Detection of point mutations in the dystrophin gene

The dystrophin gene has been localised to Xp 21.1. Mutations of this gene can lead to the clinical manifestations of Duchenne and Becker muscular dystrophies (DMD/BMD). In the majority of DMD and BMD patients the disease-causing mutation is a deletion detectable by southern analysis or multiplex PCR, however in 30% of patients no deletion is observed using these conventional tests. Using PCR amplification of cDNA it was possible to detect a deletion in the product of the dystrophin gene of one such individual affected with BMD. It was then necessary to characterise the mutation in order to determine whether this was the disease-causing mutation. The mutation was found to be a point mutation by genomic sequencing at the donor splice site consensus sequence of intron 19. This resulted in alternate splicing of exon 19, two mANA transcripts being produced one with exon 19 and the other without. This was determined sufficient to cause BMD in the family. It was then necessary to provide an easy and efficient diagnostic test in order to determine the carrier status of females and also to provide those females determined to be carriers with an accurate prenatal diagnostic test. A new technique, single strand conformation polymorphism, performed on genomic DNA provided evidence of conformational difference between the affected allele and the normal allele. Allele specific primers were also designed and optimised. These primers, 19FA and 19FC, only varied at one base, one being the "normal" allele primer and the other the "affected" allele primer. The amplified product using primers for the normal and the affected allele were then run on agarose gels and stained with ethidium bromide providing a quick and easy method of determining the carrier, affected or normal status of all family members. The localisation and characterisation of this splice site mutation involved many molecular techniques including, RNA extraction, cDNA synthesis, nested amplification, sequencing, single strand conformation polymorphism and the amplification refractory mutation system. All of these techniques can be used to isolate and characterise point or minor mutations in the 30% of Western Australian dystrophic families, who currently lack an accurate diagnostic test for their dystrophic condition.