The Co-Occurrence of Low-Frequency Pathogenic Variants in TBXA2R Exacerbating the Hemorrhagic Symptoms in Siblings with Hemophilia B

Hemophilia is an inherited clotting disorder caused by a deficiency of factor VIII (FVIII) or factor IX (FIX), both of which are serine proteases in the intrinsic pathway of the coagulation cascade.[ 1 ] In recent years, patients with hemophilia have been able to lead normal active lives by maintaining appropriate factor levels through adequate replacement therapy, tailored to the intensity of their physical activity. However, individual differences in bleeding symptoms are frequently observed among patients with hemophilia, even when presenting at the same factor level of severity.[ 2 ] Some patients may experience hemorrhages despite having high clotting activity through receiving adequate replacement therapy.[ 3 ] Platelets play a critical role in primary hemostasis and thrombosis. Inherited platelet disorders (IPD) comprise a heterogeneous group of rare diseases caused by genetic defects that affect platelet formation and/or function. The clinical presentations in patients with IPD are highly variable, ranging from almost asymptomatic to those with severe bleeding symptoms even within families with the same genetic defect.[ 4 ] Thus, the phenotype of patients with IPD often becomes apparent only after external events such as surgery or trauma. Here we identified the coexistence of pathogenic variants in F9 and TBXA2R in siblings with hemophilia B who presented with treatment-resistant hemorrhages. TBXA2R encodes the thromboxane A2 receptor (TXA2R), and its genetic abnormalities are associated with an IPD. Initially, the siblings were exclusively diagnosed and treated for hemophilia B; however, despite maintaining adequate levels of factor activity, they experienced recurrent bleeding episodes. The occurrence of treatment-resistant bleeding raised suspicion of additional hemorrhagic complications. A thorough examination revealed abnormal platelet aggregation capacity, which ultimately led to the identification of a genetic abnormality in the TBXA2R gene through whole exome sequencing (WES). This report represents the first report of cases with combined variants in the F9 and TBXA2R genes. Case 1 is a 16-year-old boy who was diagnosed with severe hemophilia B (baseline FIX <1%) at 10 months of age due to intracranial hemorrhage. He began receiving weekly regular infusions of plasma-derived FIX (pdFIX) and switched to extended half-life recombinant FIX (EHL-rFIX) at the age of 6. Despite maintaining an adequate factor level (trough >10%) through regular replacement therapy, he started experiencing recurrent joint bleeds in his left ankle at the age of 9, ultimately developing a target joint. His annual joint bleeding rate (AJBR) reached three to four episodes per year. He had tried all types of rFIX products available in Japan and consistently maintained trough levels of FIX between 15 and 20%. Nevertheless, he continued to experience frequent bleeding in his left knee joint and right ankle. Additionally, he had prolonged postoperative bleeding following phimosis surgery and left ankle synovectomy, despite maintaining adequate factor activity. It became apparent that achieving hemostasis for this patient was challenging without maintaining factor activity close to 100% during bleeding episodes or postoperative care. Case 2 is a 12-year-old boy with severe hemophilia B (baseline FIX <1%), the younger brother of case 1. He began receiving weekly infusions of pdFIX at 9 months of age and switched to EHL-rFIX at 3 years of age. Despite maintaining a trough level of FIX between 10 and 20%, he experienced recurrent bleeding in his left hip joint. Additionally, around the age of 8, he began to easily develop muscle bleeds during physical activity. Furthermore, he frequently encountered challenges with hemostasis during tooth extractions. It is necessary to administer recombinant FIX (rFIX) immediately before each extraction at higher factor activity; otherwise, bleeding cannot be controlled. Due to the treatment-refractory hemorrhage observed in this sibling, we decided to investigate whether other bleeding disorders were present in addition to hemophilia B. In case 1, the activity levels of FVIII, factor XI, factor XII, and factor XIII were found to be normal. Additionally, the FIX antigen level was approximately 20% when the activity was at 5%, indicating that the patient had cross-reactive material (CRM) reduced hemophilia B. To determine whether primary hemostatic abnormalities were involved, we performed platelet light transmission aggregometry (LTA) using the Sysmex CN-6000. The assay revealed that the secondary aggregation wave in response to ADP (2 mM) was absent in both siblings ([ Fig. 1A ]). The TEG6s Platelet Mapping assay (Haemonetics Co., Braintree, MA) also showed that clot strength induced by ADP was reduced in both siblings compared with the control ([ Fig. 1B 1–3 ]). Additionally, in case 1, reduced clot strength induced by arachidonic acid (AA) was also observed ([ Fig. 1B 2 ]). Based on these results, we suspected the presence of a platelet function disorder in the siblings and subsequently performed WES on the siblings and their nonconsanguineous parents. All participants provided informed consent for genetic analysis (IRB #E2005–9109). WES revealed a hemizygous variant in F9 (c.128G > A, p.R43Q) in both siblings and the same heterozygous variant in F9 in their mother. Additionally, a heterozygous variant in the TBXA2R (c.179G > T, p.R60L) was identified in both siblings, which was also present in their mother ([ Fig. 2A, B ]). This variant has already been reported as a pathological variant.[ 5 ] [ 6 ] [ 7 ] [ 8 ] Fig. 1 Agonist-induced platelet functional analysis. ( A ) Platelet light transmission aggregometry (LTA) in response to ADP (2 mM) in the sibling and their mother as well as a healthy control. ( B ) Profiles of TEG 6S platelet mapping assay. The figures demonstrate the clot strength induced by four substances: fibrin (MA ActF ), Kaolin-activated thromboelastography with heparinase (MA HKH ), ADP (MA ADP ) and AA (MA AA ) in healthy control ( B1 ), case 1 ( B2 ), and case 2 ( B3 ), respectively. AA, arachidonic acid; ActF, activator F; ADP, adenosine diphosphate; HKH, kaolin with heparinase; MA, maximum amplitude; R, reaction time; TEG, thromboelastography. Fig. 2 Genomic analysis of the family members. ( A ) Sanger sequence chromatographs showing TBXA2R gene variants in family members. ( B ) Pedigree for the affected family. Filled symbols represent individuals with variants in the TBXA2R gene and hashed symbols represent individuals with variants in the FIX gene. To further investigate platelet function in both siblings, we conducted a platelet function test using U46619 (9,11-Methanoepoxy Prostaglandin H2, Tocris Bioscience Cat#1932), a stable analog of TXA2 that serves as a potent TXA2 agonist. As a result, the siblings and their mother, who carried a heterozygous variant in the TBXA2R , demonstrated impaired aggregation in response to U46619 ([ Fig. 3 ]). Fig. 3 A stable analog of TXA2-induced platelet functional analysis. Platelet light transmission aggregometry (LTA) in response to U46619 (1.25 µM). The clinical and genetic information on the siblings and the mother are summarized in [ Table 1 ]. The mother, who is a carrier of hemophilia, had a FIX level of 22%, consistent with mild hemophilia B; however, she has not experienced any bleeding symptoms to date. Her only clinical manifestation has been delayed wound healing ([ Table 1 ]). Notably, the mother showed normal responses to ADP ([ Fig. 1A ]). Currently, both cases 1 and 2 are receiving regular administration of tranexamic acid in addition to their regular infusions of EHL-rFIX, which has demonstrated some degree of effectiveness.