Biochemical thrombophilia screening was performed in all family members in 2023. Due to varying levels of AT activity, measurements were carried out with four different commercially available tests, the Innovance Antithrombin and Berichrom Antithrombin III assays (Siemens Healthineers, Marburg, Germany) on a Sysmex CS-5100 analyser (Siemens Healthineers), the HemosIL Liquid Antithrombin assay (Instrumentation Laboratory, Boston, MA, USA) on a ACL TOP 700 analyser (Instrumentation Laboratory), and the STA-Stachrom assay (Diagnostica Stago, Asnières, France) on a STA-R Evolution workstation (Diagnostica Stago) (Table 1). In proband 1, AT activity varied with the different methods from reduced to normal values. In all four assays, AT activity was most reduced in Proband 3, who also had the most reduced antithrombin antigen level. At blood sampling, Proband 1 was treated with LMWH 10 000 IU subcutaneously twice a day, Proband 2 used warfarin, and Proband 3 and the unaffected mother did not take any anticoagulant drugs. In Proband 1, triple-positive antiphospholipid antibodies were present, and they were persistent after 12 weeks.

Proband 1 and 2 had previously been radiologically investigated. CT venography in proband 3 was performed and showed normal IVC. Other findings, including gracility of the vena iliaca and a significant collateral system, were interpreted as post-thrombotic changes (Fig. 1c).

All family members tested negative for the common factor V Leiden and the prothrombin G20210A mutation in the F5 and F2 genes. Whole exome sequencing (WES) was carried out in Proband 1 and 2 to identify possibly contributing factors to their IVC anomalies and a genetic cause for the observed reduced AT activity. WES revealed heterozygosity for the Budapest 3 mutation, c.391C > T, p.Leu131Phe in the SERPINC1 gene in both brothers. Several loss-of-function and missense variants with a frequency below 0.1% were observed in genes with hitherto unknown connections to the phenotype. In Proband 1, an exome-based gene panel for thrombophilia comprising 135 genes, was also analyzed in an accredited diagnostic laboratory, confirming the previous finding of the Budapest 3 mutation but with no further finding of pathogenic variants. The exome-based gene panel for vascular malformations was performed for Proband 3, with negative results.

We verified the WES results by Sanger sequencing of the entire SERPINC1 gene and included all family members. Heterozygosity for the AT Budapest 3 variant was confirmed in all probands but not in the asymptomatic mother. Of note, Proband 3 was also heterozygous for the variant, c.1063T > G (p.Phe355Val) in SERPINC1. The entire F2 and F11 genes were also Sanger sequenced and no relevant findings were detected in the three probands.