Duchenne Muscular Dystrophy

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Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that also involves gene deletion and duplication. DMD is a devastating, progressive, muscle-wasting disorder that is usually not diagnosed before the age of 3, but often results in wheelchair confinement by age 12 and death in the early twenties. Manifestations of this disease include pseudohypertrophy of muscles, joint contractures, scoliosis, respiratory compromise, cardiomyopathy, and markedly increased serum creatine kinase (CK) levels resulting from leakage of CK from diseased muscle into the bloodstream. The brain is also affected and, consequently, the IQ range of DMD patients is approx 20 points below average. However, mental abnormalities are certainly not found in all patients. The use of oral prednisone has been demonstrated to improve the strength and function of patients with DMD (100,101). This observation indicates the significance of immune-modifying factors determining the clinical expression of inherited neuromuscular diseases. Aggressive symptom management might extend the survival of DMD patients into their mid-twenties. It was once believed that patients with a milder form of the disease were affected with a distinct disorder, Becker muscular dystrophy (BMD). However, molecular analysis has demonstrated that mutations in the same gene are responsible for both disorders. BMD patients could remain ambulatory through adulthood. Individuals with pheno-types intermediate to DMD and BMD have also been described (102). The DMD gene has been mapped to Xq21, and although DMD is an X-linked recessive disorder, studies have demonstrated that 8% of DMD carrier females are mildly affected because of skewed X-inactivation (103).

8.1. GENETICS The DMD gene was one of the first genes to be cloned by positional cloning (104). The gene is extremely large, spanning approx 2300 kb, and contains 79 exons that encode the protein dystrophin. The normal protein product is not expressed in DMD patients. In skeletal muscle, dystrophin is located in the sarcolemma membrane, where it is believed to play a crucial role in linking the contractile apparatus of myocytes to the sarcolemma membrane (105). Isoforms of dystrophin, produced by the use of alternative splice sites and alternative promotors, are expressed in cardiac muscle and in the brain. The finding that dystrophin is expressed in the brain offers a possible explanation for the mental subnormality often present in DMD patients. Sixty percent of DMD patients have deletions, and 6% have duplications within the DMD gene. Although the deletions are distributed throughout the

Fig. 2. Southern blot analysis of a DMD patient (P) and a normal control (C). The probe cDMD 5b-7 in combination with the BglH digest (A) shows absence of the 2.8-, 3.3-, and 3.5-kb bands, consistent with the deletion of exons 45-47. The probe cDMD 8 with either the BgllI or the HindIII digest (B) shows the absence of all bands, consistent with the deletion of exons 48-52. The probe cDMD 9/BglII digest shows absence of the 16-kb band, and cDMD 9/HindIII digest shows the absence of the 7.8-kb and 8.3-kb bands (C); both are consistent with the deletion of exons 53 and 54. These results indicate that the deletion spans exon 45-54.

Fig. 2. Southern blot analysis of a DMD patient (P) and a normal control (C). The probe cDMD 5b-7 in combination with the BglH digest (A) shows absence of the 2.8-, 3.3-, and 3.5-kb bands, consistent with the deletion of exons 45-47. The probe cDMD 8 with either the BgllI or the HindIII digest (B) shows the absence of all bands, consistent with the deletion of exons 48-52. The probe cDMD 9/BglII digest shows absence of the 16-kb band, and cDMD 9/HindIII digest shows the absence of the 7.8-kb and 8.3-kb bands (C); both are consistent with the deletion of exons 53 and 54. These results indicate that the deletion spans exon 45-54.

DMD gene, two deletional "hot spots" have been identified. One of these regions extends over the first 20 exons, and the second region includes exons 45-53 (106). Although regions of clustered deletions suggests the presence of specific sites of breakage and recombination, such sites have not been identified. There is no correlation between the size of deletion and severity of disease. However, deletions resulting in frameshift mutations are generally associated with a severe phenotype, and most nonframe-shift mutations are associated with a BMD phenotype (107). Exceptions to this rule are noted and most commonly with those patients having deletions of exons 3-7, which produces an out-of-frame mutation but leads to either DMD or Becker phenotypes (108). Duplications are associated with severe phenotypes, as they often result in frameshift mutations or protein truncation. Point mutations that have been identified in DMD patients are distributed throughout exons 8-70 (107).

8.2. MOLECULAR DIAGNOSIS Prior to cloning of the DMD gene and identification of dystrophin, diagnosis of DMD was based primarily on clinical features, muscle biopsy, and CK levels. Carrier testing involved examination of CK levels, which is often equivocal in carriers, and linkage analysis. Current diagnostic and carrier testing strategies include direct examination of the DMD gene and/or dystrophin analysis. Direct analysis of the DMD gene is accomplished by PCR or Southern blotting techniques. The Southern-based assay typically utilizes both HindlH and Bglll restriction enzyme digestion and hybridization with the following cDNA probes: 1-2a (exons 1-9), 2b-3 (exons 10-20), 4-5a (exons 21-33), 5b-7 (exons 34-48), 8 (exons 47-52), and 9 (exons 53-59). Each exon is represented by a specific combination of bands on the autoradiogram (Fig. 2 ). The 3' end of each exon has been char acterized with regard to whether the exon ends with the first, second, or third nucleotide of a codon (109). This information, along with Southern blot results for an individual patient, can be used to determine whether or not a particular deletion will cause a shift in the translational reading frame. Southern blot detection of carrier females and patients with duplications is based on dosage differences and requires quantitative analysis. In a small percentage of DMD patients, the junction of the deletion or duplication creates a novel restriction fragment. Carrier status determination is more easily accomplished in families where a junction fragment is present.

Polymerase chain reaction analysis typically involves multiplex reactions in which the promoter region and multiple exons are amplified (Fig. 3). The multiplexes described by Kunkel and Chamberlain (110) are commonly used in the clinical laboratory. PCR detects approx 98% of the deletions detected by Southern blot analysis. However, PCR-based detection of duplications and of carrier females is difficult, and PCR analysis cannot be used to distinguish between frameshift and nonframeshift deletions. It should be noted that in the prenatal setting, the speed and smaller specimen requirements of PCR might render it the method of choice for the evaluation of at risk male fetuses. For males with a suspected diagnosis of DMD, many molecular genetics laboratories begin the analysis with the PCR-based assay. If PCR fails to detect a deletion, Southern analysis can be used for further investigation. If a deletion is detected by PCR, Southern analysis with the appropriate probes can be used to confirm the extent of the deletion, as well as to distinguish between frameshift and nonframeshift mutations. The information derived from the molecular analysis can subsequently be utilized by at risk family members.

Fig. 3. PCR analysis of the DNA from a normal control (C) and the DMD patient (P) described in Fig. 2. The PCR results indicate deletion of exons 45 and 48.

Approximately 35% of DMD patients have no detectable deletions or duplications. For families of these patients, linkage analysis can often be used for prenatal evaluation and to determine the carrier status of at risk females. Linkage analysis can involve a number of approaches; the most informative are PCR-based assays in which extragenic (CA)n repeats on the 5' and 3' ends of the DMD gene as well as intragenic (CA)n repeats are utilized (111). Although linkage analysis is highly accurate, it requires the participation of key family members, which might eliminate its utility for certain families. At-risk females who are in this situation will require alternative testing strategies for the evaluation of carrier status. This testing might include determination of CK values and dystrophin analysis. It should be noted that dystrophin analysis offers the advantage that it is not dependent on the type of mutation present in the DMD gene (107). Complete sequencing of the dystrophin gene is currently being offered in a few laboratories. This will assist in the direct identification of all mutations and holds promise in the molecular diagnosis of DMD, although these techniques remain very labor-intensive. For additional information concerning DMD testing strategies, a reference by Bushby and Anderson (111) is recommended.

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