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

Abstract

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal 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 the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting 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 DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. 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 sites.

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations

“…4 Every mutant discovered by these methods was sequenced with the ABI PRISM 377 or 310 Sequence Analyser. Some variants have been studied, on a fraction of the total sample, with a restriction enzyme specific method: the following cSNSs numbered as in Table A1, nos.…”

Spatial patterns of cystic fibrosis mutation spectra in European populations

“…Estimates of allele diversity are bracketed between two extremes: they would be minimal if all unknown mutations in a population were, in fact, the same allele, and they would be maximal if all unknown mutations were different from each other. Intensive mutation-detection efforts by means of denaturing high-performance liquid chromatography 27 have shown that chromosomes bearing previously unknown mutations carried each a new, different, unique mutation. Thus, it is likely that the 'unknown' portion of the mutation spectrum contains a wide diversity of alleles.…”

Influence of the Duplication of CFTR Exon 9 and Its Flanking Sequences on Diagnosis of Cystic Fibrosis Mutations

“…This variation eludes common PCRbased techniques for mutation detection in this region, including direct sequencing, as well as denaturing highperformance liquid chromatography. Thus, if denaturing high-performance liquid chromatography analysis is used, it is necessary to use primers that have been documented 7 to prevent the variability that T/TG repeats can cause. Using this method, however, only the beginning of exon 9 is amplified.…”