Abstract
We have designed a multiplex ligation-dependent probe amplification (MLPA) assay to simultaneously screen all 79 DMD gene exons for deletions and duplications in Duchenne and Becker muscular dystrophy (DMD/BMD) patients. We validated the assay by screening 123 unrelated patients from Serbia and Montenegro already screened using multiplex PCR. MLPA screening confirmed the presence of all previously detected deletions. In addition, we detected seven new deletions, nine duplications, one point mutation, and we were able to precisely determine the extent of all rearrangements. To facilitate MLPA-based screening in laboratories lacking specific equipment, we designed the assay such that it can also be performed using agarose gel analysis and ethidium bromide staining. The MLPA assay as described provides a simple and cheap method for deletion and duplication screening in DMD/BMD patients. The assay outperforms the Beggs and Chamberlain multiplex-PCR test, and should be considered as the method of choice for an initial DNA analysis of DMD/BMD patients.
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References
Den Dunnen JT, Grootscholten PM, Dauwerse JD et al: Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. Hum Mol Genet 1992; 1: 19–28.
Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener CA, Kunkel LM : Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987; 50: 509–517.
Den Dunnen JT, Grootscholten PM, Bakker E et al: Topography of the DMD gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989; 45: 835–847.
Oudet C, Hanauer A, Clemens P, Caskey CT, Mandel JL : Two hot spots of recombination in the DMD-gene correlate with the deletion prone regions. Hum Mol Genet 1992; 1: 599–603.
Forrest SM, Cross GS, Speer A, Gardner-Medwin D, Burn J, Davies KE : Preferential deletion of exons in Duchenne and Becker muscular dystrophies. Nature 1987; 329: 638–640.
Chamberlain JS, Gibbs RA, Ranier JE, Nga Nguyen PN, Caskey CT : Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res 1988; 23: 11141–11156.
Beggs AH, Koenig M, Boyce FM, Kunkel LM : Detection of 98-percent DMD/BMD gene deletions by polymerase chain reaction. Hum Genet 1990; 86: 45–48.
Hu X, Burghes AHM, Ray PN, Thompson MW, Murphy EG, Worton RG : Partial gene duplications in Duchenne and Becker muscular dystrophies. J Med Genet 1988; 25: 369–376.
Yau SC, Bobrow M, Mathew CG, Abbs SJ : Accurate diagnosis of carriers of deletions and duplications in Duchenne/Becker muscular dystrophy by fluorescent dosage analysis. J Med Genet 1996; 33: 550–558.
Ioannou P, Christopoulos G, Panayides K, Kleanthous M, Middleton L : Detection of Duchenne and Becker muscular dystrophy carriers by quantitative multiplex polymerase chain reaction analysis. Neurol 1992; 42: 1783–1790.
White S, Kalf M, Liu Q et al: Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am J Hum Genet 2002; 71: 365–374.
Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G : Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30: e57.
Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J : MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics 2005; 6: 29–35.
Schwartz M, Duno M : Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method. Genet Test 2004; 8: 361–367.
Miller SA, Dykes DD, PoleskY HF : A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215.
Abbs SJ, Yau SC, Mathew CG, 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; 28: 304–311.
Kunkel LM, Snyder JR, Beggs AH, Boyce FM, Feener CA : Searching for dystrophin gene deletions in patients with atypical presentations; in Lindsten J, Petterson U (eds): Etiology of Human Diseases at the DNA Level. New York: Raven Press, 1991, pp 51–60.
Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM : An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988; 2: 90–95.
Lu QL, Mann CJ, Lou F et al: Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nat Med 2003; 9: 1009–1014.
Aartsma-Rus A, Janson AA, Kaman WE et al: Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74: 83–92.
Tuffery-Giraud S, Saquet C, Chambert S et al: The role of muscle biopsy in analysis of the dystrophin gene in Duchenne muscular dystrophy: experience of a national referral centre. Neuromuscul Disord 2004; 14: 650–658.
Roest PAM, Roberts RG, Van Der Tuijn AC, Heikoop JC, Van Ommen GJB, Den Dunnen JT : Protein truncation test (PTT) to rapidly screen the DMD-gene for translation-terminating mutations. Neuromusc Disord 1993; 3: 391–394.
Mendell JR, Buzin CH, Feng J et al: Diagnosis of Duchenne dystrophy by enhanced detection of small mutations. Neurol 2001; 57: 645–650.
Flanigan KM, von Niederhausern A, Dunn DM, Alder J, Mendell JR, Weiss RB : Rapid direct sequence analysis of the dystrophin gene. Am J Hum Genet 2003; 72: 931–939.
Hofstra RM, Mulder IM, Vossen R et al: DGGE-based whole-gene mutation scanning of the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Hum Mutat 2004; 23: 57–66.
Acknowledgements
We thank the patients and their families for their cooperation and Dave van Heusden for expert technical assistance. Jan Schouten is director and shareholder of MRC-Holland, the producer of the MLPA kits described in this article.
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Lalic, T., Vossen, R., Coffa, J. et al. Deletion and duplication screening in the DMD gene using MLPA. Eur J Hum Genet 13, 1231–1234 (2005). https://doi.org/10.1038/sj.ejhg.5201465
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DOI: https://doi.org/10.1038/sj.ejhg.5201465
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