A novel VWF mutation associated with macrothrombocytopenia, cytoskeletal defects, and distinct functional abnormalities: A case report

Abstract

Abstract Background: von Willebrand disease type 2B (vWD 2B) is a rare inherited bleeding disorder characterized by gain-of-function mutations in the VWF gene, particularly within codons 1305–1316 of exon 28. These mutations enhance binding affinity to platelet glycoprotein (GP)Ibα and often result in macrothrombocytopenia. While structural platelet abnormalities are generally mild, certain genotypes—such as p.Val1316Met—may present with cytoskeletal disorganization reminiscent of primary hereditary thrombocytopathies. Despite ~30 known vWD 2B mutations, clinical heterogeneity suggests the existence of additional, uncharacterized variants. Methods: We report a 31-year-old male of Pakistani origin with lifelong mild mucocutaneous bleeding, including recurrent epistaxis and gingival bleeding. A family history of undifferentiated vWD and prior administration of FVIII concentrates in Pakistan were noted. Morphological, functional, immunofluorescence, and genetic analyses were performed, including platelet ultrastructure assessment, aggregometry, and sequencing of the VWF and known cytoskeletal genes causing inherited platelet disorders (ACTN1, ACTB, APRC1B, DIAPH1, FLNA, FYB1, MYH9, TPM4, TUBB1, WAS). Results: Platelet counts ranged from 22 to 50 G/L. Microscopy revealed marked anisocytosis with frequent giant platelets. Platelet granules were present but unevenly distributed. Light Transmission Aggregometry (LTA) was not possible in part due to the low platelet count. Mixing patient and healthy donor platelets 1:1 led to spontaneous aggregation without any stimulation. Large vWF multimers were not detectable, suggestive of consumptive loss due to spontaneous platelet activation. Flow cytometry confirmed normal expression of GPIb/IX and GPIIb/IIIa, as well as dense and α-granule markers. Immunofluorescence revealed diffuse, abnormal localization of filamin A and non-muscle myosin IIA (NMMIIA), suggesting cytoskeletal impairment. α- and β1-Tubulin staining was normal. No pathogenic variants were found in thrombocytopathy-related genes. Genetic analysis identified the heterozygous c.3946G>A (p.Val1316Met) mutation in exon 28 of VWF, consistent with vWD 2B. Additionally, a second heterozygous variant, c.6056A>T (p.Asp2019Val) in exon 35, was identified. This variant has not been reported in major genomic databases (ClinVar, GnomAD) so far, and is currently classified as a variant of uncertain significance (VUS). Conclusion: This case supports the phenotypic complexity of vWD 2B and highlights the potential contribution of multiple VWF variants to the patient phenotype. Here, the presence of abnormal platelet cytoskeletal features suggests a concomitant pathogenic effect of the novel p.Asp2019Val variant.