ABCMdb

PMID: 25735457

Ramalho AS, Clarke LA, Sousa M, Felicio V, Barreto C, Lopes C, Amaral MD
Comparative ex vivo, in vitro and in silico analyses of a CFTR splicing mutation: Importance of functional studies to establish disease liability of mutations.
J Cyst Fibros. 2015 Feb 27. pii: S1569-1993(15)00039-9. doi: 10.1016/j.jcf.2015.02.002., [PubMed]

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No. Mutations Sentence Comment

0 ABCC7 p.Ile1234Val Original Article 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 a,1 , Luka A. Clarke a , Marisa Sousa a , Ver&#f3;nica Felicio a , Celeste Barreto b , Carlos Lopes b , Margarida D. Amaral a,Ìe; a University of Lisboa, Faculty of Sciences, BioISI - Biosystems and Integrative Sciences Institute, Lisboa, Portugal b Hospital de Santa Maria, Lisboa, Portugal Received 9 October 2014; revised 9 January 2015; accepted 4 February 2015 Abstract The Cystic Fibrosis p.Ile1234Val missense mutation actually creates a new dual splicing site possibly used either as a new acceptor or donor. Login to comment 3 ABCC7 p.Ile1234Val Our data demonstrate that p.Ile1234Val leads to usage of the alternative splicing donor (but not acceptor) resulting in alternative CFTR transcripts lacking 18 nts of exon 22 which produce a truncated CFTR protein with residual Cl-channel function. Login to comment 31 ABCC7 p.Gly576Ala ABCC7 p.Asp648Val ABCC7 p.Asp565Gly ABCC7 p.Thr665Ser Indeed, it was demonstrated that several CFTR missense mutations also alter splicing, e.g., p.Asp565Gly and p.Gly576Ala [20], p.Asp648Val and p.Thr665Ser [21] as well as p.Gly893Gly (c.2811 G N T) [22]. Login to comment 33 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Herein, we have compared in silico prediction models with in vitro and ex vivo analyses of the CFTR mutation p.Ile1234Val or c.3700A N G (legacy name I1234V) in exon 22 [5-23] to assess the value of these approaches in assessing the consequences of alternative splicing mutations. Login to comment 35 ABCC7 p.Ile1234Val Next, we assessed the RNA level in native tissues from a CF patient with the F508del/p.Ile1234Val genotype and our data evidenced the occurrence of alternative CFTR transcripts, corresponding to usage of the cryptic splice donor that is preferentially used vs the normal one. Login to comment 36 ABCC7 p.Ile1234Val Finally, to elucidate how p.Ile1234Val affects splicing and to functionally characterize the CFTR protein resulting from the alternatively spliced transcript, we performed in vitro studies, using a mini-gene. Login to comment 37 ABCC7 p.Ile1234Val Our data demonstrate that p.Ile1234Val does in fact affect splicing, resulting in the same alternative transcripts and generation of a truncated CFTR protein which is only residually processed and exhibiting reduced Cl-channel function. Login to comment 38 ABCC7 p.Ile1234Val We conclude that the major defect associated with p.Ile1234Val is alternative splicing, but this was not adequately predicted by existing in silico prediction models. Login to comment 42 ABCC7 p.Ile1234Val Patient, genotype nomenclature, nasal brushing and rectal biopsies RNA was collected from nasal and colon epithelial cells of a CF patient compound heterozygous for the [p.Ile1234Val (c.3700A N G)] + [p.Phe508del (c.1521_1523delCTT)] CFTR mutations. Login to comment 49 ABCC7 p.Ile1234Val Transcript analyses The impact of p.Ile1234Val (c.3700A N G) mutation on pre-mRNA splicing was determined by RT-PCR in the region of exons 19-23 using the primers I1R and I1L (see Supplementary Table S1) as described [27]. Login to comment 52 ABCC7 p.Ile1234Val To distinguish between transcripts resulting from both alleles an RT-PCR was done in the exon 22-23 region using the primers F2R(ex22) and I1L(ex23) (see Supplementary Table S1), as before [27] followed by hydrolysis with the restriction enzyme DdeI (New England BioLabs, Ipswich, USA) to distinguish between putative full length transcripts resulting from p.Phe508del and p.Ile1234Val alleles. Login to comment 53 ABCC7 p.Ile1234Val Indeed, only transcripts resulting from the p.Ile1234Val allele (full length or alternatively spliced) possess the DdeI restriction site (C|TNAG) which is absent in p.Phe508del transcripts. Login to comment 54 ABCC7 p.Ile1234Val Thus, the p.Ile1234Val transcripts will be hydrolysed into two fragments of: a) 164 nts and 75 nts (alternatively spliced transcripts, 239 bp); or b) 182 bp and 75 bp (normally spliced, full length transcripts, 257 bp) whereas from the p.Phe508del transcripts (257 nts) will not be hydrolysed by DdeI (see Supplementary Fig. S1). Login to comment 55 ABCC7 p.Ile1234Val Quantitative real time PCR was performed essentially as described [28] using primers designed using Primer3 software (http://frodo.wi.mit.edu/) and specific for the full length (5'-CAGTAAGTCCTGGCCAGAGG-3') and alternatively spliced (5'-CATTTCCTTCTCAGTGGGCCT-3') products of the p.Ile1234Val allele (common reverse primer: 5'-GCTTTCC TCCACTGTTGCAAA-3'). Login to comment 59 ABCC7 p.Ile1234Val In silico analysis of splicing consensus values and ESE prediction The normal and mutated sequences containing the exon 22 and IVS21 and IVS22 boarding regions (~100 nt each) were submitted to six independent splicing signal prediction open source programmes: Spliceview2 [29]; NNSPLICE3 [30], Human Splicing Finder4 [31]; NetGene2 World Wide Web Server5 [32]; Alternative Splice Site Predictor (ASSP) 6 [33]; and Spliceport7 [34], in order to determine the potential of usage for the new acceptor and donor created by the p.Ile1234Val (c.3700A N G) mutation. Login to comment 67 ABCC7 p.Ile1234Val Site-directed mutagenesis was also used to introduce all mutations analysed here into the CFTR-IVS22art mini-gene or pNUT p.Ile1234Val-CFTR cDNA (see primers in Table S3). Login to comment 69 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Stable CFTR-expressing BHK cells Baby hamster kidney (BHK) cells, grown in DMEM F12, with 5% FBS, 1% of PenStrep were transfected with the two pNUT-CFTR-IVS22art mini-gene constructs: wt and the p.Ile1234Val (c.3700A N G) mutant and also all CFTR minigene variants analysed as well as with the p.Ile1234Val- CFTRcDNA pNUT, to produce novel BHK cell lines as before [36]. Login to comment 93 ABCC7 p.Ile1234Val Clinical data from the CF patient A female CF patient with the genotype p.Phe508del (c.1521_1523delCTT)/p.Ile1234Val (c.3700A N G) was the index case for this study. Login to comment 99 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Processing and functional analysis of p.Ile1234Val CFTR mutant Given that the p.Ile1234Val (c.3700A N G) mutation was described [5] as a missense mutation, we first determined how this mutation affected protein processing and function. Login to comment 100 ABCC7 p.Ile1234Val To this end, we produced a BHK stable cell line expressing the p.Ile1234Val-cDNA-CFTR mutant from which we analysed CFTR protein by Western blot (WB). Login to comment 101 ABCC7 p.Ile1234Val Results (Fig. 1A) clearly show that the p.Ile1234Val cDNA-CFTR mutant is fully processed to its mature, fully-glycosylated form (band C), just like wt-cDNA-CFTR. Login to comment 102 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val To assess p.Ile1234Val-cDNA-CFTR Cl-channel activity, cells expressing this CFTR mutant were analysed by the iodide efflux technique (Fig. 1B) and data showed that again p.Ile1234Val-cDNA-CFTR presented Cl-channel function similar to wt-CFTR. Login to comment 113 ABCC7 p.Ile1234Val Patient Age at diagnosis 5 Age (years) 25 Sex F Genotype [p.Phe508del] + [p.Ile1234Val] Sweat test (mEq/L)a 115 FVC % predicted 81% FEV1% predicted 69% Bacteria Sa, Pa Pancreatic status PS Nasal polyposis No FVC, forced vital capacity; FEV1, forced expiratory volume in 1 s; Pa; Pseudomonas aeruginosa. Login to comment 120 ABCC7 p.Ile1234Val Processing and functional analysis of the p.Ile1234Val-CFTR mutant protein. Login to comment 121 ABCC7 p.Ile1234Val 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. Login to comment 123 ABCC7 p.Ile1234Val Lane 1 - wt-CFTR, lane 2 - F508del-CFTR and lane 3 - BHK I1234V-cDNA CFTR. Login to comment 124 ABCC7 p.Ile1234Val B) Results from functional assessment of I1234V-cDNA CFTR by the iodide efflux technique using Forskolin and Genistein as CFTR agonists. Login to comment 127 ABCC7 p.Ile1234Val Analysis of the p.Ile1234Val mutation that corresponds to the 3700A N G alteration at the CFTR nucleotide sequence. Login to comment 130 ABCC7 p.Ile1234Val 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 18 nts 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 231 nts of exon 22 will be produced. Login to comment 136 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val 3.4. Effect of CFTR p.Ile1234Val mutation at the mRNA level of a CF patient with [p.Phe508del] + [p.Ile1234Val] genotype In order to determine whether the splice sites created by the c.3700A N G mutation are used in vivo, CFTR transcripts from native tissues (nasal cells and colonic tissue) of the index CF patient were analysed by RT-PCR in the region of the mutation, i.e., ex19-ex23. Login to comment 144 ABCC7 p.Ile1234Val To distinguish the transcripts resulting from each allele (p.Phe508del and p.Ile1234Val), we next performed RT-PCR in the ex22-ex23 region (see Materials and methods and Fig. S1B and C) followed by hydrolysed with the restriction enzyme DdeI. Login to comment 145 ABCC7 p.Ile1234Val The enzyme has a recognition site in transcripts resulting from the p.Ile1234Val allele but not in F508del transcripts. Login to comment 146 ABCC7 p.Ile1234Val The putative I1234V normal spliced (full-length 257 bp) would originate fragments of 182 bp and 75 bp; and the alternative transcripts (239 bp) would generate fragments of 164 bp and 75 bp. Login to comment 151 ABCC7 p.Ile1234Val Relative quantitative transcript analysis by real-time PCR analysis (Table 3) indicates that the levels of transcripts in patients' cells are present in a ratio of between 32:68 and 48:52 (F508del(full): I1234V(altspliced)), based on data using primer pairs specific for the I1234V splice variant and the F508del mutation, respectively. Login to comment 152 ABCC7 p.Ile1234Val These data suggest that two CFTR transcripts are present: the F508del transcript and the alternatively spliced transcript derived from the p.Ile1234Val allele. Login to comment 153 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val With the F508del specific primer the amounts of both transcripts are similar, but with the primers specific for the I1234V splice variant we seem to obtain more of the splice I1234V derived transcripts. Login to comment 154 ABCC7 p.Ile1234Val However this may be explained by the higher efficiency of PCR amplification for fragments with a smaller length as the case of the I1234V alternative spliced fragment (less 18 nts). Login to comment 155 ABCC7 p.Ile1234Val In the cellular models we could observe 100% of full-length transcripts for the cells expressing the IVS22 mini-gene and almost 100% of alternative splice transcripts for the I1234V IVS22 mini-gene. Login to comment 180 ABCC7 p.Ile1234Val 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. Login to comment 185 ABCC7 p.Ile1234Val The enzyme has a recognition site in transcripts resulting from the p.Ile1234Val allele but not in F508del transcripts. Login to comment 186 ABCC7 p.Ile1234Val The alternative transcripts from the p.Ile1234Val allele (239 nts fragment) will be hydrolysed to two fragments of 164 nts and 75 nts. Login to comment 187 ABCC7 p.Ile1234Val In case full-length transcripts from the p.Ile1234Val allele are produced (257 nts), they will be hydrolysed by DdeI enzyme into two fragments of 182 nts and 75 nts. Login to comment 189 ABCC7 p.Ile1234Val 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. Login to comment 190 ABCC7 p.Ile1234Val Table 3 Relative abundance of CFTR transcripts quantified by real-time PCR in a patient with the F508del/I1234V genotype. Login to comment 191 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val Relative transcript abundance (of 100%) Sample Full length Alternatively spliced F508del/I1234V (NB) - I1234V primer set 32.1% (=non altern. Login to comment 192 ABCC7 p.Ile1234Val ABCC7 p.Ile1234Val spliced) 67.9% F508del/I1234V (NB) - F508del primer set 47.6% 52.4% (=non F508del) IVS22 (BHK) 100% 0% IVS22 + I1234V (BHK) 2.4% 97.6% wt/wt (NB) 100% 0% F508del/F508del (NB)ߤ 100% 0% 7 revert the splicing defect caused by the 3700A N G mutation. Login to comment 197 ABCC7 p.Ile1234Val RNA from BHK cells stably expressing the IVS22art-CFTR mini-gene (wt, mutant (p.Ile1234Val or 3700A N G) and mutant plus "fish revertant" (3704G N A)) was analysed by RT-PCR in the region of ex22 to ex23 (primers F2R and I1L). Login to comment 200 ABCC7 p.Ile1234Val Importantly, data also show that the mutant IVS22art-CFTR mini-gene containing c.3700A N G (p.Ile1234Val) resulted in alternative splicing, yielding only one PCR fragment of smaller size that of wt-IVS22art (Fig. 4A, lanes 3 and 2, respectively). Login to comment 202 ABCC7 p.Ile1234Val This mini-gene was thus able to mimic the in vivo splicing alteration produced by the c.3700A N G (p.Ile1234Val) mutation. Login to comment 208 ABCC7 p.Ile1234Val Characterization of the p.Ile1234Val (c.3700A N G) using the IVS22art CFTR mini-gene. Login to comment 224 ABCC7 p.Ile1234Val This protein is indeed significantly different from the one resulting from the p.Ile1234Val cDNA simply as a missense mutation (Fig. 1B, lane 3). Login to comment 225 ABCC7 p.Ile1234Val ABCC7 p.Ser1235Asn The CFTR protein resulting from the "fish revertant" (c.3704G N A) which reverts the splicing of IVS22, is also correctly processed (Fig. 4B, lane 3), despite the fact that it has two missense mutations (p.Ile1234Val-c.3700A N G and p.Ser1235Asn-c.3704G N A). Login to comment 232 ABCC7 p.Ile1234Val In contrast, the p.Ile1234Val missense protein has normal function when compared to wt-CFTR. Login to comment 242 ABCC7 p.Ile1234Val In summary, the p.Ile1234Val (c.3700A N G) mutation alters CFTR splicing producing alternative transcripts lacking 18 nts, which are then translated into a protein lacking 6 aa that is only partially processed as most does not traffic to the PM, where it has reduced function as Cl-channel. Login to comment 266 ABCC7 p.Ile1234Val We focussed on the c.3700A N G mutation, for which the cDNA based expression models would lead to the classification of the I1234V protein as non-CF causing, since this variant, if solely considered as the cDNA isoleucine to valine amino acid change, is normally processed and functional, as we also show here (Fig. 1). Login to comment 269 ABCC7 p.Ile1234Val In contrast, using our mini-gene strategy that allows the in vitro analysis of the consequences of CFTR mutations both at the mRNA and protein levels, we demonstrate that the p.Ile1234Val/c.3700A N G mutation is in fact a splicing mutation which results in the production of CFTR transcripts lacking 18 nts of exon 22 leading to the production of a truncated protein lacking 6 amino acids at the N-terminal of the CFTR NBD2 domain (Fig. 3, Table 3). Login to comment 297 ABCC7 p.Ile1234Val ABCC7 p.Ile175Val For these mutations we performed an analysis with the NNS software and found that 3 of these sites were recognized as splice sites, but only 2 (the GU created by the I175V and the GU created by I1234V) had higher scores than the normal splice site (see Table S14). Login to comment