Molecular and genetic verification of von Willebrand disease type 2N

Abstract

Introduction. Von Willebrand disease (vWD) is caused by von Willebrand factor (vWF) dysfunction resulting from pathogenic variants in the vWF gene coding the vWF protein. vWD type 2N is of particular interest, as it is characterized by almost normal vWF antigen level (Ag:vWF) and vWF loss of ability to bind FVIII and protect it from premature clearance, which leads to a low FVIII coagulation activity (FVIII:C). Therefore, the same phenotype occurs in patients with 2N type of vWD and hemophilia A.Aim — to identify patients with 2N type vWD using molecular genetic methods.Methods. Data from the medical histories of vWD patients were used. The major parameter in consideration was FVIII:C to vWF:Ag ratio, which is expected to be below 0.7 in type 2N of vWD. Pathogenic variants in exons and exon-intron junctions of the vWF gene were identified by Sanger sequencing. Due to recessive inheritance of type 2N, verification of the 2N vWD diagnosis required the identification of two pathogenic variants.Results. Three patients were considered as suffering from type 2N of vWD according to hemostasis parameters (FVIII:C/vWF:Ag < 0.7). One patient with a preliminary hemophilia A diagnosis was included after sequencing of the F8 gene, which showed no alterations, so 2N type of vWD was suspected. In all cases, sequencing of the relevant functional regions of the vWF gene led to verification of vWD type 2N. One woman (patient # 4) had a homozygous pathogenic variant p.Arg854Gln (c.2561 G>A) associated with type 2N vWD. One woman (patient # 3) was a compound heterozygote for the pathogenic variant p.Arg816Trp (c.2446 C>T) associated with type 2N and a newly described insertion c.2098_2099insG, that leads to a frameshift. The woman with FVIII:C/vWF:Ag < 0.7 (patient # 1) and the patient # 2 with preliminary hemophilia А diagnosis were both compound heterozygotes for the same combination of pathogenic variants — c.2435delC and p.Thr791Met (c.2372 C>T). Pathogenic variant p.Thr791Met is associated with type 2N, while the deletion c.2435delC should lead to allele disabling.Conclusion. Molecular methods allow more precise differentiation of type 2N from other types of vWD and hemophilia A.