R117H and IVS8-5T cystic fibrosis mutation detection by restriction enzyme digestion

The number of thymines in a thymine repeat in intron 8 (IVS8) next to the exon 9 of the cystic fibrosis gene (CFTR) affects the efficiency of this site to act as a splice acceptor site and the subsequent inclusion or skipping of exon 9 into the CFTR protein. Another source of genetic variation, responsible for mild cystic fibrosis (CF) and/or congenital bilateral absence of the vas deferens, is the mutation R117H, located in exon 4 of CFTR. Both these genetic variants can be detected using restriction site-generating PCR amplification. The severity of the CF phenotype is partly dependent on the IVS8 background on which R117H occurs; thus, it is important to be able to test clinically for both these variants. A method using restriction digestion of PCR product is presented.