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Michal Zucker, Amalia Biron Research Institute of Thrombosis and Hemostasis, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel. Tel.: +972 3 5302105; fax: +972 3 5351568. E-mail: email@example.com
See also Duga S, Asselta R. Mutations in disguise. This issue, pp 1973–6.
Summary. Background: Point mutations within exons are frequently defined as missense mutations. In the factor (F)XI gene, three point mutations, c.616C>T in exon 7, c.1060G>A in exon 10 and c.1693G>A in exon 14 were reported as missense mutations P188S, G336R and E547K, respectively, according to their exonic positions. Surprisingly, expression of the three mutations in cells yielded substantially higher FXI antigen levels than was expected from the plasma of patients bearing these mutations. Objectives: To test the possibility that the three mutations, albeit their positions within exons, cause splicing defects. Methods and results: Platelet mRNA analysis of a heterozygous patient revealed that the c.1693A mutation caused aberrant splicing. Platelet mRNA of a second compound heterozygote for c.616T and c.1060A mutations was undetectable suggesting its degradation. Cells transfected with a c.616T minigene favored production of an aberrantly spliced mRNA that skips exon 7. Cells transfected with a mutated minigene spanning exons 8–10 exhibited a significant decrease in the amount of normally spliced mRNA. In silico analysis revealed that the three mutations are located within sequences of exonic splicing enhancers (ESEs) that bind special proteins and are potentially important for correct splicing. Compensatory mutations created near the natural mutations corrected the putative function of ESEs thereby restoring normal splicing of exons 7 and 10. Conclusions: The present findings define a new mechanism of mutations in F11 and underscore the need to perform expression studies and mRNA analysis of point mutations before stating that they are missense mutations.
Factor (F)XI deficiency is an injury-related bleeding disorder that has a worldwide distribution and is common in Jews. Two mutations, c.403G>T and c.901T>C predicting E117X and F283L, respectively (residue numbers devoid of signal peptide), account for approximately 98% of mutant alleles that cause FXI deficiency in Jews . About 190 other mutations have been identified in different populations (http://www.isth.org; http://www.factorXI.org). Among these mutations, 18 were defined as splicing mutations and 126 mutations were defined as missense mutations although most of the latter were not analyzed at the mRNA level nor were they expressed in cells.
Normal splicing depends on the recognition of the 3′ and the 5′ splice sites by spliceosome components. Inclusion of exons in the mature mRNA also depends on two major groups of cis-acting regulatory sequences: exonic and intronic splicing regulators. Among these are specific 6–8 exonic nucleotide sequences termed exonic splicing enhancers (ESEs) that are located in exons at varying distances from the splice sites [2,3]. These ESEs bind serine-arginine-rich proteins (SR-proteins) that regulate the function of flanking splice sites . It was suggested that most exons contain at least one functional ESE site [2,3]. Mutations in ESEs can affect the binding of SR-proteins to these ESEs, leading to failure of splicing and exon skipping or inclusion of intron segments in mRNA . Therefore, apparent missense mutations in ESE sites can in reality be splicing mutations [5,6]. ESEs are particularly important for exons that have weak natural splice sites that display less homology with the consensus sequences . In the F11 gene, we noticed that 50% of the acceptor splice sites are weak yielding potency scores below 0.4 which is the threshold score determined by the splice-site predictor program of the Berkeley Drosophila Genome Project (BDGP). A 1.0 score represents the maximal score . The potential implication is that these weak acceptor sites are susceptible to aberrant splicing resulting in degradation of mRNA. Indeed, a recent study showed multiple aberrant splicing events in wild-type FXI pre-mRNA of human liver and platelets . Moreover, splicing at such weak splice sites can be further weakened or strengthened by mutations in ESEs. Unlike the acceptor sites, the donor splice sites of the F11 gene are strong, with potency scores that range from 0.8 to 1.0.
In the present study, we characterized in two patients with FXI deficiency three-point mutations in exons 7, 10 and 14 of the F11 gene. All three mutations were previously reported and were defined as missense mutations [9–15]. Surprisingly, expression of cDNA of these mutations in baby hamster kidney (BHK) cells yielded higher FXI levels than expected from the plasma levels in the patients. This discrepancy could stem from the fact that we expressed normally spliced cDNA in the cells as missense mutations whereas in the patients, the mutations could have caused abnormal splicing. The present finding prompted us to investigate a potential splicing defect caused by aberrant ESE functions related to the three mutations. We herein present evidence that these mutations, albeit their location in the middle of exons, give rise to FXI deficiency by affecting, at least in part, ESEs thereby impairing mRNA splicing.
Two patients with known FXI deficiency were examined. Patient 1, a 62-year-old white woman from the United States, had moderate FXI deficiency with a plasma FXI activity of 22% and FXI antigen of 20%. She had a history of menorrhagia, life-long bruising, a severe postpartum hemorrhage and bleeding after tooth extractions. Her ristocetin cofactor level was 120% and von Willebrand factor (VWF) antigen level was 182%. Patient 2, a 23-year-old white woman of French origin, had severe FXI deficiency with plasma FXI activity of 6% and antigenicity of 6%. She was diagnosed after a finding of prolonged partial thromboplastin time (aPTT). She had a negative history of bleeding and underwent an uneventful appendectomy, dental extraction and a cesarean section. The two patients were not of Jewish origin. Both consented to be examined and the Institutional Review Board of the Sheba Medical Center approved the study.
DNA was extracted from leukocytes using a standard procedure . Amplifications of all exons and flanking exon–intron boundaries of the F11 gene were performed by polymerase chain reaction (PCR) followed by direct sequencing of amplified fragments by an automatic sequencer (ABI, Foster City, CA, USA).
Assays of FXI activity and antigen level
FXI activity was determined by an aPTT-based assay using severe FXI-deficient plasma as a substrate (FXI activity < 1%) and 1:10 to 1:320 dilutions of reference normal plasma for construction of a standard curve. A 1:5 dilution of patients’ plasma yielded an assay sensitivity of < 1%. FXI antigen was measured in patients’ plasma samples and in media and lysates of transfected cells using ELISA. This assay was carried out as previously reported .
Analysis of platelet mRNA
Platelets were separated from blood by 10-min centrifugation at 800 ×g yielding platelet-rich plasma, and then by 5 min centrifugation at 14 000 ×g. The platelet pellet was resuspended in RNAlater reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Platelet mRNA was produced using the RNeasy mini kit (Qiagen) and then it was reverse transcribed to cDNA using the Moloney Murine Leukemia Virus (MMLV; Promega, Madison WI, USA) and random primers (Promega). Splicing patterns of the cDNA from the patients’ and control platelets were compared by PCR and agarose gel-electrophoresis of the products.
Expression of wild-type and mutant FXI
Expression of normal and mutant FXI cDNA in BHK cells was performed as described previously . Briefly, human FXI cDNA in pBR322 vector, kindly provided by Dr Dominic Chung from the University of Washington, Seattle, USA, was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA). Mutations were introduced into the pcDNA3-FXI vector by QuickChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) using specific primers. The presence of the desired mutations was verified by sequence analysis. Two stable clones of wild-type (WT) and two stable clones of mutated pcDNA3-FXI were created. These clones (1 μg) were used for three transfections of BHK cells using Lipofectamine reagent (Gibco, Paisley, UK) and selected by a medium containing 0.7 mg mL−1 neomycin (Gibco). For co-transfections, two pcDNA3-FXI plasmids, 0.5 μg each, were used simultaneously. Stably transfected cells (5 × 106 cells mL−1) were grown for 24 h in 7 mL of serum-free medium (Optimem 1; Gibco) and thereafter, the medium was collected. COS cells were transiently transfected with 1 μg of either WT or mutated pcDNA3-FXI using Lipofectamine reagent. Cells were grown for 48 h and, thereafter, medium was collected for antigen measurements.
ESEfinder (http://rulai.cshl.edu/tools/ESE2/)  is a web resource that uses experimentally defined scoring matrices to identify possible ESEs in DNA sequences. ESEs responsive to the human serine-rich (SR) proteins SF2/ASF, SC35, SRp40 and SRp55 were sought in the regions of exons 7, 10 and 14 where the mutations of the patients were located.
In vitro splicing analysis
The ExonTrap system (MoBiTec GmBH, Gottingen, Germany) was used to analyze the splicing patterns of the mutated transcripts vs. the wild-type transcript . Genomic control DNA was used as a template for PCR amplification of a 2470-bp segment that includes exon 7 and parts of its flanking introns. This was carried out by using a forward primer in intron 6 that contained an XhoI restriction site and a backward primer in intron 7. The PCR product was digested by XhoI and XbaI and cloned into XhoI- and XbaI-digested ExonTrap vector. The c.616T mutation in exon 7 was introduced into the ExonTrap vector using the QuickChange™ site-directed mutagenesis kit according to the manufacturer’s instructions using specific primers. The same procedure was performed for the c.1060A mutation in exon 10 for which the minigene included 900 bp comprising exons 8 through exon 10 and segments of introns 7 and 10. The correct sequences of WT and mutant plasmids were verified by sequence analysis. The constructs were transiently transfected into COS cells using Lipofectamine reagent. Forty-eight hours after transfection, total RNA was extracted from the cells using the RNeasy mini kit and then reverse transcribed into cDNA using MMLV. Splicing patterns of the mRNA from the transfected cells were revealed by PCR, agarose gel-electrophoresis of the products and real-time PCR.
Real-time PCR analysis
Amplification reactions of the minigene mRNA were carried out using the LightCycler 480 SYBR Green I Master (Molecular Probes Inc., Eugene, OR, USA) and consisted of 45 cycles of 95 °C for 10 s and 60 °C for 15 s. Each sample was tested four times. A standard curve for each pair of primers was established using four dilutions of cDNA samples: 1:50, 1:250, 1:500 and 1:1000. As a reference gene, we used PCR amplification of the first exon in the ExonTrap vector that was expected to be produced in the same amount by both WT and mutant COS cells. To detect the normally spliced product in the minigene expressing exon 7, we used a forward primer complementary to the first exon in the vector and a reverse primer complementary in part to the last exon of the vector and in part to the end of exon 7. To detect the normally spliced product in the minigene expressing exons 8–10, we used a forward primer complementary in part to the end of exon 8 and in part to the beginning of exon 9 and a reverse primer complementary in part to the end of exon 9 and in part to the beginning of exon 10. We compared the ratio between the target and the reference in WT and mutant minigenes. The results were analyzed using the LightCycler 480 Software 1.5.0 SP3 (Roche diagnostics GmbH, Mannheim, Germany). Results of all experiments were analyzed for statistically significant differences using Student’s t-test. The sequences of all primers used throughout all experiments are available upon request.
Sequences of all exons and flanking exon–intron boundaries of the F11 gene revealed a c.1693G>A substitution in exon 14 of patient 1 and two mutations in patient 2, a c.616C>T substitution in exon 7 and a c.1060G>A substitution in exon 10. All three mutations were reported previously in other FXI-deficient patients and were defined as missense mutations [9–15] predicting an E547K substitution in patient 1 and P188S and G336R substitutions in patient 2. In the current nomenclature, the signal peptide is included and thus these mutations are designated as E565K, P206S and G354R, respectively.
Expression of the three apparent missense mutations in cells
Expressions of the three mutations were carried out by introducing each into normally spliced cDNA followed by their transfections into BHK cells. Surprisingly, FXI antigen levels in the media of transfected cells of the three mutants were substantially higher than expected from the plasma levels in the patients (Table 1). In the heterozygous patient 1, the plasma FXI antigen level was 20% of normal whereas in the medium of BHK cells only harboring the mutant cDNA, the level was 74% compared with WT cells. Cells expressing the missense mutation E547K presented no secretion defect (Table 1). In the compound heterozygous patient 2, the plasma FXI antigen level was 6% of normal whereas in the medium of cells harboring both P188S and G336R mutations, the antigen level was 68% of WT cells. Cells expressing missense mutations P188S or G336R presented a mild secretion defect, however, this defect could not explain the very low FXI antigen level (6%) in the patient’s plasma. Similar discrepancies were detected when the three mutations were expressed using transient transfections in COS cells indicating that the phenomenon was not cell-type dependent (COS cells expressing P188S or G336R secreted 90% and 81%, respectively).
Table 1. Expression levels of recombinant wild-type and mutant factor (F)XIs in baby hamster kidney (BHK) cells
Secretion index (%)‡
Predicted amino acid substitution*
In cell lysates (ng mL−1)†
In cell media (ng mL−1)†
*Residue numbering does not include the signal peptide. †Results are given as mean ± SEM of three transfections. ‡Proportion of FXI in cell media vs. cell lysates.
1693A (exon 14)
128 ± 14
119 ± 14
616T (exon 7)
140 ± 11
68 ± 10
1060A (exon 10)
180 ± 97
54 ± 29
616T + 1060A
P188S + G336R
270 ± 40
108 ± 13
225 ± 13
160 ± 21
The discrepancy between the measurements of FXI antigen in the patients’ plasma and media of cells carrying the mutations prompted us to examine a possible effect of the mutations in exons 7, 10 and 14 on F11 mRNA splicing.
Findings in patient 1
The BDGP splice site program  indicated that while all F11 donor sites were strong, seven out of the 14 acceptor sites were very weak (Table 2), with six having a score below the 0.4 threshold and one, in exon 13, having a score close to the threshold (0.45). Because the acceptor site of exon 14 was among the very weak sites, we sought ESEs in this exon using the ESEfinder program. Five ESEs were identified in normal exon 14, with the c.1693A mutation introducing an additional ESE which binds SF2/ASF splicing protein (Fig. 1A).
Table 2. F11 splice site potency scores*
Donor splice site
Acceptor splice site
*Scores were determined using the BDGP splicing predictor program .
Platelet mRNA of patient 1 was reverse transcribed to yield cDNA. PCR amplification of a cDNA segment containing exon 13 through exon 15 of F11 yielded normally spliced cDNA in both patient and control cDNA. Notably, however, the amount of the normally spliced cDNA was about twofold higher in the control than in the patient’s sample (Fig. 1B, upper panel). The sequence of the normally spliced cDNA was confirmed in both samples by sequencing and did not contain the c.1693A mutation. This result was expected because the patient is a heterozygote for the mutation.
Nested PCR amplifications of a cDNA spanning exon 13 through the middle of intron 14 revealed a clear segment of 919 bp in the patient but not in the control cDNA (Fig. 1B, middle panel). Sequencing of the 919-bp fragment revealed an aberrantly spliced cDNA which skipped exon 14 and included segments of intron 13 and intron 14 (Fig. 1C). Interestingly, introns 13 and 14 include several identical segments which can explain the aberrant splicing at this position, although there is no consensus splice site in the skipping point of this aberrantly spliced product.
For the control gene, platelet integrin αIIb, the cDNA was similarly displayed in control and patient’s platelets (Fig. 1B, lower panel). This indicated that similar amounts of mRNA were extracted from the patient and control platelets.
Findings in patient 2
The effect of the c.616T mutation on splicing. Patient 2 was a compound heterozygote for c.616C>T substitution in exon 7 and c.1060G>A substitution in exon 10. For exon 7, an ESE was predicted to interact with the SC35 splicing protein (Fig. 2A). This ESE spans nucleotides 612 through 619. The c.616T mutation located within this ESE was predicted to abolish the binding of SC35.
As no F11 mRNA was detected in the patient’s platelets, we evaluated the effect of the mutation on the splicing process by an in vitro splicing procedure using an ExonTrap vector. COS cells were transfected with a vector containing WT or mutated exon 7. COS cells expressing WT exon 7 produced the 405 bp normally spliced mRNA (Fig. 2B). COS cells harboring the c.616T mutation located in the middle of exon 7 produced also an aberrantly spliced mRNA of 245 bp that skipped exon 7 (Fig. 2B). The sequence of each product was verified by sequencing. Real-time PCR revealed that the mutation caused a significantly decreased amount of normally spliced mRNA (P < 0.03; Fig. 2B, lower panel).
The effect of the c.1060A mutation on splicing. In exon 10, three ESEs flanking the site of the c.1060A mutation were identified of which two were predicted to loose their ability to bind SF2/ASF and SRp40 because of the mutation (Fig. 3A). While the acceptor site of exon 10 had a high score (0.83, Table 2), exon 9 which is only 88 bp away from exon 10, has a very weak acceptor site with a score of < 0.2. Therefore, the correct splicing of exon 9 could be dependent on the normal ESEs’ function in exon 10.
Reversely transcribed mRNA of control platelets clearly displayed F11 cDNA. In contrast, reversely transcribed mRNA from platelets of patient 2 failed to yield F11 cDNA in repeated experiments (Fig. 3B, upper panel). Integrin αIIb cDNA was similarly displayed both in control and patient’s platelet indicating unimpaired extraction of mRNA from the patient’s platelets (Fig. 3B, lower panel).
As no F11 mRNA was available for examination of splicing, we performed in vitro splicing by creating a minigene comprising exons 8, 9 and normal or mutated exon 10 including parts of their flanking introns. The minigenes were expressed in COS cells. PCR amplification of the mRNA of this minigene yielded several mRNA products both in WT and mutant cDNAs. Sequence analysis of splicing products identified the normal splicing product (625 bp), an aberrantly spliced product in which intron 9 was incorporated (713 bp) and an aberrantly spliced product that skips exon 9 (462 bp). Real-time PCR revealed that the c.1060A mutation caused a significant decrease in the production of the normally spliced mRNA (Fig. 3C). Further, real-time PCR displayed a significantly increased amount of the 462-bp aberrantly spliced mRNA (data not shown).
Compensatory mutations restore normal splicing. Because the mutations were predicted to impair the splicing process by disrupting the function of ESEs, we assumed that it would be possible to restore normal splicing by correcting the dysfunctional ESEs . For this purpose, we used the ESEfinder program to analyze whether changes in the other nucleotides of the respective ESEs would restore the binding of SR proteins in the presence of the mutations. Figure 4 shows that the c.616T natural mutation in exon 7 imparted an SR protein binding score that was below the threshold, whereas the WT sequence displayed an above-threshold score. By introducing an in silico T to G alteration at position 612, the SR protein binding score was predictably restored (Fig. 4, upper panel).
A similar correction was observed for ESEs containing the c.1060A mutation in exon 10. For the two ESEs that bind SF2/ASF and SRp40 proteins, respectively, an in vitro introduction of an A to C alteration at position 1057 corrected the binding scores for the two proteins (Fig. 4, lower panel).
Based on these predictions, we carried out experiments to test restoration of normal splicing. COS cells were transfected with the ExonTrap vector containing WT exon 7, c.616T-mutated exon 7 or exon 7 that harbored the natural mutation along with the c.612G compensatory mutation. Figure 2B shows that the compensatory mutation restored normal splicing. Real-time PCR of the products confirmed that the normal splicing of exon 7 was favored by the WT cells and by cells harboring both native mutation and the compensatory mutation but not by cells only harboring the native mutation (Fig. 2B).
For exon 10, COS cells were transfected with the ExonTrap vector containing the WT exon 10, the mutated exon 10 or exon 10 that harbored the natural mutation as well as an A to C compensatory alteration at position 1057. PCR amplification of these minigenes yielded several mRNA products. Real-time PCR of the normally spliced mRNA product revealed that the native mutation reduced significantly the amount of the normally spliced mRNA and the compensatory mutation restored normal splicing (Fig. 3C).
An alternative approach was to introduce a nucleotide substitution that results in a synonymous change while still reducing the ESE scores of all the splicing proteins. However, in silico analysis indicated that it was impossible to introduce a synonymous nucleotide change that would reduce both the SRp40 and the SF2/ASF binding scores as occurring in the presence of the native mutation.
Normal splicing requires correct recognition of the 5′ and the 3′ splice sites at the boundaries of exons and introns. For certain exons, particularly those with weak splice sites, ESEs are essential for normal splicing [2,3]. Such ESEs are involved in the exon splicing process by binding SR proteins that regulate splicing [4,20,21]. Point mutations within ESEs were shown to impair pre-mRNA splicing, usually by causing skipping of the mutated exon [2,3,22]. Such defects in splicing were observed in genes of tissue factor, integrins αIIb and β3, fibrinogen and other genes [19,21–31]. In the present study, this mechanism is shown for the first time in the F11 gene. We showed that the F11 gene is particularly prone to abnormal splicing because of weak acceptor sites. Indeed, natural alternative splicing events were shown to occur in wild-type human F11 . This implies that normal splicing of F11 mRNA is dependent on intact ESEs. There are also intronic sequences (namely ISEs) that can affect splicing but in the present study we focused on exonic splicing sequences because the apparent missense mutations were within exons.
We described three-point mutations in exons 7, 10 and 14 of F11 that were reported as missense mutations based solely on their positions within exons [9–15]. Expression of these mutations in cells yielded peculiar phenotypes that were different from the phenotype of affected patients harboring these mutations. Other patients harboring the same mutations were also shown to have lower levels of plasma FXI than those observed in our expression studies [10–15].
Patient 1 was heterozygous for a c.1693G>A mutation in exon 14, and had a plasma FXI antigen level of 20%. Patient 2 had a FXI antigen level of 6% resulting from two point mutations in exon 7 (c.616C>T) and exon 10 (c.1060G>A). Cells expressing these mutations produced FXI antigen at levels that were substantially higher than predicted by the corresponding plasma levels (Table 1). The discrepancy between the patients’ phenotypes and the results obtained in cells expressing these mutations was surprising because transfected cells usually exhibit phenotypes that are similar to the phenotypes of patients harboring the same mutations [17,32–35]. In the FXI mutation database 126 out of 192 mutations were defined as missense mutations. Among these 126 apparent missense mutations, only 36 mutations were expressed in cells. None of the cells expressing missense mutations exhibited different FXI secretion than was predicted from the patients’ phenotype. Thus, of 39 missense mutations expressed in cells (including those presented herein) only three (∼8%) displayed a discrepancy between the patients’ and cell phenotypes. This discrepancy could be explained, at least in part, by a splicing abnormality elicited by mutated ESEs located in exons 7, 10 and 14.
In platelets of patient 1, the normal exon 14 representing the normal allele of this heterozygous patient was detectable, as expected, at about half the amount compared with the control (Fig. 1B). Moreover, an aberrantly spliced product, that skipped exon 14 and contained segments of introns 13 and 14, was only detected in the patient mRNA. These findings suggest that as a result of the mutation causing a splicing abnormality, one of the aberrant splicing products comprised skipping of the mutated exon 14 and insertion of parts of introns 13 and 14 which resulted in an alteration of the reading frame.
In the platelets of patient 2, F11 mRNA was undetectable, whereas mRNA of a control gene was identified (Fig. 3B) suggesting that the F11 mRNA was degraded. Because we could not detect mRNA in the platelets of patient 2, we constructed minigenes of control and mutated exons 7 and 10 and examined their in vitro splicing in COS cells. Both the c.616T mutation in exon 7 and the c.1060A mutation in exon 10 were associated with limited correct splicing (Figs 2B and 3C). Skipping of exon 7 was also shown in wild-type FXI pre-mRNA of human liver and platelets , suggesting a similar splicing for liver and platelets. This aberrant splicing alters the reading frame, thus introducing a premature termination codon in exon 10. Compensatory mutations in both minigenes were predicted to restore the ESEs function (Fig. 4) and indeed were shown to restore normal splicing of both exons (Figs 2B and 3C).
Patient 2 had a 6% FXI antigen level which presumably stemmed from small amounts of normally spliced mRNA that was translated to P188S and G336R. Expression of mutant FXI proteins in BHK cells revealed a partial secretion defect for both mutations (Table 1). Skipping of each one of the exons involved (exons 7, 9 or 10) alters the reading frame. Conceivably, the plasma levels of FXI reflect both a splicing defect and a secretion defect. In other patients harboring the same mutations, different degrees of splicing defects and secretion of FXI are predicted to yield different levels of plasma FXI.
Collectively, the present study indicates for the first time that point mutations within exons of F11 can cause splicing defects emanating from mutations in ESEs. Our findings have several implications for defining mutations in F11 and other clotting factor genes: (i) apparent missense mutations should not be defined or modeled as such, as long as they have not been expressed in cells and examined at the mRNA level; (ii) apparent missense mutations that are not expressed in cells should be defined by the nucleotide alteration and by the predicted but not validated amino acid change; and (iii) a discrepancy between protein levels in patients harboring apparent missense mutations and levels found in cells expressing the mutations can be an indication that ESEs-related splicing defects are involved.
Disclosure of Conflict of Interest
The authors state that they have no conflict of interest.