Background: The 5T allele of the polyT tract located within intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is a variant that in trans with a severe CFTR mutation can result in normal phenotype, congenital bilateral absence of vas deferens (CBAVD), or mild cystic fibrosis. The 5T allele has been associated with the skipping of exon 9, a process that seems to be influenced by an adjacent 9-13TG tandem repeat. The 12- or 13TG repeats are often associated with an abnormal phenotype. We present here a single-step method for direct haplotyping of the TG repeats in 5T carriers.
Method: The method is based on a single-step PCR, using a fluorescently labeled forward primer and a reverse allele-specific primer matching the 5T allele. We validated the test in 30 control samples of known 5T-poly(TG) haplotype and then used this method to evaluate 57 clinical samples.
Results: The expected TG genotypes were obtained for all 5T control samples, and no nonspecific amplification of either the 7T or 9T alleles was detected. In our 5T-positive collection 9 of 9 (100%) CBAVD patients, 6 of 12 (50.0%) chronic pancreatitis patients, and 12 of 36 (33.3%) individuals undergoing assisted reproduction showed 5T-12TG haplotype.
Conclusions: Our method is an accurate, specific, and simple tool to characterize the 5T poly(TG) haplotype. Our results confirm the high frequency of 5T-12TG in CBAVD patients and do not preclude a potential effect also in pancreatitis. This assay can be useful in assessment of the disease risk in 5T carriers.