A novel genetic variant in <scp> <i>PTGS1</i> </scp> affects N?glycosylation of cyclooxygenase?1 causing a dominant?negative effect on platelet function and bleeding diathesis

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