Rapid detection of cystic fibrosis transmembrane conductance regulator gene IVS8 5T variant by real-time PCR.

Abstract

Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasian populations and is caused by mutations in the CF transmembrane conductance regulator ( CFTR ) gene. The CFTR gene encodes a transmembrane protein that forms a cAMP-regulated chloride channel. Wide variations in disease manifestations are observed among affected CF patients, and a multitude of disease-causing mutations have been found in the CFTR gene (1). In 97% of men with CF, bilateral congenital absence of the vas deferens (CBAVD) blocks the transport of spermatozoa from testicular structures to the distal genital tract, causing azoospermia (2). Infertility attributable to CBAVD does not necessarily coincide with other manifestations of CF. CBAVD accounts for ∼1–2% of all male infertility and at least 6% of the cases of obstructive azoospermia. Isolated CBAVD patients carry either a CF mutation (ΔF508 in 16–83% of cases and R117H in 6–29% of cases) and/or a 5T variant in intron 8 (12–47% of patients), supporting the hypothesis that CBAVD represents a mild, primary genital form of CF (2)(3)(4)(5)(6)(7). Three length variations of a polythymidine (polyT) tract within the splice acceptor site in intron 8 of the CFTR gene (GenBank accession no. M55106) have been associated with variable efficiency of exon 9 splicing (8). On the basis of the increased frequency (compared with the general population) of the five-thymidine (5T) variant [vs seven or nine thymidines (7T or 9T)] in CBAVD patients, the 5T variant was classified as a CBAVD mutation (7). Currently available but time-consuming methods for 5T/7T/9T genotyping include PCR amplification followed by acrylamide gel electrophoresis (9)(10), detection of mRNA length (2), restriction endonuclease digestion (7), single-strand conformation polymorphism analysis and direct sequencing (11), capillary zone electrophoresis (12), and …