215 The Novel Exon 52 Cys2771Phe Variant Disrupts von Willebrand Factor Dimerization in Familial Type 2A von Willebrand Disease

Introduction/Objective Type 2A von Willebrand disease (vWD) arises from qualitative defects in von Willebrand factor (vWF) that result in a reduction of high-molecular-weight multimers (HMWM). Approximately 70-80% of type 2A variants cluster in exon 28 of the VWF gene, which encodes the A1 and A2 domains. In contrast, variants in the C-terminal cysteine knot (CTCK) domain (exon 52) are rare, comprising <10% of type 2A variants. In this domain, three amino acids—vWF Cys2771, Cys2773, and Cys2811—form disulfide bonds that connect two vWF units together to form a dimer that is the building block for larger vWF multimers. Cys2771 forms reciprocal interchain disulfide bonds with Cys2773 of the opposing subunit (and vice-versa), the first step that dimerizes vWF monomers in the endoplasmic reticulum. Cys2811 on the first subunit forms a disulfide bond with Cys2811 on the second unit to protect the first two bonds from reduction, thereby rendering dimerization irreversible. Variants at these locations are thought to disrupt dimerization (a pre-requisite for multimerization) and multimer assembly. We report a previously undescribed exon 52 missense change that produces a classic type 2A phenotype in three related individuals, expanding the genetic spectrum and providing additional mechanistic insight into type 2A vWD. Methods/Case Report A 21-year-old male with a lifelong history of easy bruising and prolonged bleeding following minor trauma presented for hematologic evaluation. Platelet count, prothrombin and activated partial thromboplastin times, and fibrinogen level were normal. The PFA-100 platelet function screen failed to achieve aperture closure with both ADP and epinephrine. Plasma vWF activity using the VWF:GPIbM assay was reduced (23% of normal, reference ≥50%), with normal vWF antigen (109%) and factor VIII activity (92%). vWF multimer analysis demonstrated near-complete absence of HMWM. An additional fast-migrating band not seen in normal control plasma was present, likely representing a vWF monomer. Next-generation sequencing of VWF identified a novel heterozygous variant (c.8312G>T, p.Cys2771Phe) in exon 52 that encodes the CTCK domain, which was confirmed by Sanger sequencing. No other pathogenic, likely pathogenic, or variants of uncertain significance were identified in the VWF or GP1BA genes. In silico computational tools predicted the variant to be damaging. Both sons (ages 2 and 1) carried the same variant and exhibited similar clinical and laboratory findings, consistent with autosomal-dominant inheritance. Results NA Conclusion In this report, we describe three patients with the novel single nucleotide substitution c.8312G>T in exon 52 of the VWF gene. This converts cysteine to phenylalanine at amino acid position 2771, which abolishes two of the three interchain disulfide bonds required to maintain the integrity of the vWF dimer, impairing subsequent multimer assembly—biochemically explaining the absent HMWM and bleeding phenotype observed. The identification of Cys2771Phe broadens the catalogue of CTCK-domain variants associated with type 2A vWD, highlights the value of integrating phenotypic assays, multimer analysis, and molecular genetics in bleeding-disorder evaluations, and enhances understanding of vWF biosynthesis and the pathophysiology of type 2A vWD. As many VWF sequencing assays only cover exon 28, panels restricted to this region may miss functionally significant vWF variants, resulting in false-negative interpretations; as such routine inclusion of exon 52 in vWF sequencing panels will improve detection of rare but clinically significant variants and inform family counselling and management.