Expanding the continuum of antithrombin deficiencynew insights on this severe thrombophilia via the comprehensive analysis of cases in atypical or emerging clinical scenarios

Abstract

INTRODUCTION. Antithrombin is an anticoagulant serpin that plays a key role in haemostasis. Antithrombin deficiency (activity<80%) is a severe thrombophilic state. More than 400 variants have been described in SERPINC1, the gene encoding antithrombin. Characterization of many of these variants has generated plenty of knowledge. AIMS. To decipher new mechanisms involved in antithrombin deficiency by the characterization of cases in atypical/emergent scenarios, with four specific lines of research: 1. To identify and characterize cases with transient antithrombin deficiency. 2. To assess the frequency of inferior vena cava defects in patients homozygous for antithrombin Budapest 3 (p.Leu131Phe). 3. To characterize a family with antithrombin deficiency and a strong history of embryo-foetal deaths. 4. To analyse the association between inherited thrombophilias and COVID-19 complications. METHODS. Study of a cohort of 444 unrelated cases with antithrombin deficiency, recruited from 1998 to 2021. Plasma antithrombin was characterized by functional and biochemical methods. SERPINC1 was studied by Sanger&apos;s or next generation sequencing and multiplex ligation-dependent probe amplification. In line of research 1, we defined cases with transient antithrombin deficiency as those that alternated normal and defective functional values of antithrombin. In line 2, we selected cases homozygous for antithrombin Budapest 3, from 1118 patients (index cases and relatives) from two different cohorts (Spain, N=692; Hungary, N=426) for abdominal image exam. In line 3, we focused on a family with antithrombin deficiency and a strong history of embryo-foetal deaths. In line 4, two designs were followed to identify cases with thrombophilia and SARS-CoV-2 infection: 1) retrospective analysis of SARS-CoV-2 infection in subjects with prior diagnosis of antithrombin deficiency (N=331), and 2) whole exome sequencing of patients <75 years old hospitalized with SARS-CoV-2 pneumonia (N=87). RESULTS. The results of each line of research are summarized: 1. A total of 139 cases from our cohort presented transient antithrombin deficiency, and 61 (43.9%) of them had molecular defects: 48 had SERPINC1 variants, and 13 had hypoglycosylation. Two main mechanisms explained transient deficiency: the limitation of current functional methods to detect some variants and the influence of external factors on the pathogenic consequences of these mutations. 2. Twenty-four of 61 homozygous Budapest 3 subjects recruited had an available image exam. Atresia of the inferior vena cava system was observed in 17 (70.8%) of these cases. Short tandem repeat analysis supported the specific association of the vascular malformation with the SERPINC1 variant. 3. Two different mutations were identified in this family: a severe quantitative variant (c.1154-14G>A), and antithrombin Budapest 3. Molecular characterization of a dead embryo revealed a compound heterozygosis for the variants, which may have led to extremely severe antithrombin deficiency and embryonic lethality. 4. Fifteen patients with thrombophilia and SARS-CoV-2 infection were identified. Increased at-admission levels of D-dimer were found in patients with severe thrombophilia. Patients with prior thrombotic history (N=5), all on anticoagulant therapy, did not develop symptomatic trombosis during infection. In contrast, one newly identified protein C-deficient patient, without thrombotic history, had a pulmonary embolism two days after hospital admission. CONCLUSIONS. Our work demonstrated that antithrombin deficiency is underestimated. We have expanded the clinical and molecular continuum of antithrombin deficiency: from the surreptitious behaviour of transient deficiencies to vascular malformations and embryonic death in the most extreme cases with compound SERPINC1 defects. Novel forms and mechanisms of antithrombin deficiency have been identified. Finally, SARS-CoV-2 infection has been confirmed as an emergent prothrombotic trigger for which patients with this and other inherited thrombophilias may be a high-risk population. Altogether, this work highlights the importance of the molecular analysis of antithrombin deficiency, not only for a better understanding of thrombosis biology, but also in terms of clinical implications.