Molecular and Biological Characterization of Transient Antithrombin Deficiency: A New Concept in Congenital Thrombophilia

Antithrombin deficiency, mainly but not exclusively due to SERPINC1 gene variants, is a major thrombophilia that is significantly associated to early-onset venous thromboembolism. The diagnostic algorithm of antithrombin deficiency relies on the classical biochemical-molecular sequence, adopted for all thrombophilic states. Therefore, genetic analysis of SERPINC1 is restricted to cases with confirmed antithrombin deficiency, that is, cases with at least two positive findings by using functional assays or with other relatives carrying this disorder. This strategy has enabled the identification of gene variants in up to 80% of cases with antithrombin deficiency and rendered plenty of both biochemical and genetic knowledge about antithrombin. Furthermore, defects of N-glycosylation underlie a proportion of cases with antithrombin deficiency that is not explained by SERPINC1 variants.

Nevertheless, diagnosis of antithrombin deficiency still encloses some uncertain puzzling questions. Although the prevalence of antithrombin deficiency is tremendously low, the use of high-throughput nucleotide sequencing for genetic analysis of SERPINC1 in consecutive patients with thromboembolic events suggests that antithrombin deficiency might be and underestimated disorder that surreptitiously increases thrombotic risk. Additionally, false negative results by using functional methods for antithrombin deficiency screening have been reported, all involving type II deficiencies.

The present study aimed to identify gene defects and mechanisms involved in a specific type of antithrombin deficiency that might be elusive to an easy diagnosis to the classic diagnostic strategy. We addressed this aim with an original approach, the selection of cases with at least a positive finding by functional methods that however was not confirmed by a second analysis in other laboratory or in other sample from the same patient: what we have called transient antithrombin deficiency.

This work included a total of 444 consecutive unrelated subjects, referred to our centre from more than 20 European hospitals during 23 years (1998-2021), with potential antithrombin deficiency, based on at least one positive functional assay performed at the hospital of origin. At least a new sample from all patients was delivered to our centre, so a new functional assay (a uniform anti-FXa assay for all recruited samples) was carried out for validation.

By performing a full clinical, biological and characterization, a genetic defect was observed in 84.6% of 305 cases with constitutive deficiency, those with consistent deficiency in all tested samples: 248 in SERPINC1 and 10 had N-glycosylation defects. These results are fully compatible with those obtained from other large cohorts, supporting the high success rate of identification of a SERPINC1 genetic variant in these patients, and confirmed the relevance of disorders of glycosylation among cases with not SERPINC1 defect. But more interestingly, our study identified a molecular basis explaining antithrombin deficiency in 43.9% of 139 cases who had normal antithrombin activity in at least one sample, what we called transient antithrombin deficiency. These 61 cases, all with thrombosis, had missense SERPINC1 mutations (N=48), with two recurrent mutations (p.Ala416Ser, antithrombin Cambridge II, N=15 and p.Val30Glu, antithrombin Dublin, N=12) or N-glycosylation defects (N=13). Two mechanisms explained transient deficiency: the limitation of current functional methods to detect some variants, and the influence of external factors (conformational stress, generation of thrombin, alcohol intake) on the pathogenic consequences of these mutations.

Our study supports that antithrombin deficiency is underestimated and cases with moderate risk of thrombosis may be missed if only functional methods and classical diagnostic algorithms are used. Furthermore, although we must take into consideration that most cases with transient deficiency are explained by acquired deficiency and laboratory mistakes, this work shows new evidences supporting that the pathogenic effect of a gene defect may be modulated by external factors, changing the paradigm of congenital thrombophilia and making it also a transient disorder.