Deletion Screening and Point Mutation Analysis in Regions of the Duchenne/Becker Muscular Dystrophy Gene

Duchenne muscular dystrophy (DMD) is the commonest dystrophy with a birth incidence of one in 3000-3500 males, and approximately one third of all cases result from a new mutation. Affected males present with progressive muscle weakness and die as a result of respiratory or cardiac involvement in their late teens or early twenties. Becker muscular dystrophy (BMD) is a mild variety of the disease, and the most useful criterion for differentiating the two types is the age when patients become wheelchair bound. The gene is located on Xp21, and consists of more than 65 exons encoding a protein 3685 amino acids long, named dystrophin. About 65% of affected boys have a small submicroscopic gene deletion, heterogeneous in both the specific region and number of exons missing, that can be detected by Southern blot analysis using cDNA probes, and/or by sequence amplification with the polymerase chain reaction. Partial gene duplication accounts for the mutation in about 6% of the patients, while in the remainder point mutations are suspected An aim of this project was to screen DMD/BMD patients for deletions in exons 30 to 47 of the dystrophin gene covered by cDNA probe 5b-7, on Bglll digested DNA, and at the 3' end of the gene using the restriction enzymes Hind III and Bgl II in combination with cDNA probes 9, 10 and 11-14. Seventeen DMD/BMD patients were studied with cDNA 5b-7 and one deletion was detected having the distal end at exon 44 while the proximal end was in exon 20, in the region of cDNA probe 4-5a. Five cDNA deletions were detected having both end-points in the distal part of (lie dystrophin gene in a panel of thirty-six DMD/BMD affected males showing no deletion or duplication with the rest of the cDNA probes. Also, two cases of deletions which started in the region of cDNA 8 were identified to extend in the region of cDNA 9, with one of the two having the 3' end into the region of cDNA 10. No deletions were found with cDNA 11-14. Taking advantage of the different size of deletions detected, some of the Bgl II genomic fragments were related to Hind III genomic fragments of known orientation, and the order of the Hind III fragments in the region of cDNA 10 was rearranged. Also, the correlation between deletion and phenotype was examined and found to fit the reading-frame model proposed to explain the clinical difference in severity between DMD and BMD patients. For diagnostic purposes the detection of the molecular pathology of the disease can confirm the diagnosis of DMD/BMD in sporadic cases and offer direct accurate prenatal diagnosis in the family without the necessity for linkage analysis that requires DNA samples from key relatives. As Southern analysis takes five to ten days for results to be obtained assessment of the value and reliability of the polymerase chain reaction in performing deletion detection screening at multiple sites simultaneously was the next aim of the present study. Simultaneous amplification by PCR of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51 of the dystrophin gene in 118 DMD/BMD DNA samples identified a mutation in 48. 3% of the patients and detected 86% of the deletions previously revealed by Southern analysis. Discrepancies were not found between the results obtained by the two methods of analysis. On the contrary, the problem of weak hybridisation was circumvented in two cases allowing the accurate mapping of the deletion end-points. Postnatal-detection screening was also performed in two patients and both results were confirmed by Southern analysis. The ability of PCR to amplify DNA fragments from small starting amounts of not necessarily good quality DNA permitted the amplification of DNA extracted from haematoxylin and eosin stained tissue sections, as well as the detection of a deletion in an individual whose DNA failed to produce detectable signal by Southern analysis because of degradation. Speed, sensitivity, specificity and efficiency characterise the method of multiplex amplification with the polymerase chain reaction, and make it ideal in the analysis of mutations in routine clinical practice as an initial screen to detect the molecular pathology of the disease. The final aim of this project was to scan regions of the DMD/BMD gene in affected males whose molecular pathology was still unknown, for new polymorphic sites or mutations that may account for the development of the disease and to compare and assess the different methodologies for mutational screening. (Abstract shortened by ProQuest.)