A novel homozygous splice-site variant in VPS33B identified as a cause of bleeding

Abstract

BACKGROUND: Mild-to-moderate bleeding disorders are underdiagnosed, with many individuals classified as having no clear cause for their bleeding. Accurate diagnosis remains challenging and requires comprehensive clinical assessment, detailed laboratory testing, and advanced genetic testing. OBJECTIVES: This study aimed to elucidate the relevance of an integrated approach for diagnosing patients with mild-to-moderate bleeding disorders by reevaluating 2 cases previously labeled with an undefined cause. METHODS: Two young siblings with unexplained bleeding tendencies were referred for evaluation. Bleeding score were assessed using the International Society on Thrombosis and Haemostasis bleeding assessment tool questionnaire. Platelet phenotyping included blood count, blood film, coagulation tests, light transmission aggregometry, flow cytometry, western blotting, and transmission electron microscopy. Genetic analysis involved whole-exome sequencing (WES) and pathogenicity assessment of candidate variants. RESULTS: Both siblings exhibited mild-to-moderate bleeding (bleeding score, 11 and 3, respectively). Blood count, coagulation factor tests, and light transmission aggregometry response to several agonists were normal. Blood film showed large and hypogranular platelets. Flow cytometry revealed reduced ?-granule secretion upon ADP and TRAP6 stimulation. WES identified a homozygous variant in VPS33B (c.1225+5G>C) predicted to disrupt splicing. Immunoblotting confirmed null VPS33B expression and reduced von Willebrand factor levels. Transmission electron microscopy showed abnormal vacuole content and significantly reduced ?-granules, while ?-granules and mitochondria remained intact. CONCLUSION: This study reports a novel homozygous variant in VPS33B, leading to platelet dysfunction and bleeding diathesis based on ?-granule defect and syndromic manifestations. Our findings highlight the importance of an integrated diagnostic approach, including WES, and expand the clinical and genetic spectrum of arthrogryposis, renal dysfunction, and cholestasis syndrome.