The CF-modifying gene EHF promotes p.Phe508del-CFTR residual function by altering protein glycosylation and trafficking in epithelial cells

Abstract

The three-base-pair deletion c.1521_1523delCTT (p.Phe508del, F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent disease-causing lesion in cystic fibrosis (CF). The CFTR gene encodes a chloride and bicarbonate channel at the apical membrane of epithelial cells. Altered ion transport of CFTR-expressing epithelia can be used to differentiate manifestations of the so-called CF basic defect. Recently, an 11p13 region has been described as a CF modifier by the North American CF Genetic Modifier Study Consortium. Selecting the epithelial-specific transcription factor EHF (ets homologous factor) as the likely candidate gene on 11p13, we have genotyped two intragenic microsatellites in EHF to replicate the 11p13 finding in the patient cohort of the European CF Twin and Sibling Study. We could observe an association of rare EHF haplotypes among homozygotes for c.1521_1523delCTT in CFTR, which exhibit a CF-untypical manifestation of the CF basic defect such as CFTR-mediated residual chloride secretion and low response to amiloride. We have reviewed transcriptome data obtained from intestinal epithelial samples of homozygotes for c.1521_1523delCTT in CFTR, which were stratified for their EHF genetic background. Transcripts that were upregulated among homozygotes for c.1521_1523delCTT in CFTR, who carry two rare EHF alleles, were enriched for genes that alter protein glycosylation and trafficking, both mechanisms being pivotal for the effective targeting of fully functional p.Phe508del-CFTR to the apical membrane of epithelial cells. We conclude that EHF modifies the CF phenotype by altering capabilities of the epithelial cell to correctly process the folding and trafficking of mutant p.Phe508del-CFTR.

Figure 1

Allele distribution at the EHF locus. Markers EHFSat1 and EHFSat2 were genotyped on 101 CF families with a total of 171 patients who are homozygous for c.1521_1523delCTT in CFTR. Alleles at EHFSat1 and EHFSat2 were scored using an invariant set of control samples in arbitrary repeat units of the repetitive (TG)n-repeat motif. EHFsat1-EHFsat2 haplotypes of CF patients were reconstructed using FAMHAP. (a) Map of EHF, retrieved from http://www.ncbi.nlm.nih.gov/. EHFSat1 corresponds to the polymorphic sequence starting at position 6071; primers used for amplification: 5′-TGTTGGGTCAGAGTGAATGG-3′ and 5′-ATCTCCCTGCTACCCACCTT-3′. EHFSat2 corresponds to the polymorphic sequence starting at position 24 984; primers used for amplification: 5′-GGCAGTGGGATATCAGTCCA-3′ and 5′-GCTTATTGTCCATACCCAAATCG-3′; (b–d) Allele distribution for EHFSat1 (b), EHFSat2 (c) and marker combination EHFSat1-EHFSat2 (d) of the 171 homozygotes for c.1521_1523delCTT in CFTR from 101 CF families. (e and f) Allele distribution for marker combination EHFSat1-EHFSat2 among patient subsets stratified for response to amiloride in nasal potential difference measurement ((e): case population: 13 unrelated homozygotes for c.1521_1523delCTT in CFTR, which display a response of 27 mV or less to amiloride in NPD; reference population: 17 homozygotes for c.1521_1523delCTT in CFTR, which display a response of 28 mV or more to amiloride in NPD) and manifestation of DIDS-insensitive residual chloride secretion in intestinal current measurement ((f): case population: 9 unrelated homozygotes for c.1521_1523delCTT in CFTR, which display DIDS-insensitive residual chloride secretion in ICM; reference population: 14 unrelated homozygotes for c.1521_1523delCTT in CFTR, which do not display any residual chloride secretion in ICM).

Figure 2

EHF-dependent gene regulation in relation to p.Phe508del-CFTR biosynthesis, trafficking and post-translational modification. Mature, fully glycosylated and functional p.Phe508del-CFTR—emphasized as a green line at the apical membrane (AM) – reaches the apical membrane through trafficking pathways^, illustrated as green arrows (a). These encompass biosynthesis and insertion into the lipid bilayer (IA), utilization of the ER-associated folding pathway (ERAF), passage through the ER, ERGIC (IB) and Golgi-compartments, post-translational modifications (PTMs) and finally, transport of mature p.Phe508del-CFTR to subapically localized vesicles (IC) and to the AM (ID). The alternative CFTR maturation pathway that bypasses the ERGIC and Golgi compartments is shown (II). In contrast, pathways that lead to degradation of p.Phe508del-CFTR^, are depicted in orange. These encompass the ER-associated degradation pathway ER-associated degradation (ERAD) that leads to degradation in the proteasome (PR) and the retrograde traffic of endosomes from the subapical compartment (III) toward the lysosome (LY). EHF-dependent differentially regulated genes whose products have been annotated to partake in any of these pathways crucial to p.Phe508del-CFTR biosynthesis, maturation and trafficking are shown in b–j. Gene products for which the subcellular localization cannot be specified are listed in j. Forty trafficking and maturation genes whose expression are upregulated among the nine p.Phe508del-CFTR homozygous carriers of at least one frequent EHF allele are shown in orange. Fifty-eight trafficking and maturation genes whose expression are upregulated among the seven p.Phe508del-CFTR homozygous carriers of two rare EHF alleles are shown in green.