar]
  16. Izai K., Uchida Y., Orii T., Yamamoto S., Hashimoto T. Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. I. Purification and properties of very-long-chain acyl-coenzyme A dehydrogenase. J Biol Chem. 1992 Jan 15;267(2):1027–1033. [PubMed] [Google Scholar]
  17. Kamijo T., Wanders R. J., Saudubray J. M., Aoyama T., Komiyama A., Hashimoto T. Mitochondrial trifunctional protein deficiency. Catalytic heterogeneity of the mutant enzyme in two patients. J Clin Invest. 1994 Apr;93(4):1740–1747. doi: 10.1172/JCI117158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lim S. K., Sigmund C. D., Gross K. W., Maquat L. E. Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products. Mol Cell Biol. 1992 Mar;12(3):1149–1161. doi: 10.1128/mcb.12.3.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Matsubara Y., Indo Y., Naito E., Ozasa H., Glassberg R., Vockley J., Ikeda Y., Kraus J., Tanaka K. Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family. J Biol Chem. 1989 Sep 25;264(27):16321–16331. [PubMed] [Google Scholar]
  21. Niwa H., Yamamura K., Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991 Dec 15;108(2):193–199. doi: 10.1016/0378-1119(91)90434-d. [DOI] [PubMed] [Google Scholar]
  22. Ogilvie I., Pourfarzam M., Jackson S., Stockdale C., Bartlett K., Turnbull D. M. Very long-chain acyl coenzyme A dehydrogenase deficiency presenting with exercise-induced myoglobinuria. Neurology. 1994 Mar;44(3 Pt 1):467–473. doi: 10.1212/wnl.44.3_part_1.467. [DOI] [PubMed] [Google Scholar]
  23. Saijo T., Welch W. J., Tanaka K. Intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD). Demonstration of impaired transfer of K304E-variant MCAD from its complex with hsp60 to the native tetramer. J Biol Chem. 1994 Feb 11;269(6):4401–4408. [PubMed] [Google Scholar]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Shapiro M. B., Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987 Sep 11;15(17):7155–7174. doi: 10.1093/nar/15.17.7155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tanaka K., Yokota I., Coates P. M., Strauss A. W., Kelly D. P., Zhang Z., Gregersen N., Andresen B. S., Matsubara Y., Curtis D. Mutations in the medium chain acyl-CoA dehydrogenase (MCAD) gene. Hum Mutat. 1992;1(4):271–279. doi: 10.1002/humu.1380010402. [DOI] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Uchida Y., Izai K., Orii T., Hashimoto T. Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. J Biol Chem. 1992 Jan 15;267(2):1034–1041. [PubMed] [Google Scholar]
  29. Verity M. A., Turnbull D. M. Assay of acyl-CoA dehydrogenase activity in frozen muscle biopsies: application to medium-chain acyl-CoA dehydrogenase deficiency. Biochem Med Metab Biol. 1993 Jun;49(3):351–362. doi: 10.1006/bmmb.1993.1036. [DOI] [PubMed] [Google Scholar]
  30. Yamaguchi S., Indo Y., Coates P. M., Hashimoto T., Tanaka K. Identification of very-long-chain acyl-CoA dehydrogenase deficiency in three patients previously diagnosed with long-chain acyl-CoA dehydrogenase deficiency. Pediatr Res. 1993 Jul;34(1):111–113. doi: 10.1203/00006450-199307000-00025. [DOI] [PubMed] [Google Scholar]
  31. Yokota I., Coates P. M., Hale D. E., Rinaldo P., Tanaka K. 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. Am J Hum Genet. 1991 Dec;49(6):1280–1291. [PMC free article] [PubMed] [Google Scholar]
  32. Yokota I., Saijo T., Vockley J., Tanaka K. Impaired tetramer assembly of variant medium-chain acyl-coenzyme A dehydrogenase with a glutamate or aspartate substitution for lysine 304 causing instability of the protein. J Biol Chem. 1992 Dec 25;267(36):26004–26010. [PubMed] [Google Scholar]
  33. Ziegler A., Hagmann J., Kiefer B., Nagamine Y. Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism. J Biol Chem. 1990 Dec 5;265(34):21194–21201. [PubMed] [Google Scholar]

Articles from American Journal of Human Genetics are provided here courtesy of American Society of Human Genetics

RESOURCES