Clinical Utility of Testing and Available Testing

The most important impact of DNA testing for DMD/BMD has been on presymptomatic diagnosis and the correct diagnosis of patients with DMD/BMD-like symptoms, as well as for carrier detection and prenatal diagnosis. The western immunoblot is the most sensitive test for the diagnosis of DMD or BMD, but requires a muscle biopsy from the patient. Proteins from the muscle biopsy specimen are separated by electrophoresis, transferred to a membrane, and incubated with antibodies against specific dystrophin domains. Dystrophin from unaffected patients is 427kDa in size. The complete absence of dystrophin is very specific for the diagnosis of DMD, whereas the presence of an altered molecular weight form or reduced amount of dys-trophin, or both, is consistent with BMD. It must be emphasized, however, that the clinical diagnosis and phenotype predictions should never be made exclusively on the basis of dystrophin western blot analysis. Patients with markedly reduced dystrophin levels may have the milder BMD phe-notype. Furthermore, western blot analysis is generally not sensitive enough to detect female carriers.

Southern blot analysis using a full-length dystrophin cDNA clone as a probe detects 65% of deletions in affected DMD/BMD patients, as well as gene duplications. The cDNA probes detect the site of the mutation, so meiotic recombination events are irrelevant. Therefore, the chance of diagnostic error is greatly reduced. Diagnostic strategies are based on initially identifying deletions in the affected patient.13 The deletions are simply detected by examination of Southern blots for the presence or absence of each exon containing genomic restriction fragments, which hybridize to the cDNA probe. However, Southern blot analysis requires the use of a radioisotope, is labor intensive, and is time-consuming.

Before performing Southern blot analysis with the DMD cDNA probes, most laboratories perform an initial deletion screen using multiplex PCR14 to amplify specific deletion-prone exons within the DMD gene. Deletion of specific exons from a patient's sample results in absence of the corresponding ethidium bromide-stained amplification products by gel electrophoresis analysis (Figure 7-1). Mul-

Exon 45 Exon 19 Exon 8

Figure 7-1. Multiplex DNA amplification of DMD exons 8,13,19,45, and 47. Lane 1: normal control; lane 2: DMD patient with exon 13 deletion; lane 3: DMD patient with exon 8 and 13 deletions.

Exon 45 Exon 19 Exon 8

Figure 7-1. Multiplex DNA amplification of DMD exons 8,13,19,45, and 47. Lane 1: normal control; lane 2: DMD patient with exon 13 deletion; lane 3: DMD patient with exon 8 and 13 deletions.

tiplex PCR, using primer sets for about 20 different exons, detects approximately 98% of the deletions in the DMD gene. In contrast to Southern blot analysis, which may require several cDNA hybridizations and take several weeks to complete, multiplex PCR testing can be completed in one day. This makes the technique ideal for prenatal diagnosis, when time is critical.

The identification of a deletion in a DMD patient not only confirms the diagnosis but also allows accurate carrier testing for other family members. Carrier status is determined by gene dosage analysis, which is used to assess whether a female at risk of being a carrier exhibits no reduction or 50% reduction in band intensity in those bands that are deleted for the affected male relative. A 50% reduction (single-copy intensity) for the deleted band or bands on the autoradiograph indicates a deletion on one of her X chromosomes and confirms her carrier status. Either Southern blot or quantitative PCR can be used for gene dosage analysis. Dosage determinations permit direct carrier analysis and eliminate the inherent problems of Southern blot analysis without gene dosage (recombinations, noninformative meioses, unavailability of family members, and spontaneous mutations). This is important since unlike affected males, carrier females are generally asymptomatic, and creatine phosphokinase (CPK) is elevated in only approximately two thirds of known carriers.

In 35% of families with undefined mutations, linkage analysis using microsatellite markers must be used for carrier detection and prenatal diagnosis. Microsatellite markers, which correspond to short tandem repeats (di-, tri-, or tetranucleotides) and tend to be highly polymorphic in repeat number, have been found in several locations in the DMD gene and have significantly improved linkage analysis for DMD/BMD.15 The method relies on the co-inheritance of the disease gene with those DNA microsatellite sequence variations known to be located within the disease gene. Thus, even when the responsible gene mutation remains unknown, the linkage technique allows the mutation to be traced through an affected family for prediction of carrier or disease risk.

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