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American Journal of Human Genetics logoLink to American Journal of Human Genetics
. 1995 Aug;57(2):311–320.

The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test.

R J Gardner 1, M Bobrow 1, R G Roberts 1
PMCID: PMC1801547  PMID: 7668256

Abstract

The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frame-shifting deletions in the dystrophin gene that are detectable by a multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be a mutation in the ORF. We believe that reverse-transcription--PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion.

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Selected References

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

  1. Abbs S., Yau S. C., Clark S., Mathew C. G., Bobrow M. A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods. J Med Genet. 1991 May;28(5):304–311. doi: 10.1136/jmg.28.5.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beggs A. H., Koenig M., Boyce F. M., Kunkel L. M. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum Genet. 1990 Nov;86(1):45–48. doi: 10.1007/BF00205170. [DOI] [PubMed] [Google Scholar]
  3. Boyd Y., Fraser N. J. Methylation patterns at the hypervariable X-chromosome locus DXS255 (M27 beta): correlation with X-inactivation status. Genomics. 1990 Jun;7(2):182–187. doi: 10.1016/0888-7543(90)90539-7. [DOI] [PubMed] [Google Scholar]
  4. Bulman D. E., Gangopadhyay S. B., Bebchuck K. G., Worton R. G., Ray P. N. Point mutation in the human dystrophin gene: identification through western blot analysis. Genomics. 1991 Jun;10(2):457–460. doi: 10.1016/0888-7543(91)90332-9. [DOI] [PubMed] [Google Scholar]
  5. Byers T. J., Lidov H. G., Kunkel L. M. An alternative dystrophin transcript specific to peripheral nerve. Nat Genet. 1993 May;4(1):77–81. doi: 10.1038/ng0593-77. [DOI] [PubMed] [Google Scholar]
  6. Chamberlain J. S., Gibbs R. A., Ranier J. E., Nguyen P. N., Caskey C. T. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res. 1988 Dec 9;16(23):11141–11156. doi: 10.1093/nar/16.23.11141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [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. Den Dunnen J. T., Grootscholten P. M., Bakker E., Blonden L. A., Ginjaar H. B., Wapenaar M. C., van Paassen H. M., van Broeckhoven C., Pearson P. L., van Ommen G. J. 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. 1989 Dec;45(6):835–847. [PMC free article] [PubMed] [Google Scholar]
  10. Forrest S. M., Dahl H. H., Howells D. W., Dianzani I., Cotton R. G. Mutation detection in phenylketonuria by using chemical cleavage of mismatch: importance of using probes from both normal and patient samples. Am J Hum Genet. 1991 Jul;49(1):175–183. [PMC free article] [PubMed] [Google Scholar]
  11. Green P. M., Bentley D. R., Mibashan R. S., Nilsson I. M., Giannelli F. Molecular pathology of haemophilia B. EMBO J. 1989 Apr;8(4):1067–1072. doi: 10.1002/j.1460-2075.1989.tb03474.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hodgson S., Hart K., Abbs S., Heckmatt J., Rodillo E., Bobrow M., Dubowitz V. Correlation of clinical and deletion data in Duchenne and Becker muscular dystrophy. J Med Genet. 1989 Nov;26(11):682–693. doi: 10.1136/jmg.26.11.682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu X. Y., Ray P. N., Murphy E. G., Thompson M. W., Worton R. G. Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotypegenotype correlation. Am J Hum Genet. 1990 Apr;46(4):682–695. [PMC free article] [PubMed] [Google Scholar]
  14. Kilimann M. W., Pizzuti A., Grompe M., Caskey C. T. Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR. Hum Genet. 1992 May;89(3):253–258. doi: 10.1007/BF00220535. [DOI] [PubMed] [Google Scholar]
  15. Koenig M., Hoffman E. P., Bertelson C. J., Monaco A. P., Feener C., Kunkel L. M. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987 Jul 31;50(3):509–517. doi: 10.1016/0092-8674(87)90504-6. [DOI] [PubMed] [Google Scholar]
  16. Koenig M., Monaco A. P., Kunkel L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 1988 Apr 22;53(2):219–228. doi: 10.1016/0092-8674(88)90383-2. [DOI] [PubMed] [Google Scholar]
  17. Lederfein D., Levy Z., Augier N., Mornet D., Morris G., Fuchs O., Yaffe D., Nudel U. A 71-kilodalton protein is a major product of the Duchenne muscular dystrophy gene in brain and other nonmuscle tissues. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5346–5350. doi: 10.1073/pnas.89.12.5346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lenk U., Hanke R., Thiele H., Speer A. Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in DMD patients. Hum Mol Genet. 1993 Nov;2(11):1877–1881. doi: 10.1093/hmg/2.11.1877. [DOI] [PubMed] [Google Scholar]
  19. McIntosh I., Hamosh A., Dietz H. C. Nonsense mutations and diminished mRNA levels. Nat Genet. 1993 Jul;4(3):219–219. doi: 10.1038/ng0793-219. [DOI] [PubMed] [Google Scholar]
  20. Monaco A. P., Bertelson C. J., Liechti-Gallati S., Moser H., Kunkel L. M. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics. 1988 Jan;2(1):90–95. doi: 10.1016/0888-7543(88)90113-9. [DOI] [PubMed] [Google Scholar]
  21. Powell S. M., Petersen G. M., Krush A. J., Booker S., Jen J., Giardiello F. M., Hamilton S. R., Vogelstein B., Kinzler K. W. Molecular diagnosis of familial adenomatous polyposis. N Engl J Med. 1993 Dec 30;329(27):1982–1987. doi: 10.1056/NEJM199312303292702. [DOI] [PubMed] [Google Scholar]
  22. Prior T. W., Papp A. C., Snyder P. J., Burghes A. H., Bartolo C., Sedra M. S., Western L. M., Mendell J. R. A missense mutation in the dystrophin gene in a Duchenne muscular dystrophy patient. Nat Genet. 1993 Aug;4(4):357–360. doi: 10.1038/ng0893-357. [DOI] [PubMed] [Google Scholar]
  23. Roberts R. G., Barby T. F., Manners E., Bobrow M., Bentley D. R. Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am J Hum Genet. 1991 Aug;49(2):298–310. [PMC free article] [PubMed] [Google Scholar]
  24. Roberts R. G., Bobrow M., Bentley D. R. Point mutations in the dystrophin gene. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2331–2335. doi: 10.1073/pnas.89.6.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roberts R. G., Coffey A. J., Bobrow M., Bentley D. R. Exon structure of the human dystrophin gene. Genomics. 1993 May;16(2):536–538. doi: 10.1006/geno.1993.1225. [DOI] [PubMed] [Google Scholar]
  26. Roberts R. G., Gardner R. J., Bobrow M. Searching for the 1 in 2,400,000: a review of dystrophin gene point mutations. Hum Mutat. 1994;4(1):1–11. doi: 10.1002/humu.1380040102. [DOI] [PubMed] [Google Scholar]
  27. Roest P. A., Roberts R. G., Sugino S., van Ommen G. J., den Dunnen J. T. Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum Mol Genet. 1993 Oct;2(10):1719–1721. doi: 10.1093/hmg/2.10.1719. [DOI] [PubMed] [Google Scholar]
  28. Vainzof M., Passos-Bueno M. R., Takata R. I., Pavanello R. de C., Zatz M. Intrafamilial variability in dystrophin abundance correlated with difference in the severity of the phenotype. J Neurol Sci. 1993 Oct;119(1):38–42. doi: 10.1016/0022-510x(93)90189-6. [DOI] [PubMed] [Google Scholar]

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