Hemophilia B caused by an F9 mutant, initially classified as variant of unknown significance, which results in a qualitative defect and impaired thrombin generation. (A) Pedigree with inherited F9 C.287A>G mutant resulting in p.(Gln96Arg) variant. (B-C) The p.(Gln96Arg) variant is located in the EGF-1 domain and introduces a positively charged arginine within the calcium ion binding cavity. (D) Other deficiencies or abnormalities in proteins contributing to the severe bleeding phenotype were excluded by quantitative mass spectrometry–based screening of plasma proteins corresponding to the tier 1 diagnostic gene list,²⁵ updated and endorsed at the International Society of Thrombosis and Hemostasis 2023 conference. Normal concentration of coagulation and fibrinolysis proteins in the plasma was confirmed, except for mild FXI deficiency (39 U/dL) identified in 1 sibling (III.1). (E) Thrombin-generation profile with plasma from the carrier (II.I). (F-G) Thrombin generation with plasma from siblings (III.I) and (III.II) before and 1 hour after 25 U/kg Alprolix as prophylaxis. (H) Inhibitor testing by mixing normal plasma pool and patient plasma (III.II) followed by thrombin generation with (I) lag time and (J) peak height. Data were obtained by calibrated automated thrombography (CAT) assay with 1 pM TF for coagulation initiation. Dashed lines indicate the peak height in the control condition (pooled normal human plasma).