Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations

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Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations Anabela S. Ramalho, Luka A. Clarke, Marisa Sousa, Verónica Felicio, Celeste Barreto, Carlos Lopes, Margarida D. Amaral Journal of Cystic Fibrosis Volume 15, Issue 1, Pages 21-33 (January 2016) DOI: 10.1016/j.jcf.2015.02.002 Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Processing and functional analysis of the p.Ile1234Val-CFTR mutant protein. A) BHK cells stably expressing wt, F508del (legacy name) and I1234V (legacy name) CFTR cDNA constructs (without introns) were analysed by WB using the 596 Ab specific to the epitope from aa 1204–1211 in the NBD2 domain of CFTR. Equal amounts of total protein lysate (30μg) were loaded into each lane. Lane 1 — wt-CFTR, lane 2 — F508del-CFTR and lane 3 — BHK I1234V-cDNA CFTR. B) Results from functional assessment of I1234V-cDNA CFTR by the iodide efflux technique using Forskolin and Genistein as CFTR agonists. The line at the top of the graph corresponds to the period (4min) of activation of CFTR with the agonists (see Materials and methods). Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Analysis of the p.Ile1234Val mutation that corresponds to the 3700A>G alteration at the CFTR nucleotide sequence. A) The alteration of A to G at position 3700 showed in grey results in the creation of a cryptic acceptor splice site (AG) indicated at top left and of a cryptic donor splice site (GT) indicated at lower right. B) Scheme of splicing possibilities that may occur when 3700A>G is present in the pre-mRNA. From top to bottom: i) full-length transcripts with the p.Ile1234Val missense mutation will be obtained if wt splice sites are used instead of the cryptic ones; ii) if the cryptic donor is used instead of the normal IVS22 donor, an aberrant transcript lacking the last 18nts of exon 22 will be produced; iii) if the cryptic acceptor is used instead of the normal IVS21 acceptor, an alternative transcript lacking the first 231nts of exon 22 will be produced. Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 Effect of the c.3700A>G mutation on CFTR transcripts in vivo. A) RT-PCR analysis of CFTR transcripts extracted from nasal brushing cells (NB) and colonic tissues from rectal biopsies (RB) from index CF patient (with the [p.Ile1234Val]+[p.Phe508del] genotype) and carriers using I1L (exon 23) and I1R (exon 19) primer. Lane 1 — DNA ladder 100bp from Invitrogen, lane 2 — RNA from nasal cells of the index patient, lane 3 — RNA from nasal cells of one p.Phe508del-carrier, lane 4 — RNA from nasal cells of a healthy individual, lane 5 — RNA from colonic tissue of the index patient, lane 6 — RNA from one p.Phe508del-homozygous patient and lane 7 — Negative control. B) Partial sequence obtained by sequencing of the lower band (after isolation and purification) observed in lane 3 of Fig. 3A agarose gel. This fragment lacks the last 18nts of exon 22 as expected if new donor splice site in exon 22 was used. C) Hydrolysis analysis with DdeI restriction enzyme of index patient transcripts amplified by RT-PCR with F2R (exon 22) and I1L (exon 23) primers. The enzyme has a recognition site in transcripts resulting from the p.Ile1234Val allele but not in F508del transcripts. The alternative transcripts from the p.Ile1234Val allele (239nts fragment) will be hydrolysed to two fragments of 164nts and 75nts. In case full-length transcripts from the p.Ile1234Val allele are produced (257nts), they will be hydrolysed by DdeI enzyme into two fragments of 182nts and 75nts. The F508del full-length transcripts (257nts) will not be hydrolysed by this enzyme. Lane 1 — DNA ladder — hyperladder V from Bioline, lane 2 — Products resulting from hydrolysis with DdeI restriction enzyme of the PCR products shown in lane 3, Lane 3 — RT-PCR products of transcripts extracted from nasal of the [p.Ile1234Val]+[p.Phe508del] patient with F2R and I1L primers. Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 4 Characterization of the p.Ile1234Val (c.3700A>G) using the IVS22art CFTR mini-gene. A) RT-PCR analysis of IVS22art splicing in wt and 3700A>G mutant CFTR, with the forward primer in exon 22 (F2R) and the reverse in exon 23 (I1L). Lane 1 — mRNA from BHK cells expressing the pNUT CFTR wt, lane 2 — mRNA from BHK cells expressing the CFTR IVS22art mini-gene, lane 3 — mRNA from BHK cells expressing the 3700A>G-CFTR IVS22art mini-gene, lane 4 — mRNA from BHK cells expressing the 3700A>G+3704G>A IVS22art mini-gene, lane 5 — negative control (CN) and lane 6 — 100bp gene ruler DNA ladder from Thermo Scientific. B) Immunoblot analysis of the protein produced by BHK cells transfected with the pNUT CFTR IVS22art mini-genes. The anti-CFTR antibody used (M3A7) recognized an epitope at the C-terminus of the protein. Lane 1 — total protein lysates from BHK cells expressing the pNUT CFTR IVS22art mini-gene, lane 3 — total protein lysates from BHK cells expressing the pNUT 3700A>G-CFTR IVS22art mini-gene, lane 4 — total protein lysates from BHK cells expressing the pNUT 3700A>G+3704G>A, CFTR IVS22art mini-gene. C) Functional assay by iodide efflux assay of the CFTR protein resulting from mini-genes expressed (as indicated in the figure) in stably transfected BHK cells. Cells were stimulated with forskolin (10μM) and genistein (50μM) in the period indicated by the black solid line above graph of time-course iodide efflux measurements. Data are means±SEM (n=4–5) at each point. Where error bars are not visible, the symbol has obscured them. D) Graph summarizing data of I− efflux peak magnitude generated by the different CFTR-mutants mini-gene expressed as a percentage of wt-CFTR activity. Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 5 Immunocytochemistry analysis of the CFTR protein resulting from CFTR mini-genes expressed in BHK cells. CFTR labelling is observed in the top panels, labelling with the membrane marker wheat germ agglutinin on the bottom panels. See this picture in colors and the merged images at online supplementary material (Fig. S4). Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 6 Effect of IVS22 donor strength in splicing. Here we interchange the sequences of the normal donor in IVS22 with the cryptic donor in ex22 to determine their influence on the splicing of this intron. Two positions in the normal IVS22 donor (GUGAGA — in light grey is the nucleotide +3 that will be changed to A and in dark grey is the nucleotide +6 that will be changed to U) were mutated to obtained the sequence of the new donor site (GUAAGU) and vice versa. The normal donor +3 position corresponds to the 3702 position in the new donor and the +6 corresponds to the 3705 position. All the mutants are in the 3700A>G background and the RNA analysed was extracted from BHK expressing the different CFTR mini-gene variants. Lanes 1–4 correspond to the alterations in the cryptic donor and lanes 5–7 correspond to alterations in the normal donor. Lane 1 — mRNA from 3700A>G plus 3702A>G CFTR IVS22art mini-gene, lane 2 — mRNA from 3700A>G plus 3702A>G plus 3705U>A CFTR IVS22art mini-gene, lane 3 — from 3700A>G CFTR IVS22art mini-gene, lane 4 — from 3700A>G plus 3705U>A CFTR IVS22art mini-gene, lane 5 — mRNA from 3700A>G CFTR IVS22art +3G>A mini-gene, lane 6 — mRNA from 3700A>G CFTR IVS22art +6A>U mini-gene, lane 7 — mRNA from 3700A>G CFTR IVS22art +3G>A and +6A>U mini-gene, lane 8 — negative control (CN) and lane 9 — 1Kb DNA ladder from Invitrogen. Journal of Cystic Fibrosis 2016 15, 21-33DOI: (10.1016/j.jcf.2015.02.002) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions