Application of pyrosequencing to the identification of sequence variations in the cystic fibrosis transmembrane conductance regulator gene

Background: A high number of mutations associated with cystic fibrosis have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, most of which are rare and, therefore, hamper extensive molecular diagnosis. In couples undergoing prenatal diagnosis where no mutation is found in one or both partners, additional analysis of intragenic polymorphisms may allow for the identification of fetal alleles associated with cystic fibrosis. Methods: We developed novel, rapid and accurate assays for CFTR genotype determination using pyrosequencing technology; a simple, automated and reliable technique with low cost. Results: Assays were optimized for the identification of the seven most frequent CFTR mutations (p.DeltaF508, p.N1303K, p.G542X, c.2183AA>G, c.1717-1G>A, p.W1282X, p.R1162X) in the Italian population and two common intragenic polymorphisms (rs213950 and rs1800136). Blind validation on 15 known control samples, typed for each sequence variation, allowed correct identification of all 135 genotypes. Conclusions: We demonstrated that this procedure is highly specific for the identification of individual CFTR sequence variations associated with cystic fibrosis, allowing both population screening and prenatal diagnosis. Clin Chem Lab Med 2009;47:1051-4.