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Human Molecular Genetics, 2003, Vol. 12, No. 16 2031-2040
DOI: 10.1093/hmg/ddg215
© 2003 Oxford University Press

Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene

Isabel Aznarez1,2, Elayne M. Chan1,2, Julian Zielenski1, Benjamin J. Blencowe2,3 and Lap-Chee Tsui1,2,4,*

1Genetics and Genomics Biology Program, The Hospital for Sick Children, Toronto, Canada, M5G 1X8, 2Department of Molecular and Medical Genetics, University of Toronto, Canada, M5S 1A8, 3Banting and Best Department of Medical Research, CH Best Institute, University of Toronto, Canada, M5G 1L6 and 4The University of Hong Kong, Pokfulam Road, Hong Kong

Received April 18, 2003; Accepted June 19, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2{alpha} and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. In its classic form CF is characterized by elevated concentration of sweat electrolytes, exocrine pancreatic insufficiency and obstructive lung disease (1). In addition, most male CF patients suffer from infertility due to bilateral absence of the vas deferens (2). There is considerable heterogeneity in disease severity and progression (3,4). The common molecular defect in CF is, however, due to mutations in the cystic fibrosis transmembrane-conductance regulator (CFTR) gene (5–7). The gene contains 27 exons, spanning ~200 kb of genomic DNA on human chromosome 7 (8). To date, there are more than 1100 putative mutations reported in the CFTR gene and 708 of them affect single nucleotides in the protein-coding region (http://www.genet.sickkids.on.ca/cftr/). These single nucleotide changes include missense and nonsense mutations, and single nucleotide variants. There are also mutations that affect the splicing of the CFTR gene either by directly disrupting splice sites or by creating new ones. Varied levels of alternative splicing have been detected for some of the splicing mutations (9–11). These differences are most likely due to varied utilization of intragenic splicing elements by transacting splicing factors (12,13).

Cis-acting elements required for splicing in mammalian cells include short and poorly conserved consensus sequences, which specify the 5' splice site, branch site and the 3' splice site. The 3' splice site is preceded by a polypyrimidine tract of variable length. The branch point is typically located 18–40 nucleotides upstream of the polypyrimidine tract (14). These splicing signals are essential but do not contain sufficient information to specify correct splicing patterns and they alone cannot be distinguished from numerous surrounding cryptic splice sites of similar strength (15). Their efficient recognition, as well as their regulated usage, requires sequences within exons and introns called enhancers and silencers (15). Exonic splicing enhancers (ESEs) are present in most, if not all, mammalian exons and are important for promoting selection of correct, adjacent splice sites (15). Pre-mRNA splicing requires the interaction of these critical cis-acting sequences with factors that form the spliceosome. The spliceosome is a highly complex RNA/protein machine consisting of five small nuclear (sn)RNAs and between 50 and 100 core proteins (16). A large set of protein splicing factors contains domains rich in alternating arginine (R) and serine (S) residues (RS domains), which function to promote constitutive and regulated splicing. These proteins include members of the ‘SR family’, which share a related domain organization consisting of one or two N-terminal RNA recognition motifs and a C-terminal RS domain. Other RS motif proteins have a different domain organization and are generally referred to as ‘SR-related’ proteins (16). Changes in the level of SR and SR-related proteins have recently been proposed to modify the effect of splicing mutations (17).

Our interest in studying splicing of the CFTR pre-mRNA originated from our inability to identify mutation(s) in a subset of CF patients, following mutation screening in the 5'UTR, coding and flanking intronic sequences of the CFTR gene (18). Therefore, to enhance our chances of identifying molecular defects that could affect CFTR pre-mRNA splicing, in the present study we have performed transcript analysis on a selected group of CF patients with at least one unidentified CFTR mutation. This search resulted in the identification of a number of aberrantly spliced CFTR transcript species for which no corresponding genomic DNA sequence alteration could be identified at or around the alternative splice sites utilized. We further analyzed one such aberrantly spliced form that resulted from the selection of a cryptic 3' splice site in exon 13, located 248 nucleotides downstream of the native 3' splice site. A putative ESE located between this cryptic and the correct exon 13 3' splice site was identified and, using a mingene reporter system, was shown to be important for selection of the correct 3' splice site. Two CFTR mutations were identified that disrupt this putative ESE sequence. Five other CF mutations located near exon 13 ESE sequence were analyzed, four of which were found to also disrupt the correct splicing of exon 13. Moreover, increased expression of two splicing factors was found to modify the extent of the disruptive effect of some of the exon 13 mutations. Our studies thus provide a characterization of the effects of CF-disease-associated mutations on splicing of CFTR exon 13 and suggest that alterations in the levels of certain splicing factors have the potential to play a role in determining the severity of the phenotypic consequences of these mutations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcript analysis of CF patients
To perform our CFTR transcript analysis, we exploited EBV-transformed lymphoblastoid cell lines from 22 CF patients with at least one unknown mutation. We previously showed that the splicing patterns in lymphoblastoid cell lines were similar to those observed in nasal epithelia arguing that these cell lines are a suitable source of RNA (19). Accordingly, total RNA was extracted and seven overlapping fragments spanning the CFTR full-length transcript were analyzed by RT–PCR. Using this approach we have identified and characterized 11 alternatively spliced forms of CFTR mRNA in 14 CF patients. Five of these forms could be explained by genomic mutations. The remaining six alternative forms consisted of various aberrant splicing events including insertion of intronic sequences and partial or complete exon skipping (Table 1). The level of aberrant CFTR mRNA species ranged between 10 and 40% of the total transcript (unpublished data). Moreover, no underlying causative intragenic alterations could be attributed for these aberrant transcripts, however, the cryptic splice sites utilized in these aberrant splicing events appeared to conform to the consensus sequences at splice junctions (20). These results thus raised the possibility that these aberrant transcript events could be caused by differences in the activity of splicing factors such as SR proteins.


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  		Table 1. Aberrant CFTR mRNA transcripts with no apparent genomic alterations
 
The most common species of aberrant spliced CFTR transcript found in our study population (four out of 22 patients) was apparently due to the skipping of the first 248 nucleotides of exon 13 (21). The skipping of the first 248 nucleotides of exon 13 was previously identified by Hull et al. (21) in nasal epithelial cells suggesting that it is not lymphoblastoid cell line specific. We named this species {Delta}248. The partial skipping resulted from the selection of a cryptic 3' splice site located 248 nucleotides downstream of the native 3' splice site. The skipping causes a frameshift that would lead to a truncated polypeptide due to the introduction of a stop codon 50 nucleotides downstream of the new junction (21). Visual inspection of the sequence of exon 13 pinpointed a purine-rich sequence, (GAA(A))3 repeat, located between the native and the cryptic 3' splice site. This sequence showed an almost perfect match to the consensus of previously reported purine rich ESEs (22–24). Should this purine-rich sequence function as an ESE, a subtle change in the levels of the corresponding splicing factors could cause the {Delta}248 transcript.

Minigene analysis of an exonic splicing enhancer targeted by two disease-associated mutations in exon 13
To determine whether the purine-rich sequence functions as an ESE involved in the regulation of exon 13 splicing, two minigenes were constructed. The minigene constructs spanned exon 12, 13 and 14a of the CFTR gene. Intronic sequences located between CFTR exons 12 and 13, and between 13 and 14a, were deleted such that ~100–350 nt of flanking sequences remained (Fig. 1). A plasmid containing the IVS1 of the ß-globin gene (25) was used to clone the CFTR sequences such that strong donor and acceptor splice sites of IVS1 were located 5' of exon 12 and 3' of exon 14a sequences, respectively, to ensure efficient inclusion of these exons (Fig. 1). The minigenes were driven either by the SV40 early or the CMV promoter allowing us to determine whether altered splicing patterns could arise as a consequence of a promoter-specific effect (26). The constructs contained SV40 early polyadenylation signals. One of the constructs contains an intact ESE sequence (WT) while in the other (MT), several adenines in the sequence were simultaneously substituted with thymidines (GAAAGAAGAAA to GATTGTTGTTA) (Fig. 2A). It has been previously shown that substitution of adenines to uracils in GAA-repeat ESEs eliminates their enhancing ability (22,24). Therefore, should the purine rich sequence in exon 13 function as an ESE, the MT derivative would be expected to lead to a transcript lacking the first 248 nucleotide of exon 13 (Fig. 2B).



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  		Figure 1. Schematic representation of the CFTR gene derived minigene construct. Sequences from IVS12 and IVS13 were deleted to make the reporter of a suit-able size. The black boxes represent IVS1 ß-globin 5' and 3' splice sites, respectively. The arrow with CMV or SV40 early written inside denotes the different promoters used in the constructs. The parental constructs contain the SV40 early polyadenylation signal (25). The diagram is not to scale.

 


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  		Figure 2. RT–PCR analysis of mutations affecting an ESE in exon 13. (A) Partial exon 13 sequence (nt 2074–nt 2147). The 195 and 248 cryptic 3' splice sites are boxed. The putative polypyrimidine tracts for the 195- and 248-cryptic splice sites are underlined. The putative purine-rich ESE sequence is in bold. Nucleotide changes simultaneously introduced in a minigene reporter system are boxed. CF mutations are shown in their original nomenclature. (B) Schematic representation of the expected RT–PCR products from a minigene with an intact exon 13 ESE sequence (gray box) or a mutated exon 13 ESE sequence (black box). The expected size of the full-length transcript generated from a minigene carrying the wild-type sequence of exon 13 is 887 bp while the expected size of the transcript generated from a minigene carrying a mutated exon 13 ESE sequence is 639 bp. (C) RT–PCR analysis of COS-7 cell lines transfected with minigenes driven by the CMV promoter carrying the wild-type sequence of exon 13, WT, and the highly disrupted ESE sequence, MT, minigenes separated in a 2% agarose gel. (D) RT–PCR analysis of minigenes carrying the wild-type sequence, WT, and E656X and 2108delA mutations separated in a 2% agarose gel. (E) Densitometric analysis of the resulting PCR bands was carried out using FluorChem program. The ratio of the density of the band corresponding to the {Delta}248 transcript of each minigene over the density of the band corresponding to the wild-type transcript (wt) of each minigene was calculated. The ratio for each mutant minigene was then normalized to the WT minigene control included in each transfection experiment. The bars correspond to the means and standard error calculated for the normalized ratios from three independent transfection experiments. The splicing pattern for the WT minigene also contains bands corresponding to the {Delta}195 and {Delta}248 transcripts probably due to the usage of an exogenous promoter to CFTR gene. wt=wild-type transcript; het=heteroduplexes generated from PCR amplification; {Delta}195=transcript lacking the first 195 nucleotides of exon 13; {Delta}248=transcript lacking the first 248 nucleotides of exon 13. Similar results were obtained for the same minigenes driven by the SV40 promoter and in transfection experiments using IB3 cells.

 
Both minigenes were transfected into COS-7 and IB3 (a human bronchial epithelial derived cell line) cells. Comparing transfection experiments in two different cell types would allow us to determine the tissue-specificity of the splicing patterns. Moreover, IB3 cells, derived from an organ tissue involved in CF pathology, were most relevant in our study. For each analysis, bands corresponding to different spliced products were sequenced to establish the precise location of splice site selection. The intensity of the bands was quantified using the FluorChem program. The results of three independent transfection experiments showed that the WT minigene generated a transcript in which exon 13 was efficiently and accurately included (Fig. 2C, lane 1). Conversely, the MT minigene produced, almost exclusively, a band corresponding to the transcript lacking the first 248 nucleotides of exon 13 ({Delta}248) as expected (Fig. 2C, lane 2). Quantification of resulting products from three independent transfection experiments indicated an increase in the ratio of the {Delta}248 transcript to the wt transcript of 54-fold (Fig. 2E). These results demonstrated that the purine-rich sequence located between the native and the 248 cryptic 3' splice sites could function as an ESE to promote the accurate selection of the exon 13 3' splice site.

It was noted that two exon 13 mutations, E656X (http://www.genet.sickkids.on.ca/cftr/) and 2108delA (27), were located within the putative ESE in exon 13 (Fig. 2A). The E656X mutation corresponded to a G to T substitution at nucleotide 2098; affecting the first nucleotide of the stretch of purine-rich ESE sequence, whereas 2108delA shortened the ESE sequence by one nucleotide. RT–PCR analyses of the minigene reporter transcripts containing these mutations showed an increase in the {Delta}248 transcript when compared to the WT minigene (Fig. 2D). Quantification of resulting products from three independent transfection experiments indicated that they increased the ratio of the {Delta}248 transcript to the wt transcript by 4- and 3-fold, respectively (Fig. 2E). The decreased utilization of the correct 3' splice site caused by both of these mutations provided strong additional evidence that the putative ESE sequence functions to promote correct splicing of exon 13. These findings also appeared to be consistent with previous observations that the number of consecutive GAA(A) repeats could determine the strength of an ESE (28).

Exonic mutations causing aberrant splicing of CFTR exon 13
It has been previously shown that nucleotides in the proximity of an ESE could also be involved in the regulation of splicing (29,30). For this reason we attempted to determine whether additional nucleotides near the ESE sequence were also involved in the regulation of CFTR exon 13 splicing. Minigenes carrying five additional CF mutations were constructed. The D648V (2075A->T) (31), D651N (2083G->A) (32) and G654S (2092A->G) (http://www.genet.sickkids.on.ca/cftr/) mutations are located upstream and the E664X (2122 G->T) (33) and T655S (2125A->T) (http://www.genet.sickkids.on.ca/cftr/) mutations are located downstream of the ESE sequence (Fig. 3A).



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  		Figure 3. CFTR exonic mutations affect the splicing of exon 13. (A) Partial exon 13 sequence (nt 2074–nt 2147). The 195- and 248-cryptic splice sites are boxed. The putative polypyrimidine tracts for the 195- and 248-cryptic splice sites are underlined. The putative purine-rich ESE sequence is in bold. Nucleotide changes simultaneously introduced in a minigene reporter system are boxed. CF mutations are shown in their original nomenclature. (B) RT–PCR analysis of COS-7 cell lines transfected with minigenes driven by the CMV promoter carrying the wild-type exon 13 sequence, WT, and 2074G->T, D648V, 2076T->A, D651N and G654S mutations separated in a 2% agarose gel. (C) RT–PCR analysis of COS-7 cell lines transfected with minigenes driven by the CMV promoter carrying the wild-type exon 13 sequence, WT, and E664X, 2123A->T, 2124G->T, T665S, 2123AGA->TTT, 2123A->G/2125A->G and 2126C->A mutations separated in a 2% agarose gel. (D) Bar graph showing the increase of the {Delta}195 transcript. Densitometric analysis of the resulting PCR bands was carried out using FluorChem program. The ratio of the density of the band corresponding to the {Delta}195 transcript of each minigene over the density of the band corresponding to the wild-type transcript (wt) of each minigene was calculated. The ratio for each mutant minigene was then normalized to the WT minigene control included in each transfection experiment. The bars correspond to the means and standard error calculated for the normalized ratios from three independent transfection experiments. (E) Bar graph showing the increase of the {Delta}248 transcript. The bars correspond to the means and standard error calculated as described above from three independent transfection experiments. The splicing pattern for the WT minigene also contains bands corresponding to the {Delta}195 and {Delta}248 transcripts probably due to the usage of an exogenous promoter to CFTR gene. wt=wild-type transcript; het=heteroduplexes generated from PCR amplification; {Delta}195=transcript lacking the first 195 nucleotides of exon 13; {Delta}248=transcript lacking the first 248 nucleotides of exon 13. Similar results were obtained for the same minigenes driven by the SV40 promoter and in transfection experiments using IB3 cells.

 
Transfection of the minigene carrying D648V into COS-7 and IB3 cells showed that this mutation could cause aberrant exon 13 splicing (Fig. 3B, lane 3). Direct sequencing analysis of the aberrant band revealed that it resulted from the selection of a cryptic 3' splice site located 195 nucleotides downstream of the native 3' splice site. The skipping of the first 195 nucleotides of exon 13 ({Delta}195) would cause a deletion of 63 amino acids in the regulatory domain of the CFTR protein (34). Quantification of the resulting product showed an increase in the ratio of the {Delta}195 transcript over the wt transcript of 94-fold (Fig. 3D). The A to T substitution in the D648V mutation, located 18 nucleotides upstream of the 195 cryptic 3' splice junction, would lengthen the corresponding polypyrimidine tract, thereby improving its consensus sequence (Fig. 3A). To assess this observation, we constructed two minigenes carrying single nucleotide substitutions immediately upstream (2074G->T) and downstream (2076T->A) of the mutation (Fig. 3A). RT–PCR analyses of the transcripts resulting from transfection of these minigenes showed that the 2074G->T substitution had a similar effect to D648V (Fig. 3B, lane 2 and D), while the 2076T->A substitution abolished selection of the 195-cryptic 3' splice site (Fig. 3B, lane 4). These results therefore strongly supported the conclusion that the D648V mutation could cause aberrant exon 13 splicing by improving the polypyrimidine tract of the suboptimal 195-cryptic 3' splice site.

The minigene carrying the D651N mutation yielded two species of aberrantly spliced products. Firstly, the G to A substitution responsible for D651N, located 10 nt upstream of the 195-cryptic 3' splice site (Fig. 3A), apparently enhanced the usage of this site (Fig. 3B, lane 5) increasing the ratio of the {Delta}195 over the wt transcript by 3-fold (Fig. 3D). Secondly, the mutation also caused an increase in the selection of the 248-cryptic 3' splice site (Fig. 3B, lane 5) by 2-fold (Fig. 3E). There was, however, no clear mechanism underlying the latter effect caused by this mutation. In the case of the G654S mutation, which is caused by an A to G substitution at the 195-cryptic 3' splice site (Fig. 3A), only the wild-type splicing pattern could be detected as expected (Fig. 3B, lane 6).

Both the E664X and T665S mutations were found to cause an increase in the {Delta}248 transcript (Fig. 3C, lanes 2 and 5, respectively) by 2- and 5-fold, respectively (Fig. 3E). These results were also anticipated since these two mutations, a G to T (24 nt upstream of the 248-cryptic splice site) and an A to T (21 nt upstream of the 248-cryptic splice site) substitution, respectively, lengthened the polypyrimidine tract of the 248-cryptic 3' splice site (Fig. 3A). To support this interpretation, various minigenes carrying point and multiple nucleotide substitutions in the putative polypyrimidine tract were constructed (Fig. 3A). The results of these transfection experiments (Figs. 3C and E) added further support to the conclusion that E664X and T665S could cause skipping of the first 248 nucleotides of exon 13 by improving the polypyrimidine tract of the 248-cryptic 3' splice site.

Role of trans-acting splicing factors in exon 13 splicing
It has been previously suggested that variation in the relative levels of splicing factors could modify the effect of splicing mutations (17). Since the splicing factors hTra2{alpha} (24) and SF2/ASF (35,36) have both been shown to bind specifically purine-rich ESE sequences, they appeared to be good candidates for trans-acting factors that could affect the splicing of CFTR exon 13 (22,24,37,38). To explore this possibility, we conducted co-transfection experiments in COS7 and IB3 cell lines with constructs expressing hTra2{alpha} (24) and SF2/ASF (35,36). As shown in Figure 4A, co-transfection with the hTra2{alpha}-expressing construct could partially suppress the defective splicing caused by the E656X and 2108delA mutations (compare lanes 1 to 2 and 3 to 4, respectively). The results from three independent co-transfection experiments showed that increased expression of hTra2{alpha} resulted in the preferential usage of the correct exon 13 3' splice site over the 248-cryptic 3' splice site by 7- and 9-fold, respectively (Fig. 4C). In contrast, increased expression of hTra2{alpha} had no effect on the altered splicing of the minigene reporter transcripts containing the MT or D648V mutations (Fig. 4A and C). It appeared therefore that the nucleotide changes in the MT minigene which abolished the (GAA(A))3 repeat ESE sequence (Fig. 2A), eliminated the ability of hTra2{alpha} to bind to this region of exon 13. The D648V mutation targeted the 195-cryptic polypyrimidine tract (Fig. 3A) which is not known to function as a binding site for hTra2{alpha}, thus was not affected by over-expression of this trans-acting factor. Taken together, our data appeared to be consistent with hTra2{alpha} functioning as a splicing factor shown to bind (GAA)n ESEs such as the putative purine rich ESE sequence we have defined in CFTR exon 13.



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  		Figure 4. Splicing factors modulate the splicing phenotype of exonic splicing mutations. (A) RT–PCR analysis of COS-7 cell lines co-transfected with minigenes driven by the CMV promoter and hTra2{alpha}. Minigenes carrying E656X, 2108delA, MT and D648V mutations were transfected alone (noted by the minus sign) or co-transfected with hTra2{alpha} (noted by the plus sign). The resulting RT–PCR products were separated in a 2% agarose gel. (B) Bar graph showing the increase of the wild-type (wt) transcript. Densitometric analysis of the resulting PCR bands was carried out using FluorChem program. The ratio of the density of the band corresponding to the wild-type transcript (wt) of each minigene over the density of the band corresponding to the {Delta}195 (for D648V mutation) or {Delta}248 transcript of each minigene was calculated. The ratio for each minigene co-transfected with hTra2{alpha} was then normalized to the ratio of the corresponding minigene transfected alone. The bars correspond to the means and standard error calculated for the normalized ratios from three independent cotransfection experiments. (C) RT–PCR analysis of COS-7 cell lines co-transfected with minigenes driven by the CMV promoter and SF2/ASF. Minigenes carrying D651N, E664X and T665S mutations were transfected alone (noted by the minus sign) or co-transfected with SF2/ASF (noted by the plus sign). (D) Bar graph showing the increase of the {Delta}248 transcript. Densitometric analysis of the resulting PCR bands was carried out using FluorChem program. The ratio of the density of the band corresponding to the {Delta}248 transcript of each minigene over the density of the band corresponding to the wild-type transcript (wt) of each minigene was calculated. The ratio for each minigene cotransfected with SF2/ASF was then normalized to the ratio of the corresponding minigene transfected alone. The bars correspond to the means and standard error calculated for the normalized ratios from three independent co-transfection experiments. wt=wild-type transcript; het=heteroduplexes generated from PCR amplification; {Delta}195=transcript lacking the first 195 nucleotides of exon 13; {Delta}248=transcript lacking the first 248 nucleotides of exon 13. Similar results were obtained for the same minigenes driven by the SV40 promoter and in co-transfection experiments using IB3 cells.

 
We next investigated the effect of co-transfection of an expression plasmid for SF2/ASF with the minigene reporters containing the D651N, E664X and T665S mutations. As shown in Figure 4B, the co-transfections resulted in an increase in the level of the {Delta}248 transcript (compare lanes, 1 to 2, 3 to 4 and 5 to 6, respectively). Quantification of products from three independent co-transfection experiments showed an increase in the ratio of {Delta}248 over the wt transcript of 1.5-, 4- and 3.5-fold (Fig. 4D). These results therefore did not support a role of SF2/ASF in the correct splicing of exon 13 via the purine-rich ESE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Cystic fibrosis is a common recessive disorder caused by more than 1100 mutations in the CFTR gene. Despite the extensive mutation characterization work around the world, there is still a substantial fraction of patients with typical clinical diagnosis of CF but unknown mutations in the CFTR gene (39). The proportion of unknown CFTR mutations is higher among atypical CF patients (38). In our search for possible mutations that could affect CFTR pre-mRNA splicing, we have identified five aberrantly spliced CFTR transcripts with no apparent causative genomic alteration. The aberrant transcripts could account for 10–40% of the total CFTR transcript. Varied activity of trans-acting splicing factors may provide a plausible explanation for these aberrant species. The aberrant forms could contribute to the CF phenotype observed in milder patients with only one known CFTR mutation. In these cases, a reduction of the properly spliced transcript from the normal allele may bring the total CFTR in the secretory epithelium to below the level that is required for normal chloride conductance activity. Such variations in the level of CFTR pre-mRNA splicing may not be sufficient, however, to account for CF patients who showed no linkage to the CFTR locus, as identified in the study of Groman et al. 2002 (39).

In the present study, we have investigated the molecular mechanism underlying the generation of one of the aberrantly spliced products involving CFTR exon 13, the {Delta}248 transcript. We have identified a consensus ESE sequence that can promote the selection of the correct 3' splice site in exon 13. This mechanism has not been shown previously in the CFTR gene. Using a CFTR minigene construct, we have found that two previously reported CFTR mutations, E656X and 2108delA, located in the ESE consensus sequence could actually cause aberrant splicing of exon 13. We have also expanded the search for other relevant sequences to the splicing of exon 13 and analyzed the effect of five additional previously reported CFTR mutations, D648V, D651N, G654S, E664X and T665S. The aberrant splicing of exon 13 observed for D648V, E664X and T665S mutations is probably due to strengthening the polypyrimidine tract adjacent to one of two cryptic 3' splice sites, located at 195 and 248 nt, downstream of the native 3' splice site.

Remarkably, six out of the seven mutations that we studied cause aberrant splicing of exon 13. These results suggest that many other mutations in the 27 exons of the CFTR gene may impact on pre-mRNA splicing. This indicates that it is no longer warranted to simply classify CF and other disease-associated mutations solely on the basis of predicted amino acid changes. For example, the molecular consequence of D648V and T665S was previously predicted to cause amino acid changes that could affect the chloride channel activity of the CFTR. Subsequent studies showed that these mutations introduced into CFTR cDNA have no effect on the chloride channel activity of the corresponding expressed mutant CFTR proteins (40). However, from the present study, we predict that the CF phenotype associated with the D648V and T665S mutations is most likely due to their effect on exon 13 splicing. One may argue that knowing whether nonsense mutations affect splicing does not change the fact that these mutations are deleterious. However, recent reports showed evidence that nonsense mutations can be partially suppressed by aminoglycoside antibiotic treatment (41). Therefore, our results showing that two nonsense mutations, E656X and E664X, affect the splicing of exon 13 acquire particular importance. Recently, new molecular approaches to treat aberrant splicing events have been developed (42–44). Therefore, together with several other recent reports of exonic mutations that affect splicing (15,45–47), our study emphasizes the importance of assessing the phenotypic consequence of disease mutations also at the splicing level. In fact, one should know the true molecular consequence of all mutations through proper experimentation and data analysis.

The notion that genes other than CFTR may play a role in modifying the severity of CF disease has been considered in recent years (48,49). In light of the effects on splicing of CFTR mutations demonstrated in the present study, it is tempting to speculate that trans-acting splicing factors could function as modifiers of their splicing phenotype. The consequence of splicing defects caused by mutations in the CFTR gene could be enhanced by varied levels of splicing factors, translating into a more severe CF disease phenotype.

In our present study we show that over-expression of two different splicing factors, hTra2{alpha} and SF2/ASF, can alter the relative levels of the correct versus aberrantly spliced forms of transcripts from the minigene constructs carrying different exon 13 splicing mutations. These two factors appear to have opposite effects on the splicing of exon 13. Increased expression of hTra2{alpha} decreased the aberrant splicing of exon 13 caused by two exonic splicing mutations, E656X and 2108delA. Conversely, SF2/ASF exacerbated the effect of D651N, E664X and T665S mutations on the splicing of exon 13. That opposite effects were observed for SF2/ASF and hTra2{alpha} argues that their effects are specific and not a general consequence of over-expression of splicing factors. It is conceivable that variations in the levels and/or activities of these and other splicing factors could translate into differences in the severity of certain CF phenotype presentations. Furthermore, these results provide suggestive evidence to support our previous speculation regarding the possible contribution of trans-acting splicing factors on the disease phenotype observed in CF patients with only one known CFTR mutation.

While our results are only suggestive at this time, they nevertheless argue that splicing factors, and SR proteins in particular, are warranted for further investigation as candidates for modifiers of CF disease. It will be important in future studies to determine whether changes in the relative levels of specific splicing factors in patient cells correlate with relative levels of CFTR mRNA isoforms as well as CF disease presentation. A better understanding of the possible consequences of splicing factor variations, will help define future directions for both the diagnosis and treatment of CF.

We realize that our conclusions have only been drawn from cell-culture experiments and transfection studies using minigene constructs. Attempts to recruit tissue samples from the patients in which the various CFTR mutations were originally identified have failed so far. The mutations were reported to associate with CF phenotype [D648V (31), E656X (http://www.genet.sickkids.on.ca/cftr/), 2108delA (27), E664X (33) and T665S (http://www.genet.sickkids.on.ca/cftr/)] or with pulmonary disease [D651N (32)]. Unfortunately, in most cases only one CF patient was reported to carry one of these mutations preventing us from assessing any phenotype variation. Nevertheless, our wild-type minigene reporter efficiently and accurately spliced exon 13 in two different cell lines. Therefore we believe that our reporter construct provides a reliable model system for characterizing sequence elements involved in the splicing of exon 13.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Patients
The study population consisted of 22 patients diagnosed with CF based on the presence of clinical symptoms characteristic for the disease (1). These patients had one or two unidentified CFTR alleles following genomic DNA mutation screening (18).

CFTR transcript analysis
Total RNA was extracted from cultured lymphoblastoid cells using RNeasy extraction kit (Qiagen). First strand cDNA was synthesized from 2 to 3 µg of total RNA by oligo dT-primed reverse transcription with Superscript II Reverse Transcriptase (Invitrogen) as per manufacturer's directions. Two successive rounds of PCR were then performed to generate overlapping fragments covering the full-length CFTR transcript. Accordingly, the first round of PCR amplification [1 min at 94°C, 1 min at 57°C and 2 min at 72°C for 30 cycles using 2.5 U of AmpliTaq DNA polymerase (Boehringer Ingelheim)] generated three overlapping fragments. The second round of PCR amplification [20 s at 94°C, 20 s at 55°C and 30 s at 72°C for 30 cycles using 2.5 U of AmpliTaq DNA polymerase (Boehringer Ingelheim)], using the first round product as a template and nested primers, generated a total of seven fragments. The PCR products were analyzed by electrophoresis on 8% polyacrylamide gel. PCR products displaying altered mobility were isolated from the gel and subjected to direct DNA sequencing analysis using a Thermostable Sequenase Radiolabeled Terminator Cycle Sequencing Kit and 33P-labeled dideoxy terminators (Pharmacia-Amersham).

CFTR minigene constructs
Wild-type sequences of exons 12 (356 bp), 13 (1320 bp) and 14a (373 bp) and their corresponding flanking intronic sequences were amplified using the following primers, IVS11SalI, 5'-TTTCAGTGAGTCGACGTGGTGACC-3'; IVS12BglII/PflMI, 5'-CCATTTTATGGAGGGCAGATCAGATCTGAG-3'; IVS12BglII, 5'-AGTACAGATCTCTAGGGACC-3'; IVS13-R, 5'-TCACTGGCTTAGTAGAGGAC-3'; IVS13PflMI, 5'-GCTCCAGTAGACCATAAACTGGCTATAG-3'; IVS14XbaI, 5'-GTGTGTGCATCTAGATGTATACATCCCC-3' containing SalI, BglII and PflMI; BglII and PflMI; and PflMI and XbaI restriction sites, respectively, using as a template a bac clone containing CFTR genomic sequence spanning exons 2–15 (H_DJ0187G17). Single and multiple nucleotide substitutions were introduced in exon 13 by overlapping extension (50). The PCR program used [30 s at 94°C, 30 s at 50°C and 30 s at °C increasing the annealing temperature 0.5°C per cycle for 20 cycles and 30 s at 94°C, 30 s at 60°C and 30 s at 72°C for 10 cycles, using 1.5 U of AmpliTaq DNA polymerase (Boehringer Ingelheim)] was designed to allow the annealing of primers containing mismatches to the wild type CFTR sequence. Each resulting PCR fragment was subcloned using PCRscript amplification system (Stratagene) and retrieved using the restriction enzymes introduced in the corresponding PCR primers. The CFTR sequences were inserted in two steps into SalI and XbaI sites present in IVS 1 of the ß-globin gene which was introduced in two previously described expression plasmids driven either by the SV40 or the CMV promoter (25). First, exon 12 flanked by SalI and PflMI and exon 14a flanked by PflMI and XbaI restriction sites were simultaneously inserted into the SalI and XbaI sites of IVS1 using T4 ligase (Invitrogen). The resulting plasmid was double digested using BglII (introduced by the PCR primer for exon 12 fragment) and PflMI. In a second ligation step, exon 13 flanked by BglII and PflMI was inserted using T4 ligase (Invitrogen). The resulting CFTR minigene construct is shown in Figure 1.

Splicing factors expression constructs
The hTra2{alpha} and SF2/ASF expression plasmids contain the full-length human coding sequence (cDNA) of these genes (24,35,36).

Cell lines
COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with high glucose supplemented with 10% fetal calf serum and 1% streptomycin–penicillin antibiotics. IB3 cells were grown in LHC-8 medium with glutamine supplemented with 5% fetal calf serum and 1% streptomycin–penicillin antibiotics. The cells were grown in 10 cm tissue-culture dishes.

Transfection and co-transfection experiments
Six micrograms of total plasmid DNA were transfected into COS-7 and IB3 cells with Lipofectamine (Invitrogen). In co-transfection experiments, 3 µg of CFTR minigene construct and 3 µg of empty plasmid or hTra2{alpha} or SF2/ASF expression plasmids were used. Eighteen hours after transfection the medium was replaced with fresh complete medium. Cells were harvested 48 h after transfection. Each transfection was repeated three times. Total RNA was extracted as previously described. Oligo dT-primed cDNA synthesis was performed as previously described. PCR amplification [30 s at 94°C, 30 s at 60°C and 30 s at 72°C for 30 cycles using 1.5 U of AmpliTaq DNA polymerase (Boehringer Ingelheim)] of the fragment containing exon 13 was performed using the following primers X12-CL, 5'-AGCAGTATACAAAGATGCTG-3' and X14a-3L, 5'-GGACAGTAATATATCGAAGG-3'. The fragments were separated on a 2% agarose gel. Densitometric analysis of the resulting PCR bands was carried out using the FluorChem program. PCR products displaying altered mobility were isolated from the gel and subjected to direct DNA sequencing analysis using a Thermostable Sequenase Radiolabeled Terminator Cycle Sequencing Kit and 33P-labeled dideoxy terminators (Pharmacia-Amersham).


   ACKNOWLEDGEMENTS
 
The authors wish to thank Dr Alan Cochrane for SF2/ASF and hTra2{alpha} expression vectors and Dr Cochrane and Dr Johanna Rommens for their insightful comments on the manuscript. The work is supported by grants from the Canadian Cystic Fibrosis Foundation (CCFF), the Canadian Genetic Diseases Network, and the National Institutes of Health, USA (P50 DK49096-9) to L.-C.T. L.-C.T. is the HE Sellers Chair in Cystic Fibrosis and Distinguished Scientist of the Canadian Institutes of Health Research. I.A. is the recipient of a CCFF Studentship.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Vice Chancellor's Office, The University of Hong Kong, Pokfulam Road, Hong Kong. Tel: +852 2859 2100; Fax: +852 2858 9435; Email: tsuilc{at}hkucc.hku.hk Back


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