POSTER NO: 443

Splicing factors as modulators of the CFTR function in cells carrying CFTR splicing mutations

Malka Nissim-Rafinia, Batsheva Kerem
Hebrew University, Jerusalem, Israel

One important question in the field of human genetic is the mechanism underlying the high variability in disease expression among patients carrying the same genetic defects. The molecular basis for this variability has been suggested to involve genetic modifiers and environmental factors. We study the molecular basis for variability among cystic fibrosis (CF) patients carrying splicing mutations. This includes mutations which can lead to cryptic exon inclusion or exon skipping. A wide variability in the level of the aberrantly spliced pre mRNA, transcribed from such CF alleles, is found in respiratory epithelial cells among different patients. Furthermore, such a wide variability is also found among different organs of the same individual. The variability correlates with the variability in disease severity. We thus hypothesized that the molecular basis for the phenotypic variability involves factors known to regulate alternative splicing in vivo. Previously we have shown that the cellular and viral splicing factors can modulate the splicing pattern of minigenes carrying CFTR splicing mutations (3849+10kb C->T which can lead to inclusion of a cryptic exon or 5T which can lead to skipping of exon 9). Here we studied the effect of overexpression splicing factors on endogenous CFTR alleles carrying the 3849+10kb C->T mutation in epithelial cell lines from nasal polyps of two CF patients carrying the mutation. The analysis showed that the splicing pattern of RNA transcribed from the endogenous alleles could be modulated by over expression of splicing factors. The cellular factor ASF/SF2 led to almost a complete abolishment of the aberrantly spliced transcripts. The viral factor E4-ORF3 and the non-essential cellular factor Htra2-b 1 promoted exon inclusion and led to a significant increase in the level of aberrantly spliced transcripts. We further analyzed the effect of splicing modulation on the function of the CFTR protein using chloride efflux measurements. Our preliminary results show that the CFTR channels in the cells are inactive, similar to the inactivity found in cells carrying two inactive CFTR alleles. We further studied the effect of overexpression splicing factors on the chloride efflux in these cells. The results of the experiments and their significance for the development of new therapeutic approaches will be discussed.