ABCMdb

PMID: 12913074

Aznarez I, Chan EM, Zielenski J, Blencowe BJ, Tsui LC
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.
Hum Mol Genet. 2003 Aug 15;12(16):2031-40., 2003-08-15 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Glu656* 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. Login to comment 3 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser ABCC7 p.Glu664* ABCC7 p.Asp651Asn 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. Login to comment 4 ABCC7 p.Glu656* 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. Login to comment 5 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser ABCC7 p.Glu664* 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 30 splice sites. Login to comment 78 ABCC7 p.Glu656* 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). Login to comment 79 ABCC7 p.Glu656* 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. Login to comment 100 ABCC7 p.Asp648Val ABCC7 p.Asp651Asn The D648V (2075A!T) (31), D651N (2083G!A) (32) and G654S (2092A!G) (http://www.genet.sickkids.on. Login to comment 101 ABCC7 p.Glu664* 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). Login to comment 111 ABCC7 p.Glu656* (D) RT-PCR analysis of minigenes carrying the wild-type sequence, WT, and E656X and 2108delA mutations separated in a 2% agarose gel. Login to comment 117 ABCC7 p.Asp648Val 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). Login to comment 121 ABCC7 p.Asp648Val The A to T substitution in the D648V mutation, located 18 nucleotides upstream of the 195 cryptic 30 splice junction, would lengthen the corresponding polypyrimidine tract, thereby improving its consensus sequence (Fig. 3A). Login to comment 123 ABCC7 p.Asp648Val 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 30 splice site (Fig. 3B, lane 4). Login to comment 124 ABCC7 p.Asp648Val 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 30 splice site. Login to comment 125 ABCC7 p.Asp651Asn The minigene carrying the D651N mutation yielded two species of aberrantly spliced products. Login to comment 126 ABCC7 p.Asp651Asn Firstly, the G to A substitution responsible for D651N, located 10 nt upstream of the 195-cryptic 30 splice site (Fig. 3A), apparently enhanced the usage of this site (Fig. 3B, lane 5) increasing the ratio of the D195 over the wt transcript by 3-fold (Fig. 3D). Login to comment 130 ABCC7 p.Thr665Ser ABCC7 p.Glu664* Both the E664X and T665S mutations were found to cause an increase in the D248 transcript (Fig. 3C, lanes 2 and 5, respectively) by 2and 5-fold, respectively (Fig. 3E). Login to comment 134 ABCC7 p.Thr665Ser ABCC7 p.Glu664* 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 30 splice site. Login to comment 138 ABCC7 p.Glu656* As shown in Figure 4A, co-transfection with the hTra2a-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). Login to comment 140 ABCC7 p.Asp648Val In contrast, increased expression of hTra2a had no effect on the altered splicing of the minigene reporter transcripts containing the MT or D648V mutations (Fig. 4A and C). Login to comment 142 ABCC7 p.Asp648Val The D648V mutation targeted the 195-cryptic polypyrimidine tract (Fig. 3A) which is not known to function as a binding site for hTra2a, thus was not affected by over-expression of this trans-acting factor. Login to comment 144 ABCC7 p.Thr665Ser ABCC7 p.Glu664* ABCC7 p.Asp651Asn 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. Login to comment 162 ABCC7 p.Asp648Val ABCC7 p.Asp651Asn (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. Login to comment 163 ABCC7 p.Thr665Ser ABCC7 p.Glu664* (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. Login to comment 175 ABCC7 p.Glu656* 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. Login to comment 176 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser ABCC7 p.Glu664* ABCC7 p.Asp651Asn 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. Login to comment 177 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser ABCC7 p.Glu664* 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 30 splice sites, located at 195 and 248 nt, downstream of the native 30 splice site. Login to comment 181 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser 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. Login to comment 183 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser 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. Login to comment 186 ABCC7 p.Glu656* ABCC7 p.Glu664* Therefore, our results showing that two nonsense mutations, E656X and E664X, affect the splicing of exon 13 acquire particular importance. Login to comment 196 ABCC7 p.Asp648Val ABCC7 p.Glu656* Minigenes carrying E656X, 2108delA, MTand D648V mutations were transfected alone (noted by the minus sign) or co-transfected with hTra2a (noted by the plus sign). Login to comment 199 ABCC7 p.Asp648Val 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 D195 (for D648V mutation) or D248 transcript of each minigene was calculated. Login to comment 203 ABCC7 p.Thr665Ser ABCC7 p.Glu664* ABCC7 p.Asp651Asn 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). Login to comment 211 ABCC7 p.Glu656* Increased expression of hTra2a decreased the aberrant splicing of exon 13 caused by two exonic splicing mutations, E656X and 2108delA. Login to comment 212 ABCC7 p.Thr665Ser ABCC7 p.Glu664* ABCC7 p.Asp651Asn Conversely, SF2/ASF exacerbated the effect of D651N, E664X and T665S mutations on the splicing of exon 13. Login to comment 221 ABCC7 p.Asp648Val ABCC7 p.Thr665Ser ABCC7 p.Glu656* ABCC7 p.Glu664* ABCC7 p.Asp651Asn 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)]. Login to comment