Duchenne Muscular Dystrophy: Therapeutic Approaches to Restore Dystrophin

Abstract

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder. The first symptoms involve the lower limbs and appear between the third and fifth year. Due to weakness of the knee and hip extensors, patients rise from a sitting position using the Gower's maneuver. Muscle weakness progresses to the shoulder girdle-, upper arm and trunk-muscles and patients loose ambulation before the age of 12 (Emery, 1993). Histological changes involve variation in fiber size with atrophic and hypertrophic fibers, degeneration and regeneration of the muscle fibers, infiltration of inflammatory cells and fibrosis. The fiber necrosis results in leakage of the enzyme creatine kinase (CK), resulting in very high serum CK levels in DMD patients (20,000 to 50,000 U/L compared to 80 to 250 U/L in unaffected individuals). These levels decline as patients get older and the overall muscle mass decreases progressively. The pathology is caused by mutations in the DMD gene, which was known to be on the X chromosome long before the responsible gene was cloned due to an X-linked recessive inheritance pattern. The protein product of the gene is a 427 KDa protein called dystrophin. In the early 80s several groups were collaborating on the regional cloning of the gene responsible for DMD (Burghes et al., 1987; Monaco et al., 1985) which happened to co-localize with the locus for Becker muscular dystrophy (BMD) (Kingston et al., 1984). This is a milder disease, where patients are diagnosed in adolescence or adulthood, remain ambulant longer and survival is generally only slightly decreased (Emery, 2002). After a couple of years Monaco and colleagues confirmed that deletions in the identified locus caused DMD (Monaco et al., 1985) and BMD (Hoffman et al., 1988). In the following years, the coding sequence of the gene was identified, which turned out to occupy a huge genomic region. The complete cDNA and protein product of the DMD gene were published in 1987 (Hoffman et al., 1987; Koenig et al., 1987). The cloning of the genomic and coding sequence of the DMD gene allowed the development of tools for the molecular diagnosis of DMD. Deletions of one or more exons were found to be most common (65% of patients) and mainly localized in two hotspot regions in the gene (exons 220 and 45-53). This led Chamberlain and colleagues to develop a multiplex PCR able to detect the most frequent mutations (Chamberlain et al., 1988). This technique has been used for years, but recently multiple ligation-dependent probe amplification (MLPA) has been developed that allows an exact characterization of exons involved in deletions and duplications (Janssen et al., 2005; Schwartz & Duno, 2004). For small mutations a more