Peter van Mourik

22 Chapter 2 Statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, La Jolla California USA). Correlations were determined by Pearson correlation coefficient (Pearson’s r) and linear regression analysis. P-values were reported based on two-sided tests. RESULTS Fourteen R117H-CFTR (13 with known 7T-9T poly-T tracts and one with unknown poly-T status, of which nine compound heterozygous for F508del-CFTR), one class I (c.1679+1G>C/c.1679+1G>C), one class III (p.Phe508del/p.Ser1251Asn) and one wild type CFTR organoid culture were included in this study (Supplementary table 1). First, we assessed (residual) CFTR function using the FIS assay. Upon 0.05 μM and 0.128 μM forskolin treatment variability in FIS was observed between R117H-CFTR organoids cultures (AUC range: -88 – 586 AUC and 193 – 1452 AUC, respectively, Figure 1A and 1B). VX-770 treatment of the R117H-CFTR organoids resulted in increased swelling of all organoids compared to their non-treated counterparts with a large variability in VX-770 response (AUC range: 252 – 1922 and 602-2524 at 0.05 μM and 0.128 μM forskolin treatment, respectively, Figure 1A and 1C). Next, we determined whether differences in residual CFTR function and response to VX-770 correlated with CFTR protein expression by measuring glycosylated CFTR (C-band) expression. CFTR expression was corrected for sample loading using endogenous heat shock protein 90 (HSP90) and normalized to wild-type CFTR protein expression. As can be seen in Figures 2A and 2B, C-band protein expression was highly variable between R117H-CFTR organoid cultures (relative expression range: 0.23 – 2.00). We performed gene expression studies to quantify the allele-specific contribution of R117H-CFTR to the observed variability in CFTR function and response-to- therapy. R117H-CFTR mRNA expression results were normalized using ACTB and YWHAZ housekeeping genes and converted into relative quantities using the R117H-homozygous organoid culture as wild type organoids do not possess the R117H-CFTR allele. As expected, R117H-CFTR mRNA expression was highest in the R117H-homozygous organoid culture which possesses two alleles that contribute to the mRNA expression (Figure 2C). Clear variability in mRNA expression between organoid cultures with a single R117H-CFTR allele (relative expression range: 0.18 – 0.49) was detected. R117H-CFTR mRNA expression and CFTR C-band protein expression were strongly correlated (r=0.92, p<0.0001, Figure 2D). To investigate