Functional analysis of regulatory variants that determine the outcome of the monogenic disease cystic fibrosis

The clinical outcome of patients with Cystic Fibrosis is highly variable, even among patients who harbour the same CFTR mutation genotype, indicating that environmental and non-CFTR genetic factors such as modifying genes shape the course of the disease. Two contrasting alleles (C or T) of SNP rs7910656 on intron 2 of the FAS gene have been associated with mild and severe CF outcome.

To gain mechanistic insight into how the SNPs of FAS alter the course and the outcome of the disease, we performed in- silico analysis using ENCODE data which showed that rs7910656 is on a nucleosome free region and occupied with RNA POLII and H3k36me3. We have also identified a novel secondary promoter start site which is +4.5kb from rs7910656. Analysis into differential transcription factor occupancy of rs7910656 and controls rs2147420, rs1571019 showed that 29 TFs, mostly of immune regulating family occupied rs7910656 while 6 and 7TFs are on rs2147420and rs1571019 respectively. We also have found that the C-allele of rs7910656 is bound by 6 TFs (NFKB, STAT4, HIF1A, MAFA, NURR1 and TEAD) which are not observed for the T-allele. More so, the C-allele makes multimeric complexes with NFKB (p65, p50, C-Rel), binds with three motifs to STAT4 and binds with one motif to HIF1A, while T-allele does not bind with NFKB and HIF1A but binds with two motifs to STAT4.

We have designed probes for the TFs and have developed a nuclear extraction protocol that preserves the transcription factors in their native states (phosporylated and acetylated) and have carried out an Electrophoretic Mobility Shift Assay with biotinylated probes of p65 subunit of NFKB,C and T-allele of rs7910656. We found the robust binding of p65 subunit and have shown that C-allele binds more robustly than T-allele, which is consistent with our in-silico predictions.