Peter van Mourik

26 Chapter 2 influencing mRNA expression could impact R117H-CFTR function. Whether mRNA expression variability in the intestinal epithelium reflects variability in other tissues, e.g. airway epithelium, will require further investigation as mRNA expression differs between tissues 4 . We do not know if the observed large variability in mRNA expression in our studies might affect the utility of mRNA expression as predictive tool for CFTR function but our previous studies indicated that differences in organoid CFTR function translate well to the in vivo situation 10,15,16 . None of the R117H-CFTR subjects of which organoid responses were investigated in this study were treated with ivacaftor. As such, we could not correlate clinical disease and ivacaftor responsiveness. Exploratory correlation analyses of R117H-CFTR mRNA expression and 0.128 µm FIS with sweat chloride concentration (SCC; n=12), showed a trend towards correlation (r=-0.44, p=0.16, r=-0.40, p=0.17, supplementary figure 1J and 1K, respectively), which was absent for 0.05 µm FIS (Supplementary figure 1L). CFTR protein expression was significantly correlated to SCC (r=-0.6, p=0.03, supplementary figure 1M). Additional studies are required to determine the clinical and diagnostic utility of using CFTR mRNA expression as a potential predictor of therapy effectiveness. Interestingly, the correlation between mRNA expression and VX-770 FIS responses using 0.128 µM forskolin was absent (data not shown). A recent study by Cui et al. 17 demonstrated that high phosphorylation levels of CFTR can obscure potentiator responses, which could explain the absence of this correlation in our study due to this ceiling effect. A caveat in this study is that we have used available genetic information and did not perform additional testing to confirm the poly-T status or determine the TG repeats in the organoid cultures. It is known that the poly-T tract variant influences CFTR splicing 4,5,18 . As assays to determine on which allele the 7T tract is located are not readily available we assumed that 7T was in cis with R117H based on available literature 19,20 . Studies have demonstrated that the 5T variant results in increased exon 10 skipping, i.e. less full-length CFTR transcript, and a more severe disease phenotype than the 7T variant 1,3,21 . Our current study, however, demonstrated variable mRNA expression of R117H/7T-CFTR in subjects with this genotype as well suggesting that the amount of R117H-CFTR mRNA might be predictive of CFTR function in vivo. Our experiments were only performed in organoids expressing R117H-CFTR. It is unclear whether these results can be extrapolated to other genotypes (or R117H in cis