Sequencing of Exon 28 of von Willebrand Factor and Glycoprotein IB Genes to Identify Type 2B von Willebrand disease and platelet-type von Willebrand disease

Type 2B von Willebrand disease (vWD) and platelet-type von Willebrand disease (pt-vWD) are two rare inherited bleeding disorders that remain under-diagnosed and incorrectly treated due to their shared clinical and laboratory features. Proper diagnosis of vWD requires a careful patient and family history and a panel of vWD assays. Acquired vWD (AvWD) may also complicate diagnosis of type 2 vWD, as it may present similar laboratory findings. DNA analysis of von Willebrand factor (vWF) exon 28 and platelet glycoprotein \ba(GPIba) gene can potentially aid in diagnosis when samples lack higher molecular weight multimers. To incorporate molecular testing, DNA was extracted from 63 deidentified patients' plasma samples that closely resembled type 2B vWD/pt-vWD by Western blot multimer analysis and from two positive plasma controls used for identification of type 2A and type 2B vWD by vWF multimer analysis. DNA analyses were performed on exon 28 of vWF and the GPIba gene. ABO blood typing was also determined, as it is a genetic modifier of vWF. The two positive plasma controls were identified as type 2A with the n.4517C>T (p.Serl506Leu) mutation and as type 2B with the n.3916C>T (p.Argl306Trp) mutation. From the 63 study plasma samples, none of the samples tested positively for 2B or ptvWD mutations, two samples were type 1 vWD with n.3686T>G (p.Vall229Gly) and n. 4751A>G (p.Tyll584Cys), and two samples were found to be Type 2A vWD with n.4517C>T (p.Serl506Leu) and n.4789C>T (p.Argl597Trp) mutations. One specimen analysis was inconclusive. Three new silent polymorphisms were identified. The low mutation detection rate in this study highlights the importance of expanding the vWD coagulation panel to include assays such as collagen-binding assay (vWF:CB), Ristocetin Induced Platelet Aggregation (RIPA) and DNA analysis, as the routine coagulation assays lack sensitivity and reproducibility. The RIPA assay is more specific for diagnosis of type 2B and pt-vWD. Preanalytical sample handling is of concern in vWD diagnosis because cold storage of whole blood induces the loss of large and intermediate vWF multimers in healthy controls. AvWD laboratory findings are similar to those of vWD type 2, and require careful evaluation and analysis of the patient's bleeding history to exclude any role of vWD mutations. Due to the large size of the vWF gene and the heterogeneity of vWD presentation, diagnosis of vWD is very complex and no single assay or diagnosis approach is suitable for all patients.