M. Goossens

4 Simple and accurate assays for targeting CFTR mutations of specific geographic/ethnic origins by PCR allelic discrimination

The CFTR gene is one of the most highly mutated human genes with more than 1600 different mutated alleles reported, and besides the most frequent mutations, the deleterious effect of many variations remains unclear, which sometimes confuse molecular geneticists, particularly for intronic or exonic silent variations, which can affect mRNA splicing. Several softwares have been developed for in silico prediction of the impact of sequence variation on splicing, and they are commonly used in molecular laboratories. But even if they have proven their effectiveness for many genes, it is important to confirm by in vitro functional analysis. The aim of our study is to compare the bioinformatic predictions to results obtained with a reporter minigene and by direct analysis of mRNA from patients. On the basis of the bioinformatic predictions, more than 50 variations have been classified into four classes according to the expected effect on mRNA splicing (complete or partial exon exclusion, cryptic alternative splicing site, or no effect). Two variations from each class were tested in our minigene vector and at the mRNA level. As expected, mutations affecting consensus donor splice lead to complete exon exclusion and frequent polymorphisms located in the last nucleotides of exons did not affect mRNA splicing. More interestingly, some mutations, initially classified as splice mutations because of their location on the acceptor sites yielded an unexpected result in bioinformatics predictions, which was confirmed by minigene and mRNA analysis. This reporter minigene study will be extended to the 50 variations selected, and in cases of discrepancy, analysis of mRNA will be performed.