Mutation screening in human diseases

This project has utilised recently-published techniques for mutation detection within two genes responsible for human disease, the dystrophin gene and the adenomatous polyposis coli gene.The phenomenon of dystrophin gene mosaicism in mothers of apparent de novo cases of Duchenne and Becker muscular dystrophies (DMD/BMD) was examined by using a combination of carrier detection techniques. Germline and somatic mosaicism in the mother of a de novo case of DMD, a previously unreported state, was shown by combining evidence from linkage data obtained from dystrophin DNA and reverse-transcription PCR (RT-PCR) data from dystrophin RNA. A second mosaicism detection method was developed from the fluorescence in situ hybridisation (FISH) technique. Using this technique, somatic mosaicism was proven in a second mother of a de novo DMD case, a hitherto unreported application of the dystrophin FISH method. These results and others (including those from a BMD male) suggest that a significant number of de novo dystrophin deletions occur as a mitotic event during the early stages of embryogenesis, prior to blastocyst differentiation.Studies of the adenomatous polyposis coli (APC) gene involved the use of a PCR based heteroduplex analysis test and DNA sequencing. Thirteen novel mutations of the APC gene were discovered, and genotype-phenotype correlations were devised between the position of the mutation site and the age of onset of the disease and it's severity. With such correlations available, knowledge of the phenotype of a new patient will allow a more rapid genetic confirmation of the disease by narrowing down the areas of the gene which require screening.The de novo recurrence of a rare APC gene microdeletion in a second patient from the APC study population initiated an examination of the mechanisms of microdeletion mutagenesis.