Evaluation of screening strategies for the detection of molecular pathologies

There have been many studies aimed at the determination of the molecular pathologies responsible for genetic disease. Many strategies have been used for detection of base changes in genes which lead to disease and in this study several mutation detection protocols were compared and optimised in order to determine a suitable strategy for the detection of molecular pathologies in large multi-exonic genes. The HGPRT gene was used as a model gene and a panel of 10 known mutations present in this gene was used to optimise and compare SSCP, heteroduplex detection on hydrolink gels (HDHG) and chemical cleavage of mismatches (CCM) as a first line screening strategy for pin-pointing the mutation containing exon. Any suspicious exons were then sequenced using automated fluorescent sequencing. These protocols were then applied to the identification of unknown pathologies in the HGPRT, DMD and BRCA1 genes. HDHG was the most successful strategy for detecting changes in larger amplification products where there were fewer exons, while SSCP was most effective when the PCR products were around 200bp in size and where there were a large number of samples to be analysed. Three mutations were identified in the HGPRT gene of index cases from Lesch-Nyhan families and the mutation detection strategies coupled with linkage analysis using a tetranucleotide repeat within the HGPRT gene, were used to pin-point carriers of the mutations in these families. Four different mutations were found in seven of the breast/ovarian cancer families, including a 2bp deletion which was identified in four unrelated families, indicating the presence of a common Scottish mutation. No mutations were identified in the DMD/BMD gene of the individuals who showed no large exon deletion and it may be that the optimum strategy for the detection of mutations in this gene has not yet been identified.