Development and Evaluation of a PCR-based, Line Probe Assay for the Detection of 58 Alleles in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

Abstract

Cystic fibrosis (CF) is the most common life-limiting recessive genetic disorder in Caucasians, with an incidence of ∼1 in 3200 newborns (1). The disease is less common in Hispanic Americans (1 in 9200), African Americans (1 in 15 000), and Asian Americans (1 in 31 000) (2). The diagnosis of CF requires the presence of one or more characteristic phenotypic features, a history of CF in a sibling, or a positive newborn screening test result plus laboratory evidence of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (3). Although sweat chloride testing continues to be the laboratory test of choice, DNA testing is growing in popularity, especially in situations where sweat testing is unavailable or ambiguous. In 1999, 9.1% of patients attending CF centers in the United States had DNA analysis as a confirmatory diagnostic test (4). Furthermore, DNA testing is the only method available that can readily identify the carriers of CF mutations and, therefore, is the only tool for CF carrier screening. The gene responsible for CF, the CFTR gene, was identified by positional cloning (5)(6). Nearly 1000 putative disease-associated mutations have been reported in CFTR , but only a few dozen have a frequency >0.1% in the general population (1)(7). Thus, efficient and accurate population screening for CF requires an assay that can simultaneously detect the mutated and wild-type sequences of ∼30 CF alleles. We have developed the Research Prototype Cystic Fibrosis Assay-31, which uses multiplex PCR (8)(9) and colorimetric detection with sequence-specific oligonucleotide probes immobilized in a linear array on nylon membranes (10), to type 28 CF mutations and 3 CFTR polymorphisms [Table 1⇓ ; details of this procedure are described in a …