Introduction: Although FVIII activity (FVIII:C) correlates with phenotype in most Hemophilia A males, some have bleeding phenotypes milder than predicted by FVIII:C. Analysis of these individuals may provide additional insight into the relationship between F8 genotype and bleeding phenotype, identify additional factors that modulate FVIII, refine the classification of Hemophilia A and improve clinical management.We present a male subject who carries a c.5999G>C mutation in F8. This mutation has been reported in an individual with a severe bleeding phenotype in the EAHAD F8 Variant Database (www.factorviii-db.org). Our subject was serendipitously diagnosed at age 80, and has never had abnormal bleeding despite numerous challenges, including multiple dental extractions, adult circumcision, internal hemorrhoid ligation and cardiac catheterization. In an effort to resolve this genotype-phenotype discrepancy, we performed FVIII and von Willebrand Factor (VWF) assays, quantitated the extent of alternative splicing in F8, and screened for mutations in additional genes known to impact hemostasis. We validated a partial splicing defect that is consistent with mild Hemophilia A, but surprisingly cannot explain the lack of a bleeding phenotype.Methods: Peripheral blood was tested by one-stage FVIII:C and chromogenic FVIII assays, FVIII antigen ELISA, VWF antigen and activity assays, and von Willebrand Disease (VWD) Type 2 Normandy binding assay.To evaluate F8 splice variants, quantitative RT-PCR (qRT-PCR) was performed on RNA isolated from peripheral blood using primers that distinguish FVIII isoforms. Samples were tested in quadruplicate, with sample input normalized relative to GAPDH. Screening for exonic mutations in F8, F5, F2 and VWF genes was undertaken by Next Generation Sequencing (NGS).Results and Conclusions: In contrast to the previously reported severe c.5999G>C patient, this subject has reduced FVIII activity consistent with mild Hemophilia A measured by both FVIII activity assays. FVIII antigen assessment by ELISA gave similar results (0.35 IU/mL).To better understand FVIII:C levels in this individual, we sought to characterize F8 transcript isoforms. The c.5999G>C resides within the splice acceptor site of exon 19 and may result in two FVIII isoforms. One isoform produces a full-length FVIII containing a missense mutation (p.Gly2000Ala). The second isoform skips the 117 bp exon 19, but is predicted to retain an open reading frame; however, its location in the A3 domain likely disrupts secretion or function (Donadon et al., Haematologica 2018). The relative abundance of F8 variants evaluated by qRT-PCR demonstrated that despite a modest overall increase in F8 transcript relative to a normal control (1.6 fold higher expression of F8 at exon 18), the predominant isoform in this subject lacks exon 19, resulting in substantial depletion of the full-length isoform (0.3 relative to a normal control). These data indicate the full-length protein likely retains function, with transcript levels that explain residual FVIII activity.In an effort to explain absent bleeding phenotype, NGS was performed. The c.5999G>C F8 mutation was confirmed, while the thrombophilic Factor V Leiden and Prothrombin G20210A mutations were excluded. Sequencing also excluded mutations implicated in Type 2 Normandy VWD. Additional clinical testing may provide an explanation. VWF was elevated as indicated by VWF antigen and Ristocetin cofactor activity (>400%, >100%, respectively). Furthermore, VWF:FVIII binding activity by ELISA showed a higher than normal binding ratio (>1.42). VWF multimer analysis indicated normal distribution. Therefore, we propose elevated VWF abrogates this subject's bleeding phenotype.Altogether, our study expands our understanding of the functional consequences of this mutation by demonstrating that this mutation can result in FVIII levels predicting mild Hemophilia A. This case also underscores the complexities of predicting phenotype for mutations that result in partial splicing defects. Perhaps more intriguing is the lack of bleeding phenotype in this elderly individual with numerous hemostatic challenges, that might be related to increased VWF. These data support additional studies investigating the role of VWF in ameliorating bleeding risk in Hemophilia A. DisclosuresNo relevant conflicts of interest to declare.
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Jan 1, 2020
L Chen 
Open Access
