Characterization of deletion breakpoints in patients with dystrophinopathy carrying a deletion of exons 45–55 of the Duchenne muscular dystrophy (DMD) gene

Abstract

Deletion of exons 45-55 (del45-55) in the Duchenne muscular dystrophy gene (DMD) has gained particular interest in the field of molecular therapy, because it causes a milder phenotype than DMD, and therefore, may represent a good candidate for the goal of a multiple exon-skipping strategy. We have precisely characterized deletion breakpoints in three patients with del45-55 in DMD. Two of them were young adult males of the X-linked dilated cardiomyopathy phenotype, and the third patient revealed the mild Becker muscular dystrophy phenotype of late onset. The deletion breakpoints differed among patients. The deletion started at nt 226 604, 231 518, 117 284 in intron 44, and ended at nt 64 994, 59 314, 71 806 in intron 55, respectively. Deletion junctions showed no significant homology between the sequences adjacent to the distal and proximal end joints in these patients. Deletion breakpoints were not primarily associated with any particular sequence element, or with a matrix attachment region. However, there were several palindromic sequences and short tandem repeats at or near the breakpoints. These sequences, with a marked propensity to form secondary DNA structure intermediates, may predispose local DNA to breakage and intragenic recombination in these patients. Keywords: breakpoint; deletion; Duchenne muscular dystrophy (DMD); dystrophin; genomic sequence; X-linked dilated cardiomyopathy (XLDCM) INTRODUCTIONThe Duchenne muscular dystrophy gene (DMD) is the largest one so far identified, spanning more than 2.5 Mb and occupying roughly 0.1% of the human genome. 1 Mutations in DMD cause a devastating muscular dystrophy named Duchenne/Becker muscular dystrophy (DMD/BMD). The cardio-specific phenotype of dystrophinopathy is also known as X-linked dilated cardiomyopathy (XLDCM). 1 Intragenic deletions and duplications together account for over two-thirds of the mutations found in DMD/BMD patients. [2][3][4][5][6][7] Despite heterogeneity in both deletion size and location, two deletion 'hotspots' have been identified in DMD: a minor 'hotspot' including exons 2-19, and the major one involving exons 40-50 or 45-55. 2,4,5,7 Among deletions of variable sizes and locations found in DMD/ BMD patients, deletion of exons 45-55 (del45-55) in DMD has gained particular attention because an artificial dystrophin with del45-55 created by a multiple exon-skipping strategy could transform the DMD phenotype into the asymptomatic or milder BMD phenotype. 8 We reported three patients with del45-55 who presented with the phenotypes far from DMD. 9 Two of them were young adult males of the XLDCM phenotype. 9,10 The third patient revealed the mild BMD phenotype of late onset. 9,11 Our patients support the hypothesis that del45-55 could be a good candidate for the goal of a multiple exonskipping strategy for DMD.To elucidate the molecular basis of del45-55 in more detail, we have characterized the breakpoints of del45-55 in DMD in these three patients. To our knowledge, only two breakpoints have been precisely described in intron 44,...

Fast detection of deletion breakpoints using quantitative PCR

“…Deletions in exons 44-55 of the DMD gene have received special attention because of the mild phenotype associated with them. As also observed in other studies (Miyazaki et al , 2009), we found no repetitive sequences or significant homology between the sequences adjacent to the deletion breakpoints. However, we found two identical 15-bp fragments located 1,121 bp 5' of the deletion in intron 44 and 428 bp 3' of the deletion in intron 50.…”

“…The majority of DMD mutations involve a hemizygous deletion or duplication of one or more exons, while about a third of the mutations are formed de novo (Santos et al , 2014). The deletion of exons 45-55 of the DMD gene has received special attention because it is associated with a milder phenotype of the disorder (Miyazaki et al , 2009). MLPA designed to detect medium size deletions or duplications is generally used for mutation testing in DMD , but only provides information about deleted exons, with the exact location of the deletion breakpoints in large introns remaining undefined.…”

Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

“…Importantly, cardiomyopathy is the second leading cause of death in DMD patients, accounting for 10–40% of deaths in DMD populations [71–73]. While some BMD patients with an exon 45–55 deletion show X-linked dilated cardiomyopathy [56, 58, 64, 74], their prognoses are quite favorable and only a few of them show mild heart failure symptom [62]. These imply that cardiac dystrophin correction due to exons 45–55 skipping would effectively rescue cardiac symptoms.…”

DNA repair systems

“…The sites of rearrangements in the dystrophin gene o en contain short regions of homology with other sites on the X chromosome that may mediate intra-chromosomal pairing during meiosis -specifically, male meiosis [1551,1552]. In 2009, a recombina on-related mechanism was proposed to explain the apparent genomic instability in the two major muta on hotspots in the dystrophin gene -the regions between exons 8 and 13 and exons 45-52 [1553,1554]. In another, more recent study, comprised of DMD/BMD cases as well as pa ents with other X-linked disorders, it was 5 3 found that regions of microhomology (2-10 bp) were observed at breakpoint junc ons in about 60% of the study cases.…”