Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked, allelic, neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscle. Duchenne muscular dystrophy is the most common X-linked recessive lethal disease, with an incidence of approximately 1 in 3,500 newborns, and approximately one third of cases are the result of new mutations.1'2 Affected children are usually wheelchair bound by the age of 12 years. As the disease progresses, contractures increasingly develop, leading to asymmetrical spinal deformities. Most patients die at about 20 years of age due to pneumonia related to chronic respiratory insufficiency. The allelic disorder BMD has a milder clinical course and slower disease progression. Becker muscular dystrophy has been estimated to occur approximately one tenth as frequently as DMD, with an incidence of about 1 in 35,000. The majority of BMD patients initially experience difficulties between 5 and 15 years of age, although an onset in the third or fourth decade or even later can occur. By definition the affected patients remain ambulatory until 16 years of age or later, thus allowing clinical distinction from patients with DMD.
The DMD gene is the largest human gene isolated, spanning more than 2000 kilobases (kb) of genomic DNA, and is composed of 79 exons that encode a 14kb transcript, which is translated into a protein named dystrophin.3,4 Dystrophin is a 427kilodalton (kDa) cytoskeletal protein consisting of 4 domains: (1) an amino terminus that associates with actin or an actin-like protein, (2) a rod domain consisting of long, flexible rows of 24 alpha helical repeats, (3) a cysteine-rich region, and (4) a unique carboxy termi-nus.5 Dystrophin has been shown to be tightly associated with a large oligomeric complex of sarcolemmal glycopro-teins through its cysteine-rich domain and carboxy termi nus, while the amino-terminal domain interacts with actin or an actin-like protein.
By immunochemistry, dystrophin localizes to the cyto-plasmic face of the muscle cell membrane and at postsy-naptic membrane specializations in neurons. Dystrophin makes up only 0.002% of total muscle protein but up to 5% of the membrane skeleton. Dystrophin is found in skeletal muscle, smooth muscle, cardiac muscle, and brain. There are slightly different forms of dystrophin messenger RNA (mRNA) in different tissues due to different transcription start sites and alternative splicing. The function of dys-trophin is not known for certain, but proposed functions for the protein include important roles for the organization and stabilization of the sarcolemma and in protecting muscle fibers from contraction-induced injury. Patients with DMD have very little or no detectable dystrophin, whereas BMD patients have an altered size and/or quantity of dystrophin.6 However, the disease etiology may be more complex than a simple loss of dystrophin, because several of the dystrophin-associated proteins that interact with dystrophin also are absent. The dystrophin-associated proteins may be directly involved with the calcium flux in the dystrophic fibers. Thus, the loss of dystrophin may be the first of many steps that ultimately lead to muscular dystrophy.
Utilizing complementary DNA (cDNA) probes derived from the 14kb mRNA and multiplex polymerase chain reaction (PCR) analysis, approximately 65% of the DMD/BMD cases are due to deletions in the dystrophin gene.78 The deletions are nonrandomly distributed and occur primarily in the center (~80%) and less frequently near the 5' end (~20%) of the gene. The 200kb region covering intron 44, exon 45, and intron 45 is the major deletion breakpoint region of the gene. The majority of the larger deletions initiate at the 5' end of the gene.
There is no apparent correlation between the size or location of the deletion and the severity and progression of the disease. One of the largest deletions (35 exons) identified is in a mild BMD patient. Furthermore, sequences deleted in DMD patients often overlap with deletions in BMD patients. However, it was proposed that if a deletion disrupts the translational reading frame of the dystrophin mRNA triplet codons, then little or no dystrophin will be synthesized, resulting in the more severe disease, DMD.9 In the milder disease, BMD, the deletion maintains the translational reading frame, and a partially functional protein is produced. The reading frame hypothesis explains the phenotypic differences observed in about 92% of the DMD/BMD cases. One major exception to the reading frame hypothesis has been the identification of BMD patients with an out-of-frame deletion of exon 3 through exon 7. An alternate splicing mechanism or new cryptic translational start site may account for the production of an altered dystrophin protein and the milder phenotype in these patients. A small number of DMD patients with in-frame deletions have also been identified. The more severe phenotype in these patients may be due to the overall effect of the deletion on the protein conformation or may be the result of mRNA instability. Phenotypic variability has even been observed in several patients who share identical gene deletions. Deletion of exon 45, the most commonly observed DMD deletion, has also been associated with the BMD pheno-type. Some genetic variability may be due to other molecules involved in destruction of damaged muscle fibers, in muscle regeneration, or in the cellular response to different hormones.
The large gene size, particularly of the introns, which average 35 kb, may account for part of the high deletion rate; however, in addition to size, other factors must be involved. The observed nonrandom deletion pattern may reflect domain-associated variation in chromosomal stability. For instance, complications related to the maintenance of replication, correct transcription, and proper splicing of such a large gene may play an extremely important role.
Partial gene duplications have been identified in 5% to 8% of patients. Unlike the deletion distribution, approximately 80% of the duplications are located at the 5' end of the gene and only 20% in the central gene region. Out-of-frame duplications occur in DMD patients and in-frame duplications in BMD patients, thus suggesting that the reading-frame genotype-phenotype hypothesis also holds true for duplications.
Small mutations (point mutations and small deletions and duplications) in the dystrophin gene also have been identified in DMD patients.10 The majority of these mutations have been unique to individual patients and have resulted in a truncated dystrophin protein lacking part or all of the C-terminus. The truncated proteins are presumably unstable, and little or no dystrophin is produced. Therefore, these types of mutations provide little information on structural/functional relationships in the dys-trophin protein. The identification of DMD mutations that do not cause protein truncation may provide us with further insight into the function of dystrophin, as well as defining the essential regions and conformations necessary for dystrophin stability. A DMD missense mutation was found in the actin-binding domain.11 The patient was shown to have correctly localized dystrophin, thus indicating that an intact actin-binding domain is essential for function. The distribution of small mutations is fairly random throughout the gene sequence. However, whereas less than 5% of the gene deletions are found upstream of exon 55, more than 40% of the small mutations are located in this same region of the gene.12
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