Higher proportion of intact exon 9 CFTR mRNA transcripts in nasal epithelium compared to VAS deferens

Abstract

The cystic fibrosis gene encodes the cAMP-regulated chloride channel found in the apical membrane of secretory epithelial cells, called the cystic fibrosis transmembrane conductance regulator (CFTR). It has been well established that almost all males with cystic fibrosis (CF) are azoospermic due to atrophy or absence of structures derived from the wolffian duct. Interestingly, a higher than expected frequency of mutations in the CFTR gene has been identified in patients with congenital absence of vas deferens and patients with epididymal obstruction. In particular, these individuals have been found to have a significantly higher incidence of the 5-thymidine (5T) variant of the CFTR intron 8 polypyrimidine tract (IVS8-T tract) compared to normal or CF populations. The 5T variant results in less efficient splicing of CFTR exon 9 compared to the more common 7T and 9T variants and therefore produces less normal, full-length CFTR mRNA. The protein translated from the CFTR transcript lacking exon 9 appears to be devoid of cAMP-activated chloride conductance. The fact that these infertile males have no other clinical signs of classical CF suggests that the epithelia of the male reproductive tract may have the highest requirement for CFTR function or, alternatively, splicing of CFTR mRNA in the reproductive tract is less efficient than the other CF-associated organs. Nasal epithelia and segments of vas deferens were obtained from 24 healthy, previously vasectomized patients who presented for vasectomy reversal. Quantitative RT-PCR was performed on these specimens, with the region of CFTR cDNA spanning exon 9 amplified. For both nasal and vasal tissues, a strong positive correlation was found between the length of the IVS8-T tract and the proportion of mRNA with exon 9 intact (r=0.88, p0.001 for nasal; r=0.87, p0.001 for vas). In addition, for the same individual patient, a significantly higher level of transcripts lacking exon 9 exists within the vas deferens compared to those within nasal epithelia (p0.001, paired t-test). These findings suggest that the splicing of CFTR precursor mRNA is less efficient in vasal epithelia compared to respiratory epithelia. Thus, differential splicing efficiency between the various tissues which express CFTR provides one possible explanation for the reproductive tract abnormalities observed in infertile men with CFTR gene alterations but without other manifestations of CF.