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Identification of a Novel Molecular Variant in the PTGS1 Gene that Causes a Loss of a Glycan in the Catalytic Domain, Defects in Platelet Thromboxane A2 Synthesis, and Bleeding

1Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CB15/00055-CIBERER, Murcia, Spain, 2Blizard Institute &London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom, 3Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CB15/00055-CIBERER., Murcia, Spain, 4Unidad de Trombosis y Hemostasia. H. Universitario de Salamanca-IBSAL, Salamanca, Spain, 5Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT)/CIBERER, Madrid, Spain, 6Servicio de Hematología, Hospital San Pedro de Alcántara, Complejo Universitario de Cáceres, Caceres, Spain, 7Servicio de Hematología y Hemoterapia, Hospital Virgen de la Salud, Complejo Hospitalario de Toledo, Toledo, Spain, 8National Institutes of Health, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States

Abstract Number: PB1506

Meeting: ISTH 2020 Congress

Theme: Platelet Disorders and von Willebrand Disease » Platelet Function Disorders, Hereditary

Background: Thromboxane A2 [TxA2] is generated from arachidonic acid [AA] by cyclooxigenase-1 (COX1) and thromboxane synthase. To date, few patients with mutations in PTGS1, which encodes COX1, have been identified (< 5cases).

Aims: Characterization of a patient with aspirin-like platelet defect enrolled in the Spanish project “Functional/molecular characterization of patients with Inherited Platelet Disorder”.

Methods: The index case is a 13-year-old adopted girl of Asian origin, referred because of moderate chronic bleeding (BAT-ISTH=6). We performed regular platelet phenotyping (aggregation [LTA], western-blot [WB], immunofluorescence [IF] assays). TxA2 and eicosanoids synthesis was measured by enzyme-immunoassayand and LC-MS. Patient DNA was analyzed by HTS-gene panel. Recombinant WT and mutant COX1 generated by site directed mutagenesis were expressed in 293T HEK cells. N-glycosylation analysis included PNGase-F treatment.

Results: The index case had normal platelet size and count. LTA was absent with AA (1.6mM), but normal with U46619 (5mM) suggesting a defect in TxA2 synthesis, which was confirmed in LTA supernatants (< 10% vs.controls) as well as in agonist-stimulated whole blood (10-50%). COX1 level (WB & IF) was normal, but enzyme displayed faster electrophoretic mobility. HTS analysis revealed a heterozygous variant c.428A>G, p.Asn143Ser located in the catalytic domain of COX1 which affects one of the three predicted N-glycosylation sequons (Asn-X-Ser) of PTGS1. HEK 293T cells transfected with p.Asn143Ser mutant showed no AA-induced TxA2 synthesis (WT:500ng/mL; Asn143Ser:≈50-75ng/mL) and a COX1 band with faster electrophoretic mobility. In cells co-transfected with WT and p.Asn143Ser mutant, PNGaseF treatment results in a single migration band, confirming a defective N-glycosylation. Moreover cells transfected with p.Ser145Ala mutant generate another hypoglycosylated variant that also exhibited defective TxA2-synthesis.

Conclusions: We have identified a novel COX1 variant p.Asn143Ser which did not alter enzyme expression, but abrogated TxA2 synthesis and impaired platelet function. These data emphasize the relevance of N-glycans in the catalytic domain of this key constitutive enzyme. [PI17/01311-FMM AP172142019]

To cite this abstract in AMA style:

Palma-Barqueros V, Crescente M, Chan M, de la Morena-Barrio ME, Bastida JM, Bohdan N, Almarza E, Suarez-Varela S, Casas-Aviles I, Padilla J, Rodriguez-Alen A, Marin-Quilez A, Revilla N, Vicente V, Corral J, Edin ML, Zelding D, Lozano ML, Warner T, Rivera J. Identification of a Novel Molecular Variant in the PTGS1 Gene that Causes a Loss of a Glycan in the Catalytic Domain, Defects in Platelet Thromboxane A2 Synthesis, and Bleeding [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/identification-of-a-novel-molecular-variant-in-the-ptgs1-gene-that-causes-a-loss-of-a-glycan-in-the-catalytic-domain-defects-in-platelet-thromboxane-a2-synthesis-and-bleeding/. Accessed October 2, 2023.

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