Multiplex PCR for Identifying DMD Gene Deletions

Johan T. den Dunnen1, Alan H. Beggs2

1 Leiden University Medical Center, Leiden, The Netherlands, 2 Children's Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 9.3
DOI:  10.1002/0471142905.hg0903s49
Online Posting Date:  May, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The identification of dystrophin as the defective protein in patients with Duchenne and Becker muscular dystrophies (DMD and BMD) has allowed the development of sensitive and specific tests to establish a diagnosis and to aid in genetic counseling and prenatal diagnosis. The Basic Protocol describes three complementary multiplex PCR assays that detect 26 dystrophin gene exons. The multiplex nature of these assays allows the detection of up to ten different exons in a single reaction. At least one of these exons is missing in >95% of deletions. The Support Protocol describes preparation and storage of stock PCR reaction mixes with primers for each of the three diagnostic assays. The Alternate Protocol is a modification of the Basic Protocol for radioactive detection of duplications in males and deletions in carrier females.

Keywords: Duchenne muscular dystrophy; Becker muscular dystrophy; DMD gene; mutations; multiplex PCR

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Diagnostic Multiplex PCR to Detect DMD Gene Deletions
  • Alternate Protocol 1: Low‐Cycle‐Number Multiplex PCR with Radioactive Label to Detect Dosage Differences
  • Support Protocol 1: Preparation and Storage of Stock Diagnostic PCR Mixes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Diagnostic Multiplex PCR to Detect DMD Gene Deletions

  • Reaction mixes A, B, and C (see protocol 3)
  • 5 U/µl Taq DNA polymerase (e.g., Perkin‐Elmer Cetus AmpliTaq or Native Taq)
  • 50 ng/µl template DNA, prepared from cell lines, whole blood, or preserved tissues ( appendix 3B), or from chorionic villus biopsy (unit 8.3), amniotic fluid (unit 8.4), or other clinical specimens, in TE buffer, pH 8.0 ( appendix 2D)
  • Mineral oil
  • 1.5% (w/v) agarose minigel containing 0.5 µg/ml ethidium bromide (unit 2.7)
  • Electrophoresis buffer (TAE or TBE) containing 0.5 µg/ml ethidium bromide ( appendix 2D)
  • 10× gel loading buffer ( appendix 2D)
  • Dedicated micropipets for setting up reactions
  • Two thermal cyclers, preheated to 94°C
  • General‐use micropipets for analyzing reaction products
  • Additional reagents and equipment for electrophoresis using agarose minigels and gel photography (unit 2.7)
CAUTION: Ethidium bromide and human clinical specimens are hazardous; see appendix 2D for guidelines on handling, storage, and disposal.

Alternate Protocol 1: Low‐Cycle‐Number Multiplex PCR with Radioactive Label to Detect Dosage Differences

  • 10 mCi/ml [α‐32P]dCTP (3000 mCi/mmol)
  • Centricon 100 concentrator (Amicon)
  • Centrifuge with fixed‐angle (23° to 45°) rotor: e.g., Beckman JA‐18.1
  • Whatman 3MM filter paper
  • Gel dryer, preheated
  • Densitometer
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
CAUTION:32P is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Support Protocol 1: Preparation and Storage of Stock Diagnostic PCR Mixes

  • Oligonucleotide PCR primers (Tables 9.3.1, 9.3.2, and 9.3.3)
  • Double‐distilled, sterile H 2O
  • 10× PCR amplification buffer ( appendix 2D) containing 15 mM MgCl 2
  • 2.5 mM 4dNTP mix ( appendix 2D)
PDF or HTML at Wiley Online Library



Literature Cited

   Abbs, S., Yau, S.C., Clark, S., Mathew, C.G., and Bobrow, M. 1991. A convenient multiplex PCR system for the detection of dystrophin gene deletions: A comparative analysis with cDNA hybridization shows mistypings by both methods. J. Med. Genet. 28:304‐311.
   Arahata, K., Hoffman, E.P., Kunkel, L.M., Ishiura, S., Tsukahara, T., Ishihara, T., Sunohara, N., Nonaka, I., Ozawa, E., and Sugita, H. 1989. Dystrophin diagnosis: Comparison of dystrophin abnormalities by immunofluorescence and immunoblot analyses. Proc. Natl. Acad. Sci. U.S.A. 86:7154‐7158.
   Armour, J.A., Sismani, C., Patsalis, P.C., Cross, G. 2000. Measurement of locus copy number by hybridisation with amplifiable probes. Nucl. Acids Res. 28:605‐609.
   Beggs, A.H., Hoffman, E.P., Snyder, J.R., Arahata, K., Specht, L., Shapiro, F., Angelini, C., Sugita, H., and Kunkel, L.M. 1991. Exploring the molecular basis for variability among patients with Becker muscular dystrophy: Dystrophin gene and protein studies. Am. J. Hum. Genet. 49:54‐67.
   Beggs, A.H., Koenig, M., Boyce, F.M., and Kunkel, L.M. 1990. Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet. 86:45‐48.
   Bennett, R.R., den Dunnen, J.T., O'Brien, K.F., Darras, B.T., and Kunkel, L.M. 2001. Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing. BMC Genet. 2:17.
   Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., and Caskey, C.T. 1990. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. In PCR Protocols: A Guide to Methods and Applications (M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White, eds.) pp. 272‐281. Academic Press, San Diego, Calif.
   Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., Nguyen, P.N., and Caskey, C.T. 1988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucl. Acids Res. 16:11141‐11156.
   Chelly, J., Gilgenkrantz, H., Lambert, M., Hamard, G., Chafey, P., Recan, D., Katz, P., de la Chapelle, A., Koenig, M., and Ginjaar, I.B. 1990. Effect of dystrophin gene deletions on mRNA levels and processing in Duchenne and Becker muscular dystrophies. Cell 63:1239‐1248.
   Darras, B.T., Blattner, P., Harper, J.F., Spiro, A.J., Alter, S., and Francke, U. 1988. Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: Location of breakpoints on HindIII and BglII exon‐containing fragment maps, meiotic and mitotic origin of the mutations. Am. J. Hum. Genet. 43:620‐629.
   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., and van Ommen, G.J. 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.
   Flanigan, K.M., von Niederhausern, A., Dunn, D.M., Alder, J., Mendell, J.R., and Weiss, R.B. 2003. Rapid direct sequence analysis of the dystrophin gene. Am. J. Hum. Genet. 72:931‐939.
   Hoffman, E.P., Fischbeck, K.H., Brown, R.H., Johnson, M., Medori, R., Loike, J.D., Harris, J.B., Waterston, R., Brooke, M., and Specht, L. 1988. Characterization of dystrophin in muscle‐biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. New Engl. J. Med. 318:1363‐1368.
   Hoffman, E.P., Kunkel, L.M., Angelini, C., Clarke, A., Johnson, M., and Harris, J.B. 1989. Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology 39:1011‐1017.
   Hofstra, R.M., Mulder, I.M., Vossen, R., de Koning‐Gans, P.A., Kraak, M., Ginjaar, H.B., van der Hout, A.H., Bakker, E., Buys, C.H., van Ommen, G.J.B., van Essen, A.J., and den Dunnen, J.T. 2004. DGGE‐based whole‐gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Hum. Mutat. 23:57‐66.
   Hu, X.Y., Ray, P.N., Murphy, E.G., Thompson, M.W., and Worton, R.G. 1990. Duplicational mutation at the Duchenne muscular dystrophy locus: Its frequency, distribution, origin, and phenotype‐genotype correlation. Am. J. Hum. Genet. 46:682‐695.
   Ioannou, P., Christopoulos, G., Panayides, K., Kleanthous, M., and Middleton, L. 1992. Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis. Neurology 42:1783‐1790.
   Koenig, M., Beggs, A.H., Moyer, M., Scherpf, S., Heindrich, K., Bettecken, T., Meng, G., Muller, C.R., Lindlof, M., Kaariainen, H., de la Chapelle, A., Kiuru, A., Savontaus, M.‐L., Gilgenkrantz, H., Recan, D., Chelly, J., Kaplan, J.‐C., Covone, A.E., Archidiacono, N., Romeo, G., Liechti‐Gallati, S., Schneider, V., Braga, S., Moser, H., Darras, B.T., Murphy, P., Francke, U., Chen, J.D., Morgan, G., Denton, M., Greenberg, C.R., van Ommen, G.J.B., and Kunkel, L.M. 1989. The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion. Am. J. Hum. Genet. 45:498‐506.
   Koenig, M., Hoffman, E.P., Bertelson, C.J., Monaco, A.P., Feener, C., and Kunkel, L.M. 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.
   Kunkel, L.M., Snyder, J.R., Beggs, A.H., Boyce, F.M., and Feener, C.A. 1991. Searching for dystrophin gene deletions in patients with atypical presentations. In Etiology of Human Diseases at the DNA Level (J. Lindsten and U. Petterson, eds.) pp. 51‐60. Raven Press, New York.
   Lalic, T., Vossen, R.H.A.M., Coffa, J., Schouten, J.P., Guc‐Scekic, M., Radivojevic, D., Djurisic, M., Breuning, M.H., White, S.J., and den Dunnen, J.T. 2005. Deletion and duplication screening in the DMD gene using MLPA. Eur. J. Hum. Genet. 13:1231‐1234.
   Mendell, J.R., Buzin, C.H., Feng, J., Yan, J., Serrano, C., Sangani, D.S., Wall, C., Prior, T.W., and Sommer, S.S. 2001. Diagnosis of Duchenne dystrophy by enhanced detection of small mutations. Neurology 57:645‐650.
   Monaco, A.P., Bertelson, C.J., Liechti Gallati, S., Moser, H., and Kunkel, L.M. 1988. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2:90‐95.
   Multicenter Study Group. 1992. Diagnosis of Duchenne and Becker muscular dystrophies by polymerase chain reaction.  J. Amer. Med. Assoc. 267:2609‐2615.
   Neilan, B.A., Leigh, D.A., and McDonald, B.L. 1993. The rapid analysis of dystrophin gene deletions shows variable electrophoretic mobility. J. Med. Genet. 30:60‐61.
   Roberts, R.G., Coffey, A.J., Bobrow, M., and Bentley, D.R. 1992. Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR. Genomics 13:942‐950.
   Roest, P.A.M., Bakker, E., Fallaux, F.J., Verellen‐Dumoulin, C., Murry, C.E., and den Dunnen, J.T. 1999. New possibilities for prenatal diagnosis of muscular dystrophies: Forced myogenesis with an adenoviral MyoD‐vector. Lancet 353:727‐728.
   Rosenberg, C., Navajas, L., Vagenas, D.F., Bakker, E., Vainzof, M., Passos‐Bueno, M.R., Takata, R.I., Van Ommen, G.J.B., Zatz, M., and Den Dunne, J.T. 1998. Clinical diagnosis of heterozygous dystrophin gene deletions by fluorescence in situ hybridization. Neuromuscul. Disord. 8:447‐452.
   Schouten, J.P., McElgunn, C.J., Waaijer, R., Zwijnenburg, D., Diepvens, F., Pals, G. 2002. Relative quantification of 40 nucleic acid sequences by multiplex ligation‐dependent probe amplification. Nucl. Acids Res. 30:e57.
   Tuffery‐Giraud, S., Saquet, C., Chambert, S., Echenne, B., Cuisset, M.J., Rivier, F., Cossee, M., Philippe, C., Monnier, N., Bieth, E., Recan, D., Voelckel, A.M., Perelman, S., Lambert, J.C., Malcolm, S., and Claustres, M. 2004. The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: Experience of a national referral centre. Neuromuscul. Disord. 14:650‐658.
   Voskova‐Goldman, A., Peier, A., Caskey, C.T., Richards, C.S., and Shaffer, L.G. 1997. DMD‐specific FISH probes are diagnostically useful in the detection of female carriers of DMD gene deletions. Neurology 48:1633‐1638.
   White, S., Kalf, M., Liu, Q., Villerius, M., Engelsma, D., Kriek, M., Vollebregt, E., Bakker, B., van Ommen, G.J.B., Breuning, M.H., den Dunnen, J.T. 2002. Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am. J. Hum. Genet. 71:365‐374.
   Winnard, A.V., Mendell, J.R., Prior, T.W., Florence, J., and Burghes, A.H. 1995. Frameshift deletions of exons 3‐7 and revertant fibers in Duchenne muscular dystrophy: Mechanisms of dystrophin production. Am. J. Hum. Genet. 56:158‐166.
   Yau, S.C., Bobrow, M., Mathew, C.G., and Abbs, S.J. 1996. Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J. Med. Genet. 33:550‐558.
Key References
   Beggs et al., 1990. See above.
  The above references describe the origin and development of the multiplex assays for deletion detection.
   Chamberlain et al., 1988.
  These references validate the PCR methodology and discuss potential artifacts and pitfalls.
   Chamberlain et al., 1990.
   Abbs et al., 1991. See above.
   Multicenter Study Group, 1992. See above.
Internet Resources
  The Leiden Muscular Dystrophy pages. This Web site contains information on all aspects of Duchenne and Becker muscular dystrophy, the DMD gene and methods to perform DMD/BMD diagnosis.
PDF or HTML at Wiley Online Library