New genetic defects and mechanisms involved in antithrombin deficiency

Abstract

Antithrombin is a natural anticoagulant that plays a crucial role in hemostasis. The wide range of target procoagulant proteases and the potent inhibitory mechanism of this serpin explain that even moderate reductions of antithrombin significantly increase the risk of venous thrombosis. Thus, antithrombin deficiency is the first and the strongest thrombophilia that has been widely studied during more than 50 years. However, there are still some challenges concerning this anticoagulant, such as the factors involved in the clinical heterogeneity, to do a better characterization of structural variants involved in antithrombin deficiency, and to describe the molecular base of up to 25% of cases with no genetic defect. In this thesis we have tried to correlate genetic and clinical data in a large cohort of patients with antithrombin deficiency. OBJECTIVE The objective of this thesis is to supply new and original information of this rare disorder. Specifically, we aimed to describe the clinical impact of antithrombin deficiency. Moreover, we also want to deeply characterize structural variants involved in antithrombin deficiency, and to identify new molecular defects involved in this disorder. METHODS We have studied one of the largest cohorts of patients with this rare disorder, recruited during more than 20 years. For all patients, a complete set of functional, biochemical and molecular studies have been done and clinical data has been collected. Specific studies included recombinant expression and a novel approach for haplotype analysis. New molecular technologies, including third generation sequencing and long range PCR, have been used for characterization of molecular defects in SERPINC1. RESULTS Antithrombin deficiency causes very high risk of pediatric thrombosis (300-fold compared with the general population). In contrast, we have characterized a mild antithrombin deficiency of difficult functional diagnosis caused by p.Thr147Ala, a mutation with founder effect in African population. The study by different methods, including whole genome sequencing with nanopore technology, of the largest cohort of structural variants causing antithrombin deficiency showed that they are heterogeneous in size and type, and identified for the first time a complex structural variant and an insertion of a new SVA element. We described a detailed map of structural variants with intra and intergenic hotspots, and we found a common mechanism for formation of structural variants causing antithrombin deficiency that involved repetitive elements. CONCLUSIONS Our study supplies new and original information on the strongest thrombophilia. The high risk of pediatric thrombosis supports the screening of certain types of antithrombin deficiency in children of affected families to benefit from preventive strategies. In contrast, founder mutations expanded in different populations like the first one described by our study in African population will probably cause a mild deficiency of difficult functional diagnosis. We identified and characterized a new mechanism causing antithrombin deficiency: the insertion of a retrotransposon in an intron. Moreover, we dissected the structural variants involved in antithrombin deficiency. Our study also reveals that the combination of different methods is useful for the characterization of these gross gene defects, but nanopore sequencing is the most suitable method to fully characterize them.?