Summary
About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.
Similar content being viewed by others
References
Baumbach LL, Chamberlain JS, Ward PA, Farwell NJ, Caskey CT (1989) Molecular and clinical correlations of deletions leading to Duchenne and Becker muscular dystrophies. Neurology 39:465–474
Beggs AH, Koenig M, Boyce FM, Kunkel LM (1990) Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 86:45–48
Bulman DE, Gangopadhyay SB, Bebchuck KG, Worton RG, Ray PN (1991) Point mutation in the human dystrophin gene: identification through Western blot analysis. Genomics 10:457–460
Chamberlain JS, Caskey CT (1990) Duchenne muscular dystrophy. In: Appel SA (eds) Current neurology, vol. 10. Year Book Medical Publishers Chicago, pp 65–103
Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 16:11141–11156
Chamberlain JS, Gibbs RA, Ranier JE, Caskey CT (1990) Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In: Innis MA, Gelfand DH, Sninski JJ, White TJ (eds) PCR protocols. Academic Press, San Diego, pp 272–281
Cotton RGH, Rodrigues NR, Campbell RD (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397–4401
Den Dunnen JT, Grootscholten PM, Bakker E, Blonden LAJ, Ginjaar HB, Wapenaar MC, Paassen HMB van, Broeckhoven C van, Pearson PL, Ommen GJB van (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 45:835–847
Grompe M, Muzny DM, Caskey CT (1989) Scanning detection of mutations in human ornithine transcarbamoylase by chemical mismatch cleavage. Proc Natl Acad Sci USA 86:5888–5892
Heilig R, Lemaire C, Mandel J-L (1987) A 230kb cosmid walk in the Duchenne muscular dystrophy gene: detection of a conserved sequence and of a possible deletion prone region. Nucleic Acids Res 15:9129–9142
Koenig M, Kunkel LM (1990) Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. J Biol Chem 265:4560–4566
Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509–517
Koenig M, Monaco AP, Kunkel LM (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell 53:219–228
Marchuk D, Drumm M, Saulino A, Collins AR (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res 19:1154
Matsuo M, Masumura T, Nakajima T, Kitoh Y, Takumi T, Nishio H, Koga J, Nakamura H (1990) A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene. Biochem Biophys Res Commun 170:963–967
Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2:90–95
Roberts RG, Bentley DR, Barby TFM, Manners E, Bobrow M (1990) Direct diagnosis of carriers of Duchenne and Becker muscular dystrophy by amplification of lymphocyte RNA. Lancet 336:1523–1526
Roberts RG, Barby TFM, Manners E, Bobrow M, Bentley DR (1991) Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Hum Genet 49:298–310
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5468
Sicinski P, Geng Y, Ryder-Cock AS, Barnard EA, Darlison MG, Barnard PJ (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244:1578–1580
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kilimann, M.W., Pizzuti, A., Grompe, M. et al. Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR. Hum Genet 89, 253–258 (1992). https://doi.org/10.1007/BF00220535
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00220535