Extensive sequencing of the cystic fibrosis transmembrane regulator gene: Assay validation and unexpected benefits of developing a comprehensive test

Purpose: To develop a sequencing assay for the CFTR gene to identify mutations in patients with cystic fibrosis (CF). Methods: An automated assay format was developed to sequence all exons and splice junctional sequences, the promotor region, and parts of introns 11 and 19. Results: After validating the assay using 20 known samples, DNA of seven patients, four of whom were heterozygous for a known CIF mutation, was sequenced. Known CIF mutations were detected in seven of the eight chromosomes, and a novel missense mutation was detected in the eighth. In addition, this assay allowed 14 ambiguous results obtained using the Roche CIF gold strips to be resolved. Three false-positive diagnoses were prevented; a different mutation at the same codon was identified in two patients and confirmation was provided in the remaining nine cases. Conclusions: Sequencing of the CFTR gene provides important information for CF patients and is a valuable adjunct to a carrier screening program to resolve ambiguities in panel testing.