MOLECULAR CHARACTERIZATION OF FOUR FREQUENT POLYMORPHISMS IN THE CFTR GENE: ASSESSING ITS ROLEIN CYSTIC FIBROSIS DISEASE

Abstract

To date, more than 200 sequence polymorphisms have been identified in the CFTR gene thus far and only a minority has been characterized at the cellular localization and the protein levels. In previous studies, molecular genetic analysis of the entire coding region of the CFTR gene in patients identified four common polymorphisms: c.1540A>G is located in Exon 10 of CFTR; c.2694T>G in Exon 14a, whereas c.4404C>T and c.4521G>A are located in Exon 24. The aim of this study was therefore to examine the possible effects of these polymorphisms on subcellular localization and CFTR processing in different constructs to disclose their impact on the clinical phenotype. The subcellular localization using confocal microscopy of coding single-nucleotide polymorphisms c.1540A>G, c.2694T>G, c.4404C>T and c.4521G>A have shown correct membrane localization and normal effect on maturation of CFTR protein. Although maturation patterns were not affected, total amounts of mature CFTR protein were reduced for c.2694T>G and c.4521G>A polymorphisms. The double c.[2694T>G; 4521G>A], triple c.[1540A>G;2694T> G;4521G>A] and quadruple c.[1540A>G; 2694T>G;4404C>T; 4521G>A] polymorphisms have shown to be exclusively cytoplasmic consistent with an endoplasmic reticulum localization. Western blot analysis of CFTR protein indicated that the double and triple mutants had effects on the maturation of CFTR protein. So, more severe effect on CFTR protein was present when these polymorphisms were combined in complex alleles in cis, supporting the influence of these frequent polymorphisms on the clinical features of CF patients. These findings suggest that SNPs may be responsible for variation in the phenotypic expression of CFTR mutations.