Genetic characterization of thrombophilic subjects, with particular focus on hereditary defects of antithrombin (SERPINC1), protein C (PROC), and protein S (PROS1)

Despite significant advances in understanding coagulation and hemostatic mechanisms, the biological basis of thrombotic disorders remains incompletely elucidated, leaving many clinical questions unresolved. It remains unclear, for instance, why certain patients develop venous thromboembolism (VTE) in the absence of identifiable acquired risk factors or established hereditary thrombophilia. Moreover, even when a biochemical defect has been characterized, considerable phenotypic variability persists among patients harboring the same genetic alteration. This study aimed to investigate the presence of causative genetic variants in a cohort of patients with unprovoked VTE or VTE associated with minor transient risk factors, using a customized next-generation sequencing (NGS) panel targeting 33 genes involved in the coagulation cascade. A secondary objective was to identify variants that may influence the clinical and biochemical phenotypes of affected individuals. A cohort of 100 unrelated patients (45% women, mean age 33 years) followed for VTE at Padua University Hospital, all of whom had tested negative for hereditary and acquired thrombophilia through conventional screening, was analyzed. NGS was performed on genomic DNA extracted from peripheral whole blood, targeting the coding regions and exon-intron boundaries of all 33 genes. Identified variants were classified according to their pathogenic potential following the American College of Medical Genetics and Genomics (ACMG) guidelines and ClinGen curated annotations, when available. Most patients were Caucasian, with a mean BMI of 25.5 kg/m², and half reported a family history of VTE. The index event was unprovoked in 67% of cases, while 33% were associated with weak provoking factors. NGS analysis identified pathogenic variants (ACMG class 5) in 7% of patients, likely pathogenic variants (class 4) in 2%, and variants of uncertain significance (VUS, class 3) in 49%. Notably, SERPINC1 was altered in 3 patients (all class 5), PROS1 in 3 patients (2 class 4, 1 class 5), and PROC in only two patients (both class 3). These findings support the hypothesis that phenotypic discrepancies observed among patients with thrombophilia reflect complex genetic architectures, in which interactions among multiple prothrombotic and pro-hemorrhagic genetic factors collectively determine the clinical presentation. The identification of characteristically pro-hemorrhagic variants in patients with VTE suggests these may modulate the thrombophilic phenotype, potentially through rebalancing coagulation and hemostatic equilibria. In conclusion, it is not always possible to identify a single causative genetic defect in cases of unprovoked VTE. Indeed, such patients frequently harbor a multifactorial genetic background that, in conjunction with environmental factors, contributes to the occurrence of thrombotic events. Nevertheless, targeted NGS analysis of genes involved in the coagulation cascade represents a valuable diagnostic tool for identifying congenital defects underlying thrombotic and hemorrhagic disorders. This approach facilitates not only the detection of causative variants but also the exploration of the genetic basis of observed phenotypic heterogeneity, thereby advancing our understanding of the complex mechanisms governing hemostasis. The identification of such genetic determinants constitutes a meaningful step toward improved, personalized clinical and therapeutic management of affected patients.