A novel genetic variant in PTGS1 affects N‐glycosylation of cyclooxygenase‐1 causing a dominant‐negative effect on platelet function and bleeding diathesis (2020)

During platelet activation, arachidonic acid (AA) is released from membrane phospholipids and metabolized to thromboxane A2 (TXA2) through the actions of cyclooxygenase-1 (COX-1) and TXA2 synthase. Note, TXA2 binds to the platelet TXA2 receptor, causing shape change, secretion and platelet aggregation.1 Also, COX-1 (599aa; 70 kDa) has cyclooxygenase and peroxidase activities and it is functionally active as a homodimer, with each COX-1 monomer consisting of four highly conserved domains: an N-terminal signal peptide, a dimerization domain, a membrane-binding domain (MBD) and a large C-terminal catalytic domain2 (Figure 1A). Irreversible COX-1 inhibition by aspirin is a widely established anti-platelet therapy in cardiovascular disease.3 Despite the physiological and clinical relevance of platelet COX-1, few patients with congenital COX-1 defect (Bleeding Disorder Platelet Type 12; OMIM: 605735) have been characterized.2 In the 1970s some patients with mild bleeding diathesis, impaired aggregation and blunted TXA2 synthesis were described, but with no genetic confirmation. To date, only a few cases with uncommon genetic variants in PTGS1, the gene encoding COX-1, have been reported, without detailed study of their associated platelet phenotype. A patient with severe bleeding carried the PTGS1 common single nucleotide variants (SNVs) c.22C>T [p.Arg8Trp] and c.50C>T (p.Pro17Leu)2 (gnomAD, minor allele frequency [MAF] = 0.0730 and 0.0588, respectively). Heterozygosity for the p.Pro17Leu variant also aggravated hemorrhage in a pedigree with hemophilia A.2 Another patient showing bleeding and reduced TXA2 synthesis was heterozygous for the rare PTGS1 variants c.337C>T (p.Arg113Cys) and c.1003G>A (p.Val481Ile) (MAF = 0.0000597 and 0.00718, respectively).2 Recently, the BRIDGE Consortium reported a pedigree with an autosomal recessive novel variant c.965G>C (p.Trp322Ser) that abrogated COX-1 expression resulting in aspirin-like platelet dysfunction.4 Here, we report a patient presenting with a lifelong mild bleeding tendency and platelet dysfunction, associated to heterozygosity for the novel PTGS1 variant c.428A>G (p.Asn143Ser). The distinguished feature of this rare SNV is that it disturbs one N-glycosylation sequon at the catalytic domain of COX-1 causing the expression of a dysfunctional hypo-glycosylated COX-1 protein with an apparent dominant-negative effect. The methodological details of this investigation are provided in supplemental material. The proband was a 13-year-old teen of Asiatic origin, adopted by a Spanish family, presenting with a lifelong moderate bleeding diathesis. There was bruising and petechiae, being more frequent after minor traumas; recurrent epistaxis sometimes associated with nonsteroidal anti-inflammatory drug (NSAIDs) intake; menorrhagia, once requiring tranexamic acid and desmopressin treatment; excessive bleeding after surgical procedures such as tonsillectomy(ISTH-BAT score 6). On repeated testing, she showed normal blood cell counts including platelet number and volume (Table S1 in Appendix S1) and no visible abnormalities in blood smears. Blood biochemical and coagulation parameters were within normal ranges. No clinical signs of relevant organ/tissue, immune or cognitive dysfunction were present. Detailed platelet phenotyping was performed that showed; (a) pathological PFA-100 closure time for the collagen/epinephrine cartridge (Table S1), (b) normal platelet receptor expression, reduced (50%) agonist-induced fibrinogen binding and impaired agonist-induced α-granule (10%–30%) and δ-granule (25%–50%) secretion (P-selectin and CD63 release, respectively) (Figure S1A–C in Appendix S1), (c) proband's platelets showed absent with AA-induced aggregation and normal with U46619, a stable TXA2 analog, indicating unaffected signaling downstream the TXA2 receptor (Figure 1B); platelet aggregation with other agonist was normal or slightly reduced (Figure S1D). Also, (d) markedly reduced platelet synthesis of TXA2 in the supernatant of the LTA experiments (<10% of the control levels) (Figure S2A), and (e) reduced synthesis of all COX-1-dependent eicosanoids in whole blood stimulated with collagen or PAR1p (Figure S2B and Table S2). Overall, this clinical and platelet phenotype resembles that of the few previously reported cases with COX-1 defect.2 Analysis of proband's DNA with a HTS-gene panel,5 revealed a heterozygous variant c.428A>G (p.Asn143Ser) in the exon 5 of the PTGS1 gene (ENST00000362012), which was confirmed by Sanger sequencing. The proband's biological parents were not available to assess the inheritance pattern of the variant. This variant is not reported in gnomAD or 1000 genomes project databases, but a c.429C>G [p.Asn143Lys] change is listed in the gnomAD collection with a very low allele frequency of 3.98 x 10−6. As highlighted in Figure 1A, the novel p.Asn143Ser substitution is located at the N-terminal of the COX-1 catalytic domain and affects a highly conserved residue (Genomic Evolutionary Rate Profiling conservation score = 5.59; https://varsome.com/) (Figure S3A). Remarkably, Asn143 is one of the three predicted N-glycosylation sites in COX-1 (Figure 1A). The recent and firstly resolved human COX-1 crystal by Scilimati et al. (https://www.rcsb.org/structure/6Y3C) (Figure S3B) confirmed that Asn143 is decorated by an N-glycan chain. Changing Asn to Ser would theoretically abolish this N-glycosylation resulting in a hypo-glycosylated protein with unknown consequences in folding, secretion, stability and enzymatic activity. Following the ACMG standards, the current verdict for the p.Asn143Ser variant is of uncertain significance, despite most computational analysis in silico predict a deleterious effect (https://varsome.com/) (Table S3). Immunofluorescence analysis of platelets and leukocytes showed no apparent impairment in COX-1 expression in the proband (Figure S4). However, 8% SDS-PAGE of platelet lysates and immunoblotting analysis revealed a second COX-1 protein band with lower molecular weight in the proband, which is consistent with the predicted hypo-glycosylated COX-1 variant (Figure 1C). In contrast to these findings, a recent study has showed that recessive inheritance of the 965G>C (p.Trp322Ser) missense variant in PTGS1, results in loss of COX-1 platelet expression instead of COX-1 loss-of function.4 Strong evidence of the deleterious effect of p.Ans143Ser variant in COX-1 glycosylation and its enzyme activity was obtained in HEK 293 T cells transfected with either wild-type (Asn143) or mutated (Ser143) enzyme vector (Table S4). In this cell model, the variant Ser143 COX-1 displayed higher electrophoretic mobility than wild-type Asn143 COX-1, as was expected for a hypo-glycosylated enzyme with reduced molecular mass. Moreover, cells co-transfected with both Asn143 and Ser143 COX-1, simulating heterozygosis status, had a COX-1 double protein band similar to that observed in patient´s platelet lysates (Figure 1D). Importantly, this COX-1 double band disappeared upon treatment of the lysates from co-transfected cells with N-glycosidase F (Figure 1E). As shown in Figure 1F, cells expressing wild-type COX-1 (Asn143) responded to 500 μM AA stimulation with a notable TXB2 production whilst cells transfected with mutant Ser143 COX-1 displayed almost no TXB2 synthesis (194 ± 48.2 pg/ml), resembling untransfected cells (UNT) (169.0 ± 24.8 pg/ml). Interestingly, cells co-transfected with equal amounts of wild-type (Asn143) and mutant Ser143 COX-1 (1.5 μg of each vector) showed 70% reduced TxB2 synthesis in comparison to cells transfected with 1.5 μg of wild-type COX-1 vector (Asn143 + Ser143, 907.5 ± 888.9 pg/ml vs Asn143, 2906.9 ± 3736.1) (Figure 1F). These results were confirmed in a separate series of experiments in which cells transfected with Asn143 COX-1 vector (1.5 or 3 μg) produced TXB2 in a dose-dependent manner when stimulated with increasing concentrations of AA (0.5-10 μM), while in cells transfected with the mutant Ser143 this synthesis was abrogated. Remarkably, co-transfected cells (Asn143+Ser143) displayed higher than 50% reduction in TXB2 synthesis, as compared to cells solely transfected with an equivalent dose of Asn143 COX-1 (Figure 1G). These data strongly support that the p.Ans143Ser variant has a dominant-negative effect in COX-1 activity. To further test this, we established a second model in which HEK 293 cells were first stably transfected with either mutant Ser143 or wild-type Asn143 COX-1, and then they were transiently transfected with the alternative construct. The TXB2 synthesis was abolished in cells stably expressing Ser143 COX1 and co-expression of wild-type Asn143 COX-1 partially recovered TXB2 production, but following a non-linear dose-dependent pattern (Figure 1H). Similar results were found in the opposite cell model (Figure S5). Noteworthy, introduction in HEK 293 T cells of a different variant, p.Ser145Ala, disrupting the same consensus N-glycosylation sequence (Asn-X-Ser/Thr) in COX-1, also resulted in null AA-induced TXA2 synthesis (Figure 1F) and in the expression of the hypo-glycosylated COX-1 protein also disappearing after treatment with N- glycosidase F (Figure 1D,E). Overall, these results obtained in the patients platelets and in transfected cells reflected that the p.Asn143Ser variant has a dominant-negative effect, and its deleterious action exceeded that of simply haploinsufficiency of functional COX-1 protein. Since COX-1 is active as a homodimer, we can speculate that a mechanism contributing for such a dominant negative effect is an impaired activity of both wild-type/mutant and mutant/mutant dimers. Indeed, dimerization of glycosylated and non-glycosylated monomers of COX-2, an isoform of COX-1, has been reported and this is more energetically favorable than the dimerization between two N-glycosylated monomers.6 Additionally, it may also be possible that the loss of N-glycans reduces the steric hindrance favoring the access of AA into the catalytic domain of the non-functional mutant COX-1, as suggested by the blunted TXA2 synthesis, at low AA concentration, in cells co-transfected with equivalent levels of mutant Ser143 COX-1 and wild-type COX-1. Of note, a similar dominant negative mechanism has been described for the London variant of antithrombin.7 To our knowledge, three N-glycosylation sequons are predicted in human COX-1 at positions 67, 143 and 409,8 and the recent resolution of a human COX-1 crystal structure by Scilimati et al. (PDB:6y3) provided the first experimental evidence that Asn143 is decorated by an N-glycan chain. Our results demonstrate, for the first time, that the N-glycosylation at 143 is functionally relevant as its disruption, by mutation of Asn143 or Ser145 residues, results in a smaller and dysfunctional COX-1 variant. In agreement with our results, Otto et al9 has demonstrated that ovine COX-1 is N-glycosylated at positions Asn68, Asn144 and Asn410, the orthologous residues to the human ones, and removal of these N-glycans also abolishes the cyclooxygenase and peroxidase activity of hypo-glycosylated ovine COX-1 mutants.9 Mechanistically, glycans can influence protein folding, cellular trafficking and secretion, substrate binding affinity, proteolysis or clearance and the catalytic activity of proteins.10 Indeed, the relevance of glycosylation in the function of proteins of the haemostatic system such as FXI,11, 12 antithrombin,13 VWF10, protein S14 or the platelet P2Y12 ADP receptor,15 has been shown. In summary, this work extends the genetic spectrum of COX-1 defects, by the characterization of a patient with a novel variant c.428A>G in PTGS1. Studies performed on the patient's platelets and in cell models showed that this variant affects the post-transductional COX-1 N-glycosylation and leads to expression of a hypo-glycosylated protein with a dominant-negative effect in COX-1 enzymatic activity. The COX-1 hypo-glycosylation is unveiled as a new dominant-negative mechanism causing congenital platelet function disorder. More broadly, our study strengthens the relevance of N-glycans in human health and disease. This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) & Feder (PI17/01311, PI20/00926, PI18/00598, PI17/01966 and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019) and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica). TDW research is supported by a grant from the British Heart Foundation (PG/17/40/33028). VPB has predoctoral contract from CIBERER. MEMB has a postdoctoral contract from University of Murcia. AMQ holds a predoctoral grant from the Junta de Castilla y León. The corresponding author research on inherited platelet disorders is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Hemostasis (SETH). Verónica Palma-Barqueros, Marilena Crescente, María Eugenia de la Morena, Melissa V. Chan, Elena Almarza, Natalia Bohdan, Antonia Miñano, José Padilla, Ana Marín-Quilez, and José Rivera, performed platelet function studies. Verónica Palma-Barqueros, María Eugenia de la Morena, Elena Almarza, Cristina Mesa-Nuñez, Carlos Damian, Javier Corral, and, Irene Martínez-Martínez, participated in cell models design and assays., Marilena Crescente, Melissa V. Chan, Harriet E. Allan, Tania Maffucci, and, Timothy D. Warner, did TXA2 measurements, cell immune-staining and immunoblotting. Matthew L. Edin, and, Darryl C. Zeldin, performed eicosanoid measurements and analysis. Natalia Bohdan, and Ignacio Casas-Aviles recruited the patient and perform clinical management. Nuria Revilla, Agustín Rodríguez-Alen, Natalia Bohdan, Ignacio Casas-Aviles, Vicente Vicente, María L. Lozano, José María Bastida and José Rivera examined clinical data. Verónica Palma-Barqueros, Rocío Benito, José R. González-Porras, Jesús María Hernández-Rivas, José María Bastida & José Rivera performed and assessed molecular studies. Verónica Palma-Barqueros, Marilena Crescente, Timothy D. Warner, María L. Lozano, José María Bastida and José Rivera wrote the manuscript. All authors helped in data interpretation and critically reviewed the paper. The authors declare no conflict of interest. The views expressed in this manuscript are those of the authors and do not necessarily represent the views of the National Heart, Lung, and Blood Institute; the National Institutes of Health; or the U.S. Department of Health and Human Services. This study followed the Helsinki Declaration and had the approval of the Ethics Committees of the Instituto de Investigación Biomédica (IBSAL, Salamanca, Spain) and Hospital Universitario Reina Sofía (Murcia, Spain). The patient, and control subjects, provided written informed consent. Appendix S1: Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.