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• W2022307125 abstract "SummaryThis work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤ 10% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3′-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both. This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (≤ 10% of control) platelet content of GPIIb–IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3′-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both. Glanzmann's thrombasthenia (GT) [1Glanzmann E. Hereditare Hemorrhagische Thrombasthenie: ein Beitrag zur Pathologie der Blut Plattchen.J Kinderke. 1918; 88: 113-41Google Scholar] is a bleeding disorder appearing immediately after birth characterized by mucocutaneous hemorrhages, prolonged bleeding time, lack of platelet aggregation, either spontaneous or induced by physiological agonists, and low to absent clot retraction. The thrombasthenic phenotype is associated with low content or total absence of the platelet membrane glycoproteins GPIIb and GPIIIa [2Nurden A.T. Caen J.P. Specific roles for platelet surface glycoproteins in platelet functions.Nature. 1975; 255: 720-2Crossref PubMed Scopus (299) Google Scholar] that normally form a non-covalent, Ca2+-dependent heterodimer, also known as integrin αIIbβ3, that is a receptor for fibrinogen and other adhesive proteins [3Phillips D.R. Charo I.F. Parise L.V. Fitzgerald L.A. The platelet membrane glycoprotein IIb–IIIa complex.Blood. 1988; 71: 831-43Crossref PubMed Google Scholar, 4Pytela R.P. Pierschbacher M.D. Ginsberg M.H. Plow E.F. Ruoslhati E. Platelet membrane glycoprotein IIb/IIIa. Member of a family of Arg-Gly-Asp-specific adhesion receptors.Science. 1986; 231: 1559-62Crossref PubMed Scopus (661) Google Scholar]. A distinct feature of the GPIIb–IIIa complex is its exclusive expression in platelets, although recent reports indicate it can also be found in certain types of tumoral cells [5Chen Y.Q. Trikha M. Gao X. Bazaz R. Porter A.T. Timar J. Honn K.V. Ectopic expression of platelet integrin αIIbβ3 in tumor cells from various species and histological origin.Int J Cancer. 1997; 72: 642-8Crossref PubMed Scopus (64) Google Scholar]. The degree of platelet content of GPIIb–IIIa complexes is currently used to classify the Glanzmann's phenotypes [6Caen J.P. Glanzmann's thrombasthenia.Clin Haematol. 1972; 1: 383-92Google Scholar]. Type I GT patients show total absence of platelet GPIIb–IIIa and fibrinogen and there is no clot retraction. Type II GT patients show a platelet GPIIb–IIIa content ≤ 10–20% of normal individuals, detectable amounts of fibrinogen content, and a moderate clot retraction. The so-called GT variants may show normal or near normal (60–100%) platelet content of GPIIb–IIIa complexes that fail to enable high-affinity binding of fibrinogen in response to physiological agonists [7Nurden A.T. Rosa J.P. Fournier D. Legrand C. Didry D. Parquet A. Pidard D. A variant of Glanzmann's thrombasthenia with abnormal GPIIb–IIIa complexes in the platelet membrane.J Clin Invest. 1987; 79: 962-9Crossref PubMed Scopus (54) Google Scholar]. Knowledge of the primary DNA sequence and structural organization of the GPIIb and GPIIIa genes has made possible the molecular genetic analysis and elucidation of different types of mutations associated with thrombasthenic phenotypes [8French D.L. The molecular genetics of Glanzmann's thrombasthenia.Platelets. 1998; 9: 5-20Crossref PubMed Scopus (69) Google Scholar]. The present work aimed at elucidating the molecular genetic defect responsible for the thrombasthenic phenotype of a patient who suffered from mucocutaneous hemorrhages that appeared immediately after birth, prolonged bleeding time, and platelets of normal size and number showing minimal (<10%) aggregation either spontaneously or in response to physiological agonists. The markedly low platelet content of GPIIb–IIIa indicated it was a case of type II GT. The proband presents a homozygous G188A substitution located at the splice junction of exon 1–intron 1 of GPIIb that changes the Arg 63 for Lys. The complete sequencing of GPIIb and GPIIIa failed to detect mutations other than [G188A]GPIIb, suggesting its association with the thrombasthenic phenotype. The patient showed very low levels of platelet GPIIb mRNA and a correlation was found between levels of GPIIb mRNA and surface expression of GPIIb–IIIa in both parents who are heterozygotes for the [G188A]GPIIb mutation. The ‘exontrap’ analysis of a genomic DNA construct containing the first exons of GPIIb demonstrated that the [G188A]GPIIb mutation results in a marked reduction in the steady-state levels of mRNA and the usage of alternative splicing sites. Thus, it seems plausible to conclude that the thrombasthenic phenotype of the proband is the result of a limited availability of GPIIb mRNA caused by the G188A mutation. Murine monoclonal antibodies (mAb) specific for GPIIIa (713H7) and GPIIb (701E5) were raised in our laboratory using as antigen GPIIb–IIIa heterodimer isolated from human platelets. The anti-αvβ3 LM609 mAb was from Chemicon (Temecula, CA, USA). Fluorescein isothiocyanate-conjugated (FITC) F(ab′)2 fragment of rabbit antimouse immunoglobulin (Ig) was purchased from Dako A/S (Glostrup, Denmark). The horseradish peroxidase (HRP)-conjugated goat antimouse IgG was from BioRad Labs (Hercules, CA, USA). The patient is a 1-year-old female from Morocco with a history of frequent petechia, bruising, and mucocutaneous hemorrhages that started immediately after birth. Her parents, who are first cousins, and her older brother are asymptomatic. A first cousin of the proband, son of a paternal uncle, died as a result of some unknown bleeding disorder. The patient showed prolonged bleeding time and platelets of normal size and number. Platelet aggregation in response to ADP, thrombin, collagen and epinephrine was < 10% of normal platelets. Ristocetin-induced platelet agglutination was normal. The latter observations suggested the proband suffers of Glanzmann's thrombasthenia. Platelet-rich plasma (PRP) was obtained from venous whole blood by centrifugation at 150 × g for 20 min at room temperature. Platelets were sedimented at 1000 × g for 10 min, washed with 0.5 mm EDTA in phosphate-buffered saline (PBS), and incubated, at a density of 106 cells 100 µL−1, with the specific mAb for 30 min. After washing, the platelets were incubated in a 1 : 20 dilution of FITC-conjugated F(ab′)2 fragment of rabbit antimouse Ig for 20 min. Samples were analyzed in a Coulter (Miami, FL, USA) flow cytometer, model EPICS XL. FITC-labeled human fibrinogen (Fg) was prepared as previously described [9Arias-Salgado E.G. Butta N. González-Manchón C. Larrucea S. Ayuso M.S. Parrilla P. Competition between normal [674C] and mutant [674R]GPIIb subunits: role of the molecular chaperone BiP in the processing of GPIIb–IIIa complexes.Blood. 2001; 97: 2640-7Crossref PubMed Scopus (16) Google Scholar]. The PRP was diluted with Tyrode's buffer pH 7.4 (5 mm HEPES, 2 mm MgCl2, 0.3 mm NaH2PO4, 3 mm KCl, 134 mm NaCl, 12 mm NaHCO3, 0.1% glucose, 0.1% bovine serum albumin and 1 mm Cl2Ca) to a final concentration of 5 × 107 platelets mL−1. To induce aggregation, platelets (60 µL) were treated for 5 min at room temperature with one or more of the following activating agents: 20 mm dithiothreitol (DTT) (BRL, Life Technologies, Carlsbad, CA, USA), 20 nm phorbol 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO, USA), 200 µm adenosine 5′-diphosphate (ADP) (Sigma), 1 mm (-)epinephrine (Sigma), 2 µm platelet activating factor-16 (PAF) (Calbiochem, Nottingham, UK). Then, 7.5 µg of FITC-Fg was added and, after 15 min at room temperature, platelets were washed and resuspended in Tyrode's buffer for flow cytometry analysis. Total platelet RNA was obtained according to the method of Chomczynsky and Sacchi [10Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-9Crossref PubMed Scopus (63147) Google Scholar]. Screening for mutations was carried out by direct sequencing of polymerase chain reaction (PCR)-amplified overlapping fragments of reversed transcribed GPIIb or GPIIIa mRNAs. To detect alternative splicing forms of mRNA the PCR products were cloned in a T vector and individual clones sequenced. Genomic DNA was isolated from peripheral blood cells. Amplification of DNA was carried out with Taq polymerase according to the protocol recommended by Perkin-Elmer Roche (Branchburg, NJ, USA). Fragments of DNA encompassing one or more exons of GPIIb were amplified and sequenced as previously reported [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar]. A fragment of genomic DNA encompassing the exon 1–intron 1 junction was amplified using the oligonucleotide pair: sense, 5′-GTTGTGGAAGAAGGAAG-3′, and antisense, 5′-TATCGCAAATGGGAAACTCG-3′. Oligonucleotide pairs used for amplification and sequencing of the promoter and 3′-UTR regions of GPIIb were: sense, 5′-CAAAAGGAAAATGTGGCTATGG-3′ and antisense, 5′-TATCGCAAATGGGAAACTCG-3′, and sense, ATACTTCCTCACATGTGCTCT-3′ and antisense, 5′-TCTCCGTCTTCTGTTACACC-3′, respectively. The PCR products were used for direct sequencing of DNA using an automatic Applied Biosystems (Foster City, CA, USA) sequencer. Quantification of platelet GPIIb and GPIIIa mRNAs of the proband, both parents, and normal individuals was performed with the reverse transcriptase (RT)-PCR-based TaqMan System (Perkin-Elmer Roche) as previously described [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar]. One-step RT-PCR was carried out with MuLV (Murine Leukemia Virus)-reverse transcriptase followed by amplification with preactivated AmpliTaq Gold DNA Polymerase. The TaqMan probes R-CTCTGGCGCGTTCTTCCTCAAATTTAGC-Q, R-AGTGACCACGGAGCTGAAGCCCG-Q, and R-ATGCCCTCCCCCATGCCATCCTGCGT-Q, were used to anneal to targets located within PCR-amplified fragments from 2157 to 2288 bp of GPIIIa, from 582 to 776 bp of GPIIb, and from 2141 to 2435 bp of β-actin cDNAs, respectively. R and Q indicate the location of the reporter and quencher dyes, respectively. Amplification reactions were performed in closed optical tubes of a 96-well microplate using a combined thermal cycler/detector, the ABI 7700. Fluorescence emission data from each sample were collected once every few seconds as the PCR product was being generated. Rn represents the fluorescence signal ratio of the reporter and quencher dyes. ΔRn represents the normalized reporter signal minus the baseline signal established in the first few cycles of PCR. The threshold cycle Ct represents the cycle in which the fluorescent signal is first recorded as statistically significant above background [12Gibson U.E.M. Heid C.A. Williams P.M. A novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1775) Google Scholar]. Since this point will always occur during the exponential phase of amplification, quantification is not affected by any reaction components becoming limited in the plateau phase. In optimal conditions, Ct decreases by one cycle as the concentration of template doubles. The exontrap vector system (Mo Bi Tec GmbH, Göttingen, Germany) was used to analyze the RNA splicing pattern of fragments of normal and mutant GPIIb. The first exon of eukaryotic genes are not trapped by the exontrap vector because they lack a 3′ acceptor splice site. To circumvent this problem, we prepared a construct in which an extra exon was placed prior to exon 1 of GPIIb. This was achieved with a previously used exontrap construct of genomic DNA containing exons 2–9 of GPIIb and the adjacent intronic regions [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar]. First, this construct was partially digested with Pvu II and totally with Sma I to release a fragment extending from the 3′-end of exon 3 to the middle of intron 8. Then, a 295-bp DNA fragment encompassing exon 1 and the 90-bp 5′ of intron 1 was PCR amplified from the patient and a normal control, blunted-end, and cloned into the Pvu II–Sma I digested construct. The orientation of the inserts was verified by sequencing. The exontrap vectors containing the chimeric normal or mutant GPIIb DNA fragments were transiently transfected into CHO-IIIa cells by the calcium phosphate precipitation method and, 48 h later, total RNA was extracted and RT-PCR amplified as previously described [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar]. Expression plasmid containing mutant GPIIb cDNA was prepared as follows. Total RNA from the propositus was used for RT-PCR amplification of a 577-bp fragment of GPIIb cDNA, using the oligonucleotides sense (-16/-1): 5′-GTTGTGGAAGAAGGAAG-3′ and antisense (559/540): 5′-CGTAAATGCGGCTCAGGGTG-3′. The PCR product was subcloned in a T-vector to generate an EcoR I–Sac II fragment containing exon 1. This fragment was then ligated into the EcoR I- and Sac II-digested pcDNA3.1[-]/Myc-His B vector (Invitrogen, San Diego, CA, USA) containing the wild-type GPIIb cDNA. CHO cells stably expressing GPIIIa (CHO-IIIa) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Cells were transiently transfected by the diethyl aminoethyl (DEAE)-dextran method with 5 µg of either normal or mutated GPIIb constructs. Forty-eight hours after transfection the cells were harvested and the surface expression of GPIIb–IIIa complexes was determined by flow cytometric analysis. Platelet proteins were solubilized in lysis buffer (50 mm Tris pH 7.4, 150 mm NaCl, 1% Triton X-100, 0.05% Tween 20) supplemented with 1 mm phenylmethyl sulfonyl fluoride (PMSF) and electrophoresed on SDS−7.5% polyacrylamide gels, transferred to PVDF membranes, and then incubated with mAbs against either GPIIb or GPIIIa. The immunoreactive bands were detected with a peroxidase-conjugated goat antimouse IgG and visualized using the ECL chemiluminescent system. Quantification of the autoradiographs was performed with a calibrated densitometer model GS800 from BioRad. The platelet content of GPIIb–IIIa was estimated by measuring the binding of monoclonal antibodies directed against GPIIb or GPIIIa to platelets from the proband, her parents, and normal individuals, by flow cytometric analysis (Fig. 1). The platelets from the proband showed slightly positive fluorescence relative to the background obtained by exposure of cells only to the second fluorescent antimouse IgG, indicating a low (≤ 10%), yet detectable, content of GPIIb–IIIa complexes. Both parents showed reduced platelet fluorescence relative to the control platelets that could account for an approximate decrease of ≤ 40% in the surface content of GPIIb–GPIIIa. The estimation of platelet GPIIb–IIIa content by Western blotting analysis (Fig. 2) offered results consistent with those of flow cytometry, i.e. both parents showed reduced GPIIb–IIIa signals and the proband showed a very low platelet content of GPIIb and GPIIIa clearly visible only when the gels were overloaded with platelet protein (Fig. 2). Significant increases in fluorescence over the background were detected when the platelets from the proband were incubated with FITC-labeled fibrinogen and stimulated by either DTT or physiological agonists. This observation seems to indicate that the small amount of GPIIb–IIIa complexes showed a normal functional responsiveness.Figure 2Western blotting analysis of platelet GPIIb and GPIIIa content in a thrombasthenic patient and her parents. Platelet lysates were resolved on SDS−7.5% polyacrylamide gels under reduced (GPIIb) or non-reduced (GPIIIa) conditions and transferred to PVDF membranes. Immunoblotting with monoclonal antibodies was performed as described in Materials and methods. The experiment was repeated three times using at least two different protein concentrations from each sample. The autoradiographs were evaluated using a calibrated densitometer (GS800, BioRad). The figure shows data from a representative experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The reduced platelet content of GPIIb–IIIa in both parents suggested that the patient could have inherited a mutation from each progenitor. The higher GPIIIa than GPIIb and the normal platelet content of αvβ3 complexes in the patient (Fig. 1) suggested a limited availability of GPIIb, presumably due to a mutation(s) in the GPIIb gene. Thus, we first searched for mutations in the GPIIb gene by sequencing overlapping fragments of reversed transcribed GPIIb mRNA. Sequencing of the amplification products of mRNA GPIIb from the proband revealed the presence of a G188A substitution, the predicted effect of which is the change of arginine at position 63 by lysine in the GPIIb peptide. This mutation was not detected in platelet GPIIb transcripts from her parents. However, the sequencing of a fragment of genomic DNA encompassing the exon 1–intron 1 junction demonstrated that the proband was homozygous and both parents heterozygous for the G→A transition located at the 5′-donor splicing site (Fig. 3). This mutation was not found in 25 DNAs from healthy blood donors. Moreover, no mutations other than [G188A]GPIIb were found after complete analysis of the coding sequences of GPIIb and GPIIIa, and the promoter, 3′-UTR, and intronic flanking regions of GPIIb, in the proband and her parents. In principle, the marked reduction in the platelet GPIIb content observed in the proband and her parents could be the result of either a limited availability of GPIIb mRNA or a failure of [63K]GPIIb to complex GPIIIa and/or undergoing the trafficking pathway at normal rates. Nevertheless, the failure to amplify the mutant transcript from the parents suggested that either it was absent or present in very limited amounts. To elucidate this question, we first assessed the platelet content of GPIIb and GPIIIa mRNAs by the TaqMan procedure. The proband showed normal platelet GPIIIa mRNA content and a marked reduction (> 90%) in the GPIIb mRNA content relative to normal controls (Fig. 4). Both parents showed normal platelet GPIIIa mRNA content but the GPIIb mRNA levels were reduced by approx. 50% relative to normal controls analyzed simultaneously (Fig. 4). These results suggest that a correlation exists between the number of mutated [G188A]GPIIb alleles and the rate of surface expression of GPIIb–IIIa. The association of the thrombasthenic phenotype with reduced availability of GPIIb mRNA agrees with previous reports indicating that the availability of messenger is a rate-limiting step for the surface exposure of GPIIb–IIIa complexes. The cause of the reduced availability of [G188A]GPIIb mRNA is not apparent. Mutations within the consensus splice junction sequences or in its vicinity may force alternative splicing and/or exon skipping; therefore, we found it of interest to investigate whether the presence of the G188A mutation could alter the pattern of splicing with the result of GPIIb mRNA instability. To investigate this possibility, we cloned PCR-amplified fragments from reversed transcribed platelet GPIIb mRNA encompassing the first few exons, and the DNA sequence of 25 individual clones was determined. Of the analyzed clones, 70% corresponded to the [K63]GPIIb sequence. In 15% of the clones there was an insertion of the first seven base pairs of intron 1 including an in-frame STOP codon. These transcripts could be the result of activation of a cryptic splicing donor site located downstream of the normal consensus site. Other less represented alternative spliced forms (approx. 7% each) were also identified (Fig. 3, lower panel); in the alternative spliced form B, intron 1 was abnormally processed, whereas in form C intron 1 was normal but the first 12 base pairs of intron 3 were inserted. None of the abnormally spliced forms were detected in RNA from normal platelets. The exontrap analysis has been previously used to study GPIIb mutations associated with thrombasthenic phenotypes [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar]. However, in our case its use was limited by the lack of splicing signals for the first exon. To circumvent this problem, we prepared chimeric constructs in which additional exons were placed ahead of exon 1 of GPIIb. These constructs were transiently transfected into CHO cells stably expressing human GPIIIa. The results were normalized by the simultaneous analysis of transcripts from the construct and GPIIIa. The construct containing the splice site mutation yielded minute amounts of transcript of the expected normal spliced form of 561 bp found in cells transfected with the construct containing the normal splice site (Fig. 5). Two additional splicing forms were found only in cells transfected with the mutant construct. In one of them, the use of a nearby cryptic donor splicing site led to the incorporation of 7 bp, yielding a 568-bp transcript. Another, most abundant, form of 804 bp in size was the result of non-splicing of the chimeric intronic sequence. Figure 6 depicts the results obtained by expressing either normal [63R]GPIIb or mutant [63K]GPIIb into CHO cells stably expressing human GPIIIa. In agreement with previous reports, human recombinant GPIIIa is able to express on the cell surface associated with endogenous α subunits. Whether normal or mutated GPIIb cDNA was transfected, the rate of surface exposure of GPIIb–IIIa complexes was similar, indicating that the R63K substitution does not perturb the formation and intracellular trafficking of GPIIb–IIIa complexes. The propositus suffers from a bleeding disorder that appeared immediately after birth, diagnosed as Glanzmann's thrombasthenia (GT) based on its clinical and analytical features. The low (≤ 10%) platelet content of GPIIb–IIIa complexes assessed by flow cytometry as well as by immunoblotting confirmed the proband was indeed a type II GT. The normal platelet content of αvβ3 in the proband and her parents suggested that the thrombasthenic phenotype was caused by a deficit of GPIIb. The molecular genetic analysis of the GPIIb and GPIIIa genes revealed the presence of a G→A homozygous transition at position 188 of GPIIb that changes the consensus sequence of the splicing site at the exon 1–intron 1 junction. The predicted effect of this mutation is the R63K substitution in GPIIb. The pathogenic significance of this mutation is supported by the following observations: (i) the [G188A]GPIIb mutation was not found in a significant number of DNAs from normal blood donors, suggesting it is not a polymorphism; (ii) both parents were heterozygotes for the [G188A]GPIIb mutation and a correlation existed between availability of GPIIb mRNA, platelet GPIIb–IIIa content, and number of mutated alleles; (iii) no other mutations than [G188A]GPIIb were found in the propositus or her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3′-UTR, and intronic flanking regions of GPIIb. Since arginine and lysine are both positively charged residues, in principle no significant functional perturbation of GPIIb should be derived from their substitution. However, the possibility existed that [63K]GPIIb could complex GPIIIa less efficiently, or else that intracellular trafficking of the mutated complexes was hindered. Transient cotransfection of either normal [63R]GPIIb or mutant [63K]GPIIb into CHO cells stably expressing GPIIIa showed identical rates of surface expression of GPIIb–IIIa complexes. On the other hand, the platelets from the patient showed a low, yet significant, agonist-induced binding of fibrinogen consonant with the reduced number of surface GPIIb–IIIa complexes. These observations indicate that the thrombasthenic phenotype of the patient is caused by a limited availability of [63K]GPIIb, due to diminished steady-state levels of GPIIb mRNA. The reason for the reduced levels of platelet [63K]GPIIb mRNA is a challenging question considering that its translation product is a functional GPIIb protein and therefore there is no apparent physiological justification to have it eliminated. The first two and last three nucleotides of an exon define the recognition of 3′ and 5′ splice sites, respectively [13Mount S.M. A catalogue of splice junction sequences.Nucl Acids Res. 1982; 10: 459-72Crossref PubMed Scopus (2784) Google Scholar, 14Oshima Y. Gotoh Y. Signals for the selection of a splice site in pre-mRNA. Computer analysis of splice junction sequences and like sequences.J Mol Biol. 1987; 195: 247-59Crossref PubMed Scopus (185) Google Scholar]. Since the [G188A]GPIIb mutation alters the sequence of the 5′ donor splicing site of intron 1, we investigated whether this mutation could somehow perturb the processing of RNA so as to account for the low steady-state levels of GPIIb mRNA. The analysis of platelet GPIIb transcripts from the patient revealed the presence of alternative spliced forms as a result of usage of a cryptic splicing site, leading to insertion of intronic sequences and the appearance of a premature STOP codon. Since these forms were not detected in normal platelets, it was considered of interest to elucidate whether the reduced mRNA GPIIb was related to the unusual alternative splicing activity by using the exontrap analysis of genomic DNA fragments encompassing either the normal or mutated exon 1 of GPIIb. The exontrap analysis demonstrated unambiguously that the presence of the [G188A]GPIIb mutation provoked alternative splicing and a drastic reduction in the yield of normal transcripts, as it was observed in the patient's platelets. Although the latter observation strongly supports the etiopathogenic significance of the G188A mutation, the molecular mechanism for the reduced amount of mRNA is not apparent. In principle, two possibilities could be considered: (i) first, the splicing machinery did operate at subnormal rates on the mutated splicing site; (ii) second, a nearby intronic splicing sequence could be used preferably instead of the mutated exon AA signal (Fig. 3), with the result of insertion of an in-frame STOP codon and messenger instability. In general, mutations on mRNA splicing associate more frequently with exon skipping than with cryptic splice site usage [15Dietz H.C. Valle D. Francomano C.A. Kendzior Jr, R.J. Pyeritz R.E. Cutting G.R. The skipping of constitutive exons in vivo induced by nonsense mutations.Science. 1993; 259: 680-3Crossref PubMed Scopus (359) Google Scholar]. However, for 5′ splice site mutations cryptic splice site usage is favored provided that the sites, as in our case, are located in the vicinity and exhibit sufficient homology with the consensus sequence [16Krawczak M. Reiss J. Cooper D.N. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences.Hum Genet. 1992; 90: 41-54Crossref PubMed Scopus (1121) Google Scholar]. In a significant number of the GPIIb transcripts from the proband a cryptic splice site was used with the result of a RNA containing an intron-derived nonsense codon that most likely triggers a ‘nonsense-mediated decay’ (NMD) mechanism of RNA reduction [17Maquat L.E. When cells stop making sense: effect of nonsense codons on RNA metabolism in vertebrate cells.RNA. 1995; 1: 453-65PubMed Google Scholar]. This alternative splicing mechanism could explain the reduced number of GPIIb transcripts as well as the observed ratio of alternative spliced forms. The distinction between a normal and a premature termination codon that elicits a reduction in mRNA abundance is related to its position relative to 3′-most exon–exon junction. Thus, a termination codon mediates reduction in mRNA only if it is located at least 50–55 nucleotide positions upstream of the most-3′ exon–exon junction [18Cheng J. Belgrader P. Zhou X. Maquat L.E. Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance.Mol Cell Biol. 1994; 14: 6317-25Crossref PubMed Scopus (121) Google Scholar]. Nonsense mutations have been previously reported associated with thrombasthenic phenotypes causing either no change [19Vinciguerra C. Khelief A. Alemany M. Morle F. Grenier C. Uzan G. Guling D. Dechavanne M. Negrier C. A nonsense mutation in the GPIIb heavy chain (Ser870→STOP) impairs platelet GPIIb–IIIa expression.Br J Haematol. 1996; 95: 399-407Crossref PubMed Scopus (21) Google Scholar, 20Tomiyama Y. Kashiwagi H. Kosugi S. Shigara M. Kanayama Y. Kurata Y. Matsuzawa Y. Abnormal processing of the glycoprotein IIb transcript due to a nonsense mutation in exon 17 associated with Glanzmann's thrombasthenia.Thromb Haemost. 1995; 73: 756-62Crossref PubMed Scopus (31) Google Scholar] or marked reduction [21González-Manchón C. Fernández-Pinel M. Arias-Salgado E.G. Ferrer M. Alvarez M.V. García-Muñoz S. Ayuso M.S. Parrilla R. Molecular genetic analysis of a compound heterozygote for the GPIIb gene associated with Glanzmann's trombasthenia. Disruption of the 674–687 disulfide bridge in GPIIb prevents surface expression of GPIIb–IIIa.Blood. 1999; 93: 866-75Crossref PubMed Google Scholar, 22Kato A. Yamamoto K. Miazaki S. Jung S.M. Moroi M. Aoki N. Molecular basis for Glanzmann's thrombasthenia (GT) in a compound heterozygote with glycoprotein IIb gene. A proposal for classification of GT based on the biosynthetic pathway of glycoprotein Iib–IIIa complex.Blood. 1992; 79: 3212-8Crossref PubMed Google Scholar, 23Arias-Salgado E.G. Tao J. González-Manchón C. Butta N. Vicente V. Sanchez-Ayuso M. Parrilla R. Nonsense mutation in exon-19 of GPIIb associated with thrombasthenic phenotype. Failure of GPIIb (Δ597–1008) to form stable complexes with GPIIIa.Thromb Haemost. 2002; 87: 684-91Crossref PubMed Scopus (16) Google Scholar, 24Gu J.M. Xu W.F. Wang X.D. Wu Q.Y. Chi C.W. Ruan C.G. Identification of a nonsense mutation at amino acid 584-arginine of platelet glycoprotein IIb in patients with type I Glanzmann thrombasthenia.Br J Haematol. 1993; 83: 442-9Crossref PubMed Scopus (34) Google Scholar] in the cytoplasmic content of GPIIb or GPIIIa transcripts. Mutations in exons 2, 17, or 19 of GPIIb decreased mRNA levels [11Tao J. Arias-Salgado E.G. Gonzalez-Manchon C. Diaz-Cremades J. Ayuso M.S. Parrilla R. A novel [288delC] mutation in exon 2 of GPIIb associated with type I Glanzmann thrombasthenia.Br J Haematol. 2000; 111: 96-103Crossref PubMed Scopus (9) Google Scholar, 21González-Manchón C. Fernández-Pinel M. Arias-Salgado E.G. Ferrer M. Alvarez M.V. García-Muñoz S. Ayuso M.S. Parrilla R. Molecular genetic analysis of a compound heterozygote for the GPIIb gene associated with Glanzmann's trombasthenia. Disruption of the 674–687 disulfide bridge in GPIIb prevents surface expression of GPIIb–IIIa.Blood. 1999; 93: 866-75Crossref PubMed Google Scholar, 23Arias-Salgado E.G. Tao J. González-Manchón C. Butta N. Vicente V. Sanchez-Ayuso M. Parrilla R. Nonsense mutation in exon-19 of GPIIb associated with thrombasthenic phenotype. Failure of GPIIb (Δ597–1008) to form stable complexes with GPIIIa.Thromb Haemost. 2002; 87: 684-91Crossref PubMed Scopus (16) Google Scholar, 24Gu J.M. Xu W.F. Wang X.D. Wu Q.Y. Chi C.W. Ruan C.G. Identification of a nonsense mutation at amino acid 584-arginine of platelet glycoprotein IIb in patients with type I Glanzmann thrombasthenia.Br J Haematol. 1993; 83: 442-9Crossref PubMed Scopus (34) Google Scholar], whereas nonsense mutations described in exons 26 and 28 had no apparent effect on the amounts of mRNA [19Vinciguerra C. Khelief A. Alemany M. Morle F. Grenier C. Uzan G. Guling D. Dechavanne M. Negrier C. A nonsense mutation in the GPIIb heavy chain (Ser870→STOP) impairs platelet GPIIb–IIIa expression.Br J Haematol. 1996; 95: 399-407Crossref PubMed Scopus (21) Google Scholar, 20Tomiyama Y. Kashiwagi H. Kosugi S. Shigara M. Kanayama Y. Kurata Y. Matsuzawa Y. Abnormal processing of the glycoprotein IIb transcript due to a nonsense mutation in exon 17 associated with Glanzmann's thrombasthenia.Thromb Haemost. 1995; 73: 756-62Crossref PubMed Scopus (31) Google Scholar]. The observed differences comply with the rule of the termination codon position, i.e. the closer the nonsense codon to the 5′ end of the coding sequence, the lower the amount of transcript [25Cooper D.N. Human gene mutations affecting RNA processing and translation.Ann Med. 1993; 25: 11-7Crossref PubMed Scopus (103) Google Scholar]. Despite its physiological importance, the precise mechanism(s) for reducing the abundance of mutant messengers remains elusive [26Mitchell P. Tolervey D. mRNA turnover.Curr Opin Cell Biol. 2001; 13: 320-5Crossref PubMed Scopus (138) Google Scholar]. It is clear that the recognition of premature termination codons is dependent upon mRNA splicing and therefore it is a nuclear event. A competition appears to exist between splicing and nuclear degradation pathways. Multimeric complexes including splicing factors, nucleoproteins rich in arginine and serine (RS repeats), and shuttling proteins, bind to sites located near the splice site. Aberrant RNAs containing defective marks that escape to the cytoplasm are rapidly degraded. A complex interplay seems also to exist between mRNA turnover and translation, indicating that the process of mRNA surveillance and turnover is far from being understood. To conclude, we report the molecular genetic and functional studies of a novel splice junction mutation in GPIIb that changes R63K, associated with type II Glanzmann's thrombasthenia. The mutant mRNA encodes a normal full-length functional GPIIb protein but the diminished steady-state levels of GPIIb mRNA limit the availability of GPIIb and therefore the surface expression of GPIIb–IIIa complexes. The underlying molecular mechanism for the reduced availability of mRNA seems to be caused by the predominant usage of cryptic splicing sites located in the immediate vicinity of the mutated site. These alternative forms of processing produce an in-frame insertion of a translation termination codon that most likely activates the nonsense-mediated mRNA decay. This work was supported in part by grants from the Dirección General de Investigación (PM99-0095, PB97-1240 and SAF 2000-0127), Fondo de Investigaciones Sanitarias (96/2014), and Comunidad Autónoma de Madrid (08.4/0031/1998). R.F. was supported in part by the French Society of Pediatrics Pathology." @default.
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• W2022307125 title "A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia" @default.
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