Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (C FTR) gene. Ten years after the cloning of the CFTR gene, the complete scann ing of the 27 exons to identify known and novel mutations remains challengi ng. Rapid accurate identification of mutated alleles is important for prena tal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse -phase high performance liquid chromatography (D-HPLC) to analyse rapidly t he complete coding sequence of the CFTR gene. With 27 pairs of polymerase c hain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated the test conditions on samples from a panel of 1552 CF patients who came from France and other European c ountries and who had mutations and polymorphisms located in the various mel ting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing , plus 74 novel mutations reported here. This new technique for screening D NA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be ana lysed in less than a week). Our approach should reduce the number of untype d CF alleles in populations and thus decrease the residual risk in couples at risk of CE This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of s ites.