Fluorescent, Multiplexed, Automated, Primer-Extension Assay for 3120+1G→A and I148T Mutations in Cystic Fibrosis

Abstract

Cystic fibrosis [(CF); Online Mendelian Inheritance in Man No. 602421] is an autosomal recessive disorder characterized by chronic broncho-pulmonary disease, pancreatic insufficiency, and increased sweat electrolytes (1). The CF transmembrane conductance regulator ( CFTR ) gene was identified by three groups (2)(3)(4) and was mapped to the long arm of chromosome 7 (7q31). The CFTR contains 24 exons, encodes a protein of 1480 amino acids, functions as a chloride channel, and regulates other transport pathways. Over 900 disease-causing mutations in the CFTR gene have been identified. The American College of Medical Genetics has recommended a core mutation panel for general population CF carrier screening (5). This panel contains 25 of the known CF mutations, including 3120+1G→A and I148T . 3120+1G→A (intron 16) is the second most common CF mutation in the African American population and accounts for 9–14% of the African American CF chromosomes (6). I148T (exon 4) is the second most common CF mutation in the French Canadian population and accounts for 9.1% of the French Canadian CF chromosomes (7). Most of the 25 mutations recommended by the American College of Medical Genetics can be detected with commercially available methods [Roche Molecular Biochemicals, Applied Biosystems and Innogenetics (ABI)], but no assays test for both the 3120+1G→A and I148T mutations, and two of the three assays do not detect either mutation. Therefore, we developed a multiplex primer-extension assay that rapidly and simultaneously detects these two CF mutations using the ABI Prism® SNaPshotTM ddNTP Primer-Extension assay. We believe this is the first demonstration of the use of this assay in the literature. The method uses a dideoxynucleotide primer-extension reaction (PER) in conjunction with an ABI genetic analyzer to detect the addition of a fluorescently labeled ddNTP to the 3′ end of a sequencing primer (Fig. …