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.

Table 1 Summary of clinical and laboratory information on siblings and the mother

	Age, sex	Hemophilia B	Clinical presentation	Platelet aggregation with ADP	Platelet aggregation with TXA2	Responsible gene
Case 1	16 y, M	Severe	Recurrent joint bleeding and postoperative bleeding, difficult to achieve hemostasis with FIX replacement alone	Impaired	Impaired	Hemizygous variant (F9 p.R43Q) and heterozygous variant (TBXA2R p.R60L)
Case 2	12 y, M	Severe	Recurrent joint bleeding, muscular bleeding, and mucosal bleeding	Impaired	Impaired	Hemizygous variant (F9 p.R43Q) and heterozygous variant (TBXA2R p.R60L)
Mother	49 y, F	Carrier (FIX 22%)	Delayed wound healing	Normal	Impaired	Heterozygous variant (F9 p.R43Q) and heterozygous variant (TBXA2R p.R60L)

Abbreviations: ADP, adenosine diphosphate; TXA2, thoromboxane A2.

There is only one published study reporting a pedigree with simultaneous primary and secondary hemostatic abnormalities.[9] The study described a family whose bleeding symptoms were exacerbated by combined variants in FVIII and prostaglandin synthase-1. Despite having mild hemophilia A with 11% FVIII activity, the proband experienced bleeding from joints, the gastrointestinal tract, the nose, the mouth, and following surgical procedures, necessitating regular replacement therapy with recombinant FVIII and desmopressin acetate (DDAVP). In the three-generational pedigree, three individuals with both variants exhibited more severe bleeding manifestations than would typically be expected for mild hemophilia A alone. The authors suspected that the presence of an IPD accounted for the excessive hemorrhage observed in this family, given the severity of the proband's bleeding history and the abnormal bleeding history in family members without hemophilia A, which ultimately led to a diagnosis of IPD.

TXA2R, encoded by the TBXA2R gene, is a G protein-coupled receptor (GPCR) that plays a critical role in hemostasis and thrombosis through the activation of platelets.[10] Abnormalities in TXA2R can result in IPD characterized by defective platelet responses to TXA2 and are categorized as thromboxane receptor deficiency (MIM #614009).[5] The abnormal TXA2R disrupts the synergistic interaction between ADP and TXA2 signaling pathways, leading to impaired secondary aggregation of platelets in response to ADP stimulation.[11] [12] [13] Hirata et al first reported a Japanese patient with a homozygous p.R60L variant in the first cytoplasmic loop of the TXA2R, presenting with a mild bleeding disorder characterized by a defective platelet aggregation response to TXA2 and its analogs.[5] They found that family members who were heterozygous for the variant also exhibited mild bleeding symptoms and abnormal platelet aggregation, suggesting the possibility of a dominant-negative effect of p.R60L mutant as less than half the number of receptors are expected to be sufficient for irreversible aggregation of platelets by a TXA2 agonist. On the other hand, other reports have demonstrated that some heterozygotes for the p.R60L, D304N, W29C, c.167dupG TBXA2R substitution were asymptomatic despite exhibiting an abnormal platelet aggregation response to AA or U46619.[7] [14] [15] [16] In our case, the sibling exhibited an impaired aggregation response to both ADP and U46619. In contrast, the mother, who also carried combined variants in F9 and TBXA2R but was almost asymptomatic, showed a normal response to ADP but an impaired aggregation response to U46619. The discrepancy in reactivity to ADP and clinical phenotype between the sibling and mother might be explained as follows: 1) Since heterozygotes for the p.R60L TBXA2R variant are predicted to express both functional and nonfunctional TXA2R, the mother's response to ADP may be attributed to individual differences in residual reactivity to TXA2. 2) An additional unidentified hemostatic abnormality may modulate the phenotype of TXA2R deficiency in an autosomal dominant manner. Similarly, a possible explanation for the difference in reactivity to AA between cases 1 (impaired) and 2 (normal) is a variation in residual TXA2 activity, likely due to a dominant-negative effect. Since the concentration of AA is fixed in the TEG6s Platelet Mapping assay, it is possible that case 2 might also show impairment if a lower concentration of AA were used. Notably, the p.R60L variant in TBXA2R has been reported in six Japanese cases with bleeding tendencies and impaired platelet function.[6] [7] [8] In the gnomAD database (https://gnomad.broadinstitute.org/) and the ToMMo 54KJPN database (https://jmorp.megabank.tohoku.ac.jp/), the population frequency of the TBXA2R c.179G > T (p.R60L) variant ranges from 0.00014 globally to as high as 0.011 in Japan. Therefore, this genetic disorder may be more prevalent than currently recognized, as its phenotype could typically be mild.[6] [7] [8] If this variant coexists with another condition, however, such as hemophilia, both primary and secondary hemostasis may be compromised, potentially exacerbating the severity of bleeding.

Furthermore, the involvement of CRM may explain why the mother remains asymptomatic with 20% FIX activity, whereas the siblings required 100% FIX activity despite carrying the same TBXA2R variant. Case 1 showed CRM-positive (reduced) hemophilia B. In hemophilia B patients with the CRM-positive variant, there is a possibility that FIX replacement therapy may be less effective compared with patients with the CRM-negative variant. The reduced effectiveness in patients who showed CRM-positive may be due to the saturation of nonfunctional FIX already bound to collagen IV in the basement membrane, preventing exogenous FIX from binding to collagen IV.[17] Therefore one possible explanation for the asymptomatic clinical phenotype of the mother is skewed lyonization: most of the expressed FIX in the mother might be wild-type FIX, originating from the normal X chromosome that is not CRM-positive variant. However, we could not test the CRM status in case 2 and the mother. In addition, the significance of CRM status in bleeding control has not yet been established so it is unclear to what extent CRM is involved in the hemorrhage in this case.[18] Further investigations were needed.

While our primary focus tends to be on identifying carriers of rare monogenic variants associated with a severalfold increased risk, recent progress in genome-wide association studies revealed that most disease risks are inherently polygenic when considering the overall disease landscape.[19] [20] [21] Previous papers already implicated that additional oligogenic variants contributed to the modification of the clinical phenotype.[22] [23] Tanaka et al reported the analysis of WES data of 743 epileptic or developmental and epileptic encephalopathy (EE/DEE) cases with 2,366 controls.[23] They found accumulating evidence for the role of oligogenic and modifier variants in severe neurodevelopmental disorders and concluded that EE/DEE is not merely a simple collection of Mendelian genetic diseases, highlighting the genetic complexity associated with EE/DEE. Diseases caused by very rare deleterious variants, such as hemophilia, tend to be dismissed as monogenic diseases because of the large-effect variant for their phenotype. Our cases suggest that the low-frequency variants with intermediate penetrance may contribute to disease modification in addition to common phenotypes caused by very low-frequency, large-effect variants. For patients whose phenotype does not align with their genotype, comprehensive next-generation sequencing (NGS)-based genetic testing is useful for an accurate diagnosis.

In conclusion, we presented patients with hemophilia B who exhibited a severe bleeding phenotype associated with combined variants in the F9 and TBXA2R genes. Our cases suggest that the potential for complications arising from other bleeding disorders should be considered in patients with hemophilia who experience treatment-resistant or severe bleeding symptoms.

Authors' Contributions

M.S. and Y.M. designed the project and prepared the manuscript. K.K. conducted a platelet function test. F.S. and K.N. analyzed the data of WES. T.F., M.K., and S.O. revised the manuscript.

Publication History

Article published online:
17 April 2025

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