PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcinvestThe Journal of Clinical Investigation
 
J Clin Invest. 1994 April; 93(4): 1812–1819.
PMCID: PMC294249

Cloning and expression of mutations demonstrating intragenic complementation in mut0 methylmalonic aciduria.

Abstract

The mut0 mutation resulting in methylmalonyl CoA mutase (MCM) apoenzyme deficiency and methylmalonic aciduria is characterized by undetectable enzyme activity in cell extracts and low incorporation of propionate into cultured cells which is not stimulated by hydroxycobalamin. A mut0 fibroblast cell line (WG1681) from an African-American male infant complemented another mut0 cell line (WG 1130). Cloning and sequencing of cDNA from WG 1681 demonstrated compound heterozygosity for two novel changes at highly conserved sites: G623R and G703R. In addition, two previously described homozygous polymorphisms, H532R and V671I, were found. Hybridization of allele-specific oligonucleotides to PCR amplified MCM exons from the proband and family members identified a clinically normal mother, half-sister, and half-brother as carriers of the G703R change in cis with both polymorphisms. Transfection of each change into a mut0 cell line with very low MCM mRNA (GM1673) demonstrated a lack of stimulation of propionate uptake in the absence and presence of hydroxycobalamin. Cotransfection of each mutation with the previously identified R93H mutation of WG 1130 stimulated propionate uptake, indicating that G623R and G703R are independently capable of complementing the R93H mutation.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Rosenblatt DS, Cooper BA. Inherited disorders of vitamin B12 metabolism. Blood Rev. 1987 Sep;1(3):177–182. [PubMed]
  • Morrow G, 3rd, Barness LA, Cardinale GJ, Abeles RH, Flaks JG. Congenital methylmalonic acidemia: enzymatic evidence for two forms of the disease. Proc Natl Acad Sci U S A. 1969 May;63(1):191–197. [PubMed]
  • Willard HF, Rosenberg LE. Inherited deficiencies of human methylmalonyl CaA mutase activity: reduced affinity of mutant apoenzyme for adenosylcobalamin. Biochem Biophys Res Commun. 1977 Oct 10;78(3):927–934. [PubMed]
  • Kolhouse JF, Utley C, Fenton WA, Rosenberg LE. Immunochemical studies on cultured fibroblasts from patients with inherited methylmalonic acidemia. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7737–7741. [PubMed]
  • Ledley FD, Lumetta MR, Zoghbi HY, VanTuinen P, Ledbetter SA, Ledbetter DH. Mapping of human methylmalonyl CoA mutase (MUT) locus on chromosome 6. Am J Hum Genet. 1988 Jun;42(6):839–846. [PubMed]
  • Ledley FD, Lumetta M, Nguyen PN, Kolhouse JF, Allen RH. Molecular cloning of L-methylmalonyl-CoA mutase: gene transfer and analysis of mut cell lines. Proc Natl Acad Sci U S A. 1988 May;85(10):3518–3521. [PubMed]
  • Jansen R, Kalousek F, Fenton WA, Rosenberg LE, Ledley FD. Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. Genomics. 1989 Feb;4(2):198–205. [PubMed]
  • Nham SU, Wilkemeyer MF, Ledley FD. Structure of the human methylmalonyl-CoA mutase (MUT) locus. Genomics. 1990 Dec;8(4):710–716. [PubMed]
  • Wilkemeyer MF, Crane AM, Ledley FD. Primary structure and activity of mouse methylmalonyl-CoA mutase. Biochem J. 1990 Oct 15;271(2):449–455. [PubMed]
  • Raff ML, Crane AM, Jansen R, Ledley FD, Rosenblatt DS. Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation. J Clin Invest. 1991 Jan;87(1):203–207. [PMC free article] [PubMed]
  • Shevell MI, Matiaszuk N, Ledley FD, Rosenblatt DS. Varying neurological phenotypes among muto and mut- patients with methylmalonylCoA mutase deficiency. Am J Med Genet. 1993 Mar 1;45(5):619–624. [PubMed]
  • 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. [PubMed]
  • Wang RF, Cao WW, Johnson MG. A simplified, single tube, single buffer system for RNA-PCR. Biotechniques. 1992 May;12(5):702–704. [PubMed]
  • Jansen R, Ledley FD. Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning. Am J Hum Genet. 1990 Nov;47(5):808–814. [PubMed]
  • Yap EP, McGee JO. Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Res. 1991 Apr 11;19(7):1713–1713. [PMC free article] [PubMed]
  • Andrews E, Jansen R, Crane AM, Cholin S, McDonnell D, Ledley FD. Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae. Biochem Med Metab Biol. 1993 Oct;50(2):135–144. [PubMed]
  • MacGregor GR, Caskey CT. Construction of plasmids that express E. coli beta-galactosidase in mammalian cells. Nucleic Acids Res. 1989 Mar 25;17(6):2365–2365. [PMC free article] [PubMed]
  • Crane AM, Jansen R, Andrews ER, Ledley FD. Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria. J Clin Invest. 1992 Feb;89(2):385–391. [PMC free article] [PubMed]
  • Wilkemeyer MF, Crane AM, Ledley FD. Differential diagnosis of mut and cbl methylmalonic aciduria by DNA-mediated gene transfer in primary fibroblasts. J Clin Invest. 1991 Mar;87(3):915–918. [PMC free article] [PubMed]
  • Ledley FD, Crane AM, Lumetta M. Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. Am J Hum Genet. 1990 Mar;46(3):539–547. [PubMed]

Articles from The Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation