A Retrospective Analysis of Bleeding Phenotype and Von Willebrand Factor Exon 28 Polymorphism D1472H at a Single Institution

Introduction: The laboratory work-up of von Willebrand Disease includes measurement of Von Willebrand Factor (VWF) antigen (VWF:Ag), VWF activity (ristocetin cofactor activity, VWF:RCo), and factor VIII Activity (FVIII), where VWF:Ag and VWF:RCo below 30-40% in the appropriate clinical setting are consistent with VWD. Accurate diagnosis of type 2 VWD, marked by a qualitative deficiency in the VWF protein, is imperative for appropriate therapeutic interventions. Exon 28 sequencing is frequently pursued to confirm the diagnosis of type 2 VWD. An exon 28 polymorphism, D1472H, results in impaired VWF binding to ristocetin, affecting the clinical assay of VWF activity as measured by ristocetin cofactor activity, but not resulting in impaired VWF function in vivo. This polymorphism is common in the healthy population and does not result in a hemorrhagic phenotype. We reviewed our institution’s experience with exon 28 sequencing with regard to the D1472H polymorphism, laboratory phenotype, and ultimate clinical diagnosis.

Methods: Following IRB approval, patients who underwent exon 28 sequencing during evaluation for clinically significant bleeding between the years of 2003-2013 were identified. A retrospective chart review was conducted: patient demographics, VWF:RCo (lowest recorded), VWF:Ag (obtained on same day as the lowest VWF:RCo), and clinical diagnosis were collected. Descriptive statistics were employed to analyze the data.

Results: Seventy-three patients underwent exon 28 sequencing during their work up for symptomatic bleeding between 2003-2013. The median age at testing was 7 years (10 months – 56 years), 41% were females. Of the 73 patients tested, D1472H polymorphism results for 65 patients were available for review. Fourteen patients (21.5%) were found to be heterozygous for the D1472H polymorphism. No patients were found to be homozygous. There was no difference between the mean VWF:RCo/VWF:Ag ratio for the D1472H heterozygous group (64% [47-81%]) and the group without the polymorphism (64% [48-80%]). VWF:RCo was lower in the heterozygotes with type 1 VWD (23.5% [14.5%-32.5%]) than in the group without the polymorphism (31% [18.5%-43.5]) (Table 1).

In the group heterozygous for the D1472H polymorphism, 5/14 were diagnosed with type 1 VWD (Table 2). Two were diagnosed with type 2M VWD. One patient was diagnosed with type 1 VWD and mild hemophilia, 1 had type 1 VWD and Factor XI deficiency, 1 had type 2B VWD, 1 had chronic thrombocytopenia. Three patients did not have bleeding disorders.

Conclusion: At our institution, heterozygosity for the exon 28 D1472H polymorphism was not associated with decreased VWF:RCo or VWF:RCo/VWF:Ag ratios as compared to patients without this polymorphism in all patients tested. For patients diagnosed with type 1 VWD, the VWF:RCo was lower in the group heterozygous for the D1472H mutation. These patients had sub-normal levels of VWF:Ag and VWF:RCo in the setting of clinically significant bleeding. Thus, the provisional diagnosis of VWD was appropriate despite the potential effect of the polymorphism on the VWF:RCo. These results are inconsistent with previously reported studies where presence of the D1472 polymorphism resulted in lower VWF:RCo/VWF:Ag ratios and inaccurate diagnosis of VWD. This study highlights the challenges associated with the laboratory diagnosis of VWD and the importance of continued research into the development of reliable VWD testing that more closely mimics the protein’s in vivoactivity.