Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

Comparative Genomics of X-linked Muscular Dystrophies: The Golden Retriever Model

Duchenne muscular dystrophy (DMD) is a devastating disease that dramatically decreases the lifespan and abilities of affected young people. The primary molecular cause of the disease is the absence of functional dystrophin protein, which is critical to proper muscle function. Those with DMD vary in disease presentation and dystrophin mutation; the same causal mutation may be associated with drastically different levels of disease severity. Also contributing to this variation are the influences of additional modifying genes and/or changes in functional elements governing such modifiers. This genetic heterogeneity complicates the efficacy of treatment methods and to date medical interventions are limited to treating symptoms. Animal models of DMD have been instrumental in teasing out the intricacies of DMD disease and hold great promise for advancing knowledge of its variable presentation and treatment. This review addresses the utility of comparative genomics in elucidating the complex background behind phenotypic variation in a canine model of DMD, Golden Retriever muscular dystrophy (GRMD). This knowledge can be exploited in the development of improved, more personalized treatments for DMD patients, such as therapies that can be tailor-matched to the disease course and genomic background of individual patients.     

The role of free radicals in the pathophysiology of muscular dystrophy.

Null mutation of any one of several members of the dystrophin protein complex can cause progressive, and possibly fatal, muscle wasting. Although these muscular dystrophies arise from mutation of a single gene that is expressed primarily in muscle, the resulting pathology is complex and multisystemic, which shows a broader disruption of homeostasis than would be predicted by deletion of a single-gene product. Before the identification of the deficient proteins that underlie muscular dystrophies, such as Duchenne muscular dystrophy (DMD), oxidative stress was proposed as a major cause of the disease. Now, current knowledge supports the likelihood that interactions between the primary genetic defect and disruptions in the normal production of free radicals contribute to the pathophysiology of muscular dystrophies. In this review, we focus on the pathophysiology that results from dystrophin deficiency in humans with DMD and the mdx mouse model of DMD. Current evidence indicates three general routes through which free radical production can be disrupted in dystrophin deficiency to contribute to the ensuing pathology. First, constitutive differences in free radical production can disrupt signaling processes in muscle and other tissues and thereby exacerbate pathology. Second, tissue responses to the presence of pathology can cause a shift in free radical production that can promote cellular injury and dysfunction. Finally, behavioral differences in the affected individual can cause further changes in the production and stoichiometry of free radicals and thereby contribute to disease. Unfortunately, the complexity of the free radical-mediated processes that are perturbed in complex pathologies such as DMD will make it difficult to develop therapeutic approaches founded on systemic administration of antioxidants. More mechanistic knowledge of the specific disruptions of free radicals that underlie major features of muscular dystrophy is needed to develop more targeted and successful therapeutic approaches.     

Skewed X inactivation in a female MZ twin results in Duchenne muscular dystrophy.

One of female MZ twins presented with muscular dystrophy. Physical examination, creatine phosphokinase levels, and muscle biopsy were consistent with Duchenne muscular dystrophy (DMD). However, because of her sex she was diagnosed as having limb-girdle muscular dystrophy. With cDNA probes to the DMD gene, a gene deletion was detected in the twins and their mother. The de novo mutation which arose in the mother was shown by novel junction fragments generated by HindIII, PstI, or TaqI when probed with cDNA8. Additional evidence of a large gene deletion was given by novel SfiI junction fragments detected by probes p20, J-Bir, and J-66 on pulsed-field gel electrophoresis (PFGE). Immunoblot analysis of muscle from the affected twin showed dystrophin of normal size but of reduced amount. Immunofluorescent visualization of dystrophin revealed foci of dystrophin-positive fibers adjacent to foci of dystrophin-negative fibers. These data indicate that the affected twin is a manifesting carrier of an abnormal DMD gene, her myopathy being a direct result of underexpression of dystrophin. Cytogenetic analysis revealed normal karyotypes, eliminating the possibility of a translocation affecting DMD gene function. Both linkage analysis and DNA fingerprint analysis revealed that each twin has two different X chromosomes, eliminating the possibility of uniparental disomy as a mechanism for DMD expression. On the basis of methylation differences of the paternal and maternal X chromosomes in these MZ twins, we propose uneven lyonization (X chromosome inactivation) as the underlying mechanism for disease expression in the affected female.     

A nonsense mutation-created intraexonic splice site is active in the lymphocytes,but not in the skeletal muscle of a DMD patient

Production of semi-functional dystrophin mRNA from the dystrophin gene encoding a premature stop codon has been shown to modify the severe phenotype of Duchenne muscular dystrophy (DMD). In this study, we report the tissue-specific production of semi-functional dystrophin mRNA via activation of a nonsense mutation-created intraexonic splice acceptor site. In a DMD patient a novel nonsense mutation was identified in exon 42. In his lymphocytes semi-functional dystrophin mRNA with a 63-nucleotide deletion in exon 42 (dys-63) was found to be produced. In vitro splicing assay using hybrid minigenes disclosed that the mutation-created intraexonic splice acceptor site was activated. In his skeletal muscle cells, however, only the authentically spliced dystrophin mRNA was found. This finding identifies the modulation of the splicing of muscle dystrophin mRNA in cases of DMD as a potential target for therapeutic strategies to generate a milder phenotype for this disease.     

Novel mutations of dystrophin gene in DMD patients detected by rapid scanning in biplex exons DHPLC analysis

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.     

中国人群中抗肌萎缩蛋白基因突变类型和分布特点及其与临床症状的相关性

假性肥大型进行性肌营养不良症(Duchenne’s muscular dystrophy,DMD)是源于横纹肌的一种X-连锁隐性致死性遗传病,由编码抗肌营养不良蛋白(dystrophin)基因突变所致。为了探讨中国人群中DMD患者的dystrophin基因突变类型和分布特点及其与临床症状的相关性,文章采用Multiplex Ligation-Dependent Probe Amplification(MLPA)方法对720例DMD患者及其母亲和20例正常成年男性进行dystrophin基因分析。结果显示,检出率为64.9%(467/720),54.3%(391/720)的患者为基因缺失;10.6%(76/720)的患者为基因重复。累及Exon45-54缺失突变型占全部缺失型患者的71.9%(281/391);重复突变型累及Exon1-40占全部重复型患者82.9%(63/76);检出的患者中,DMD型和中间型营养不良症(Intermediate muscular dystrophy,IMD)型占90.6%(423/467),Becker型营养不良症(Becker muscular dystrophy,BMD)型占9.4%(44/467)。表明假肥大型肌营养不良症以dystrophin基因缺失突变为主,突变发生在整个基因中非均匀分布,存在突变热区,在缺失和重复的位置和片段长度与肌病的临床症状严重程度之间并不存在简单的相关关系。     

Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense

Dystrophin deficiency, which leads to severe and progressive muscle degeneration in patients with Duchenne muscular dystrophy (DMD), is caused by frameshifting mutations in the dystrophin gene. A relatively new therapeutic strategy is based on antisense oligonucleotides (AONs) that induce the specific skipping of a single exon, such that the reading frame is restored. This allows the synthesis of a largely functional dystrophin, associated with a milder Becker muscular dystrophy phenotype. We have previously successfully targeted 20 different DMD exons that would, theoretically, be beneficial for >75% of all patients. To further enlarge this proportion, we here studied the feasibility of double and multiexon skipping. Using a combination of AONs, double skipping of exon 43 and 44 was induced, and dystrophin synthesis was restored in myotubes from one patient affected by a nonsense mutation in exon 43. For another patient, with an exon 46-50 deletion, the therapeutic double skipping of exon 45 and 51 was achieved. Remarkably, in control myotubes, the latter combination of AONs caused the skipping of the entire stretch of exons from 45 through 51. This in-frame multiexon skipping would be therapeutic for a series of patients carrying different DMD-causing mutations. In fact, we here demonstrate its feasibility in myotubes from a patient with an exon 48-50 deletion. The application of multiexon skipping may provide a more uniform methodology for a larger group of patients with DMD.     

Analysis of a dystrophin gene deletion by amplification of mRNA isolated from DMD myotubes cultured in vitro

The most frequent causes for the X-linked muscular dystrophy of the allelic Duchenne (DMD) or Becker (BMD) type are partial deletions of the dystrophin gene. These mutations are accompanied either by disrupted or by preserved translational reading frames in mRNAs derived from the deleted genes. As a rule, the reading frame is destroyed in the more severe DMD, whereas it is preserved in the less severe BMD (M. Koenig et al., 1989, Am. J. Hum. Genet. 45, 498-506). We have analyzed in detail a deletion that was detected in a fetus at risk of DMD. The analysis of this mutation included the delineation of the altered subregion in the dystrophin mRNA. mRNA was isolated from myotubes derived from embryonic DMD myoblasts propagated in vitro. This study was based on enzymatic amplification by the polymerase chain reaction (PCR) of dystrophin mRNA and direct sequencing of the amplified cDNA. Exons 47 to 50 were found to be missing in the mRNA. The splicing of exon 46 to exon 51 resulted in a reading frameshift, indicating that this mutation is likely to be responsible for a DMD type of dystrophy. The clinical diagnosis of DMD for a 10-year-old patient in this family was compatible with the "reading frame" assumption.     

Human dystrophin gene transfer: production and expression of a functional recombinant DNA-based gene

Summary The identification and cloning of the gene responsible for Duchenne muscular dystrophy (DMD) and characterization of the protein product of the gene, dystrophin, has led to major advances in diagnostic and genetic counselling procedures for this inherited disorder. Due to its high mutation rate, however, individuals affected by DMD will continue to arise in large proportion by de novo mutations, and the search for direct therapies remains a high priority. In this respect direct genetic correction of dystrophin deficiency via grafting of healthy myoblast stem cells or direct introduction of functional DNA into diseased muscle tissue have both been proposed as potential therapeutic approaches. We describe here, the first example of the engineering and cloning of a synthetic gene encoding recombinant human dystrophin and its stable transfer to and expression in mammalian cells. This DMD gene construction represents a primary step towards evaluating direct DNA-mediated gene transfer as a potential treatment for this debilitating disorder.     

An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is a spontaneous, X-linked, progressively fatal disease of dogs and is also a homologue of Duchenne muscular dystrophy (DMD). Two-thirds of DMD patients carry detectable deletions in their dystrophin gene. The defect underlying the remaining one-third of DMD patients is undetermined. Analysis of the canine dystrophin gene in normal and GRMD dogs has failed to demonstrate any detectable loss of exons. Here, we have demonstrated a RNA processing error in GRMD that results from a single base change in the 3' consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. This is the first example of dystrophin deficiency caused by a splice-site mutation.     

Dystrophin analysis in clonal myoblasts derived from a Duchenne muscular dystrophy carrier.

Clonal myogenic cell cultures were established from a potential heterozygote for a mutant Duchenne muscular dystrophy (DMD) gene who was also heterozygous for isozymes of the X-linked enzyme glucose-6-phosphate dehydrogenase. Previous tissue culture studies of this muscle donor demonstrated equal proliferative capacity of myoblasts that had lyonized either the paternal or maternal X-chromosome, indicating that mutation of the DMD gene does not affect growth of myoblasts. If this muscle donor were a gonadal mosaic, this conclusion would be incorrect. In the present study, only those myogenic colonies expressing the glucose-6-phosphate dehydrogenase-A isozyme were found to express dystrophin, indicating that this woman was indeed a heterozygote for DMD. By documenting dystrophin deficiency in a specific population of myogenic cells from this woman, we verify our previous conclusion regarding the normal proliferative capacity of DMD myoblasts. Somatic cell testing of dystrophin expression may offer an alternative to established genetic carrier tests for those women in whom deletions of the DMD are not detectable, whose pedigree structure does not permit linkage analysis, or in whom standard phenotypic analyses are ambiguous.     

Marginal level dystrophin expression improves clinical outcome in a strain of dystrophin/utrophin double knockout mice

Inactivation of all utrophin isoforms in dystrophin-deficient mdx mice results in a strain of utrophin knockout mdx (uko/mdx) mice. Uko/mdx mice display severe clinical symptoms and die prematurely as in Duchenne muscular dystrophy (DMD) patients. Here we tested the hypothesis that marginal level dystrophin expression may improve the clinical outcome of uko/mdx mice. It is well established that mdx3cv (3cv) mice express a near-full length dystrophin protein at ～5% of the normal level. We crossed utrophin-null mutation to the 3cv background. The resulting uko/3cv mice expressed the same level of dystrophin as 3cv mice but utrophin expression was completely eliminated. Surprisingly, uko/3cv mice showed a much milder phenotype. Compared to uko/mdx mice, uko/3cv mice had significantly higher body weight and stronger specific muscle force. Most importantly, uko/3cv outlived uko/mdx mice by several folds. Our results suggest that a threshold level dystrophin expression may provide vital clinical support in a severely affected DMD mouse model. This finding may hold clinical implications in developing novel DMD therapies.     

A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.     

Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of?MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.     

Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame.     

Gene therapy for muscle disease

The molecular mechanisms of Duchenne muscular dystrophy (DMD) have been extensively investigated since the discovery of the dystrophin gene in 1986. Nonetheless, there is currently no effective treatment for DMD. Recent reports, however, indicate that adenoassociated viral (AAV) vector-mediated transfer of a functional dystrophin cDNA into the affected muscle is a promising strategy. In addition, antisense-mediated exon skipping technology has been emerging as another promising approach to restore dystrophin expression in DMD muscle. Ongoing clinical trials show restoration of dystrophin in DMD patients without serious side effects. Here, we summarize the recent progress in gene therapy, with an emphasis on exon skipping for DMD.