Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA)

Abstract

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.

Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center

“…6,8,40 Although differences in ethnicity should be taken into account, MLPA remains the first line in mutation detection in dystrophinopathy. [6][7][8][9] A total of four deep intron mutations were identified at least 285 bp from the exon/intron boundary; this was possible only because we used detection of novel dystrophin mRNA (Figure 4). This analysis provided 1% of detected mutations, and facilitated our perfect mutation detection rate.…”

“…[6][7][8][9] However, the identification of small mutations in the dystrophin gene remains challenging, because of the large number of exons and the huge size of the dystrophin gene. By combining quantitative PCR and direct sequencing technologies, the mutation detection rate has risen to 98% of DMD cases (106/108 total cases).…”

Detection of exonic copy-number changes using a highly efficient oligonucleotide-based comparative genomic hybridization-array method

“…Compared with existing methods, such as QF-PCR assays (quantitative multiplex-PCR of short fluorescent fragments, quantitative fluorescent multiplex-PCR, and semiquantitative fluorescent-PCR, developed to screen for deletions and duplications of all exons of DMD or CFTR gene) [White et al, 2002;Gatta et al, 2005;Lalic et al, 2005;Lai et al, 2006], the CGH array assay described in this study appears to be very promising to address diagnostic and research issues. First, as shown here, the CGH array enables detection of exonic deletions and duplications while providing the possibility to confine intronic breakpoints involved in the rearrangements.…”

Duchenne Muscular Dystrophy Diagnostics: It Only Gets Better but Some of the Same Challenges Remain

“…Furthermore, the quantitative multiplex PCR allowed for the determination of gene dosage and the accurate identification of DMD deletion carriers (6 ). Another major breakthrough was the development of multiplex ligationdependent probe amplification (MLPA) for the determination of gene dosage (7 ). MLPA is based on an initial probe pair ligation to the DMD exon, followed by a multiplex amplification that produces products of different lengths.…”