Caracterización molecular, funcional y clínica de la deficiencia congénita de antitrombinamejoras en el diagnóstico y en la comprensión de su fisiopatología

Résumé

Antithrombin is a key N-glycoprotein in the haemostatic system due to its anticoagulant activity, a function amplified by heparin. Antithrombin is encoded by the SERPINC1 gene. Genetic variants in SERPINC1 are responsible for more than 75% of cases of antithrombin deficiency, a dominant disorder that constitutes the main and most serious hereditary thrombophilia. The correct diagnosis of antithrombin deficiency is important not only to know the molecular basis of thrombosis and to facilitate family screening, but also because it helps in the clinical management of the patient, including the duration and type of anticoagulant treatment. Although the disorder has been known for almost 60 years, challenges remain in antithrombin deficiency. Thus, this disorder may be underestimated, due to the limitations of available, mostly functional, diagnostic methods. Moreover, the molecular basis of the clinical heterogeneity in patients with antithrombin deficiency is poorly understood. AIMS 1. To compare different methods of molecular diagnosis of antithrombin deficiency, showing their weaknesses and strengths. 2. To define the functional and clinical effect of variants in SERPINC1 affecting arginine residues potentially involved in the interaction with heparin. 3. To identify natural variants in SERPINC1 that generate new N-glycosylation sequences, and to characterise their functional consequences and clinical impact. 4. Characterisation of the functional and pathological effect of new mutations in SERPINC1 identified by non-targeted sequencing in cohorts of patients with thrombosis. METHODOLOGY - We analysed plasma antithrombin and the SERPINC1 gene in 663 patients, 588 patients with suspected antithrombin deficiency and 75 COVID patients in whom the whole exome was sequenced. - We identified SERPINC1 variants in databases (HGMD®, GnomAD and in published manuscripts), which were analysed by assessing secretion, glycosylation and function of the variant antithrombin generated by targeted mutagenesis and expressed in HEK-EBNA cells. Both plasma and recombinant antithrombins were analysed by Western blot, PNGase F digestion, functional assays with different proteases, chromogenic assays, cross-linked immunoelectrophoresis, and ELISA. Molecular study of SERPINC1 was performed by MLPA, Sanger sequencing and nanopore. RESULTS/CONCLUSIONS - Structural variants account for 5-8% of cases with antithrombin deficiency. The combination of a point mutation and a structural variant, if present, is very rare in antithrombin deficiency. - The identification of false positives by MLPA advises careful design of probes to avoid polymorphic regions and validation of any positive result. - Mutations of arginine residues in antithrombin have heterogeneous consequences, both biochemically and clinically. - We identified the third heterozygosis composed of variants affecting heparin affinity, with very high thrombotic risk. - The generation of new N-glycosylation sequences in SERPINC1 resulting from natural mutations has considerable heterogeneity in terms of occupancy, functional consequences (secretion or heparin affinity defects) and clinical implications (generating moderate or severe deficiencies). - Glycosylation efficiency could be modulated by modifying glucose availability. - There are many SERPINC1 variants identified by gene sequencing in thrombosis patients that are not easily detected by commercial functional methods. - Our results recommend the development of an antithrombin deficiency assessment system based on FVIIa inhibition.