Detection of five common CFTR mutations by rapid-cycle real-time amplification refractory mutation system PCR.

Abstract

Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations and has a carrier frequency of 1 in 25 (1). The gene involved codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane-associated protein involved in ion transport across the plasma membrane of epithelial cells. To date more than 1000 mutations have been described in this gene, and most are rare (2). By focusing on five common mutations it is possible to detect the disease-causing mutation in ∼90% of Irish patients (3). The five mutations [and the percentages of Irish (3) and worldwide (2) cases] are F508del (77.4%, 66.0%), G551D (7.1%, 1.6%), R117H (2.7%, 0.3%), 621+1 G>T (1.4%, 0.7%), and G542X (0.5%, 2.4%). Four of these fall into the severe class of mutations in which the mRNA is incorrectly spliced (621+1 G>T), or in which the protein is not synthesized (G542X) or is blocked during processing (F508del), or its regulation is blocked (G551D). Mutations in the CFTR gene can be detected by many mutation-detection systems, including single-strand conformation polymorphism analysis (4), restriction fragment length polymorphism analysis, Amplification Refractory Mutation System (ARMS) PCR (5), and more recently, real-time PCR systems using Sybr Green 1 (6) or hybridization probes (7). Currently, for mutation detection using hybridization probes on the LightCycler system, the detector probe is designed to overlie the possible site of the mutation. This can have drawbacks when multiplexing in the same detection channel because peaks can become merged, making genotyping of a sample difficult. This study aimed to improve the multiplexing capabilities of real-time PCR in its use for mutation detection. To this end we used ARMS PCR primers to selectively amplify the wild-type or mutant alleles in separate reactions. Subsequent detection of PCR products was carried out with a common set of hybridization probes. The advantage …