Application of genetic testing for the diagnosis of von Willebrand disease

Abstract

von Willebrand disease (VWD) is the most frequent inherited bleeding disorder, with an estimated symptomatic prevalence of 1 per 1000 in the general population. VWD is characterized by defects in the quantity, quality, or multimeric structure of von Willebrand factor (VWF), a glycoprotein being hemostatically essential in circulation. VWD is classified into 3 principal types: low VWF/type 1 with partial quantitative deficiency of VWF, type 3 with virtual absence of VWF, and type 2 with functional abnormalities of VWF, being classified as 2A, 2B, 2M, and 2N. A new VWD type has been officially recognized by the ISTH SSC on von Willebrand factor which has also been discussed by the joint ASH/ISTH/NHF/WFH 2021 guidelines (ie, type 1C), indicating patients with quantitative deficiency due to an enhanced VWF clearance. With the advent of next-generation sequencing technologies, the process of genetic diagnosis has substantially changed and improved accuracy. Therefore, nowadays, patients with type 3 and severe type 1 VWD can benefit from genetic testing as much as type 2 VWD. Specifically, genetic testing can be used to confirm or differentiate a VWD diagnosis, as well as to provide genetic counseling. The focus of this manuscript is to discuss the current knowledge on VWD molecular pathophysiology and the application of genetic testing for VWD diagnosis.

Keywords: NGS; VWD; VWF; genetic testing; molecular diagnosis.

FIGURE 1

Structure of the von Willebrand factor (VWF) gene, pseudogene, and protein precursor. A schematic representation of the pre-pro-VWF is presented along with its homologous repeated domain. Disulfide bonds between subunits that are involved in dimerization and multimerization are shown along with ligand binding sites. The arrows indicate the major location of mutations that cause various type 2 von Willebrand disease. Recessive type 2 von Willebrand disease variants are shown by an asterisk (*). ADAMTS13, a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13; FVIII, factor VIII; GP, glycoprotein; SP, signal peptide.

FIGURE 2

Type of von Willebrand factor genetic variants stratified for each von Willebrand disease type. The majority of types 1, 2A, 2B, 2M, and 2N variants reported so far are missense, differently from type 3 von Willebrand disease with only 25% missense variants.

FIGURE 3

von Willebrand factor (VWF) domain distribution and the type of von Willebrand disease (VWD) for all the reported variants so far. Variants of VWD types 1 and 3 were found all over the VWF domains, mainly propeptide VWF (D1-D2 domain), D′-D3, D4, and C1-C6. In type 2A, 45% of variants were in the A2 domain, and the rest were in the D1-D2 (15%), D′-D3 (20%), A1 (14%), and cystine-knot (CK) domain (6%). All variants of type 2B were in the A1 domain (85%) or D3-A1 junction (15%). Type 2M variants were located mostly in the A1 (75%) but also in the A3 domains (25%). A majority of type 2N variants were in the D′-D3 (89%), and the rest, 11%, were in the D2 domain. SP, signal peptide.

FIGURE 4

Assessment of pathogenicity and interpretation of novel variants. In the case of a novel variant, large population-based databases such as gnomAD can be used to filter out common variants. Next, the effect of variants at the protein level can be assisted using in silico tools. The study of family members is useful in understanding the pathogenicity of genetic variants and their inheritance patterns. The true effect of variants on protein production and function can be determined by cell and animal-based models. The final step is to confirm the variant influence in the studied proband using phenotypic assays to classify variant.

FIGURE 5

Genetic counseling for von Willebrand disease (VWD). VWD occurs among men and women equally. Autosomal–dominant VWD types: type 1, 2A, 2B, and 2M; autosomal recessive VWD types: type 2N and type 3. Of note, a rare type 2A phenotype, 2A(IIC), is also inherited as autosomal recessive. An autosomal-dominant VWD only requires one copy of the von Willebrand factor (VWF) gene to be altered for the condition to present. The chance of a child inheriting the variant and developing the VWD from an affected parent is 1 in 2 (50%). In autosomal recessive conditions, both copies of the VWF gene are required to be altered for the condition to be present. The chance of a child inheriting 2 copies of the altered gene and developing VWD when both parents are carriers is 1 in 4 (25%). Chr, chromosome.