Comparison of genotypes and phenotypes for von Willebrand factor gene variants using Japanese genome database

Abstract

von Willebrand disease (VWD) is a common inherited bleeding disorder. The aim of this study was to determine the predicted disease states associated with various pathogenic von Willebrand factor (VWF) variants and their phenotypes using the largest Japanese whole-genome database. Of the 5857 VWF gene variants registered in the Japanese Multi-Omics Reference Panel (jMorp), variants with the following criteria were extracted: (1) caused protein abnormalities due to genetic alterations; (2) have already been detected and included in a database, including known association with VWD; and (3) highly likely pathogenic by in silico analysis. We measured VWF activity, antigen, propeptide, and collagen binding activity in stored plasma samples obtained from heterozygous carriers of the selected variants. A total of 29 VWF variants (26 single nucleotide and 3 small insertions/deletions) were detected, and 6 of these were found in Leiden Open Mutation Database. We obtained 43 plasma samples from individuals carrying these 29 variants as heterozygous. For the 43 variant carriers, their mean age was 43.0 years, and blood group was type O in 17 (39.5%). Analysis of these plasma samples showed low VWF levels (<50%) in 6 (14.0%). Low VWF levels were found in 2 of 8 of the nonsense (25%) and 4 of 31 of the missense variants (12.9%). Taking into consideration the limitation of using stored plasma samples, analysis of the jMorp indicated that most VWF gene variants with predicted pathogenic potential did not correlate with phenotypic expression. Our results supported incomplete penetrance and variable expressivity of the VWF gene variants.

Graphical abstract

Figure 1.

Selection process of pathogenic VWF variants and related plasma samples from jMorp. The pathogenicity of the 5857 VWF variants registered in 8.3KJPN was assessed by registration in the database LOVD, in silico analysis of pathogenicity, and genetic aberrations that do not escape pathogenicity. Because some variants matched >1 criterion, the number of variants selected by each criterion was counted in order of “variant type” first, followed by “database,” and then “in silico prediction.” In total, 29 variants were considered pathogenic; and VWF:Rco, VWF:Ag, VWF:pp, and VWF:CB in 43 plasma samples obtained from the carriers of these variants were analyzed, and their relation to phenotype was investigated. CADD, combined annotation dependent depletion; InMeRF, individual meta random forest; PON-P2, Pathogenic-or-Not-Pipeline2.

Figure 2.

VWF levels for each variant. (A) Plasma samples with <50% VWF:Rco were considered diagnostic for VWD. Two of 6 samples were nonsense variants, and the remaining were missense variants. Of the 4 missense variants, 3 showed amino acid substitution to cysteine. Two missense variants, p.Asp400His and p.Arg1943Cys, were novel. (B) VWF levels for missense variants in plasma samples obtained from 3 different individuals. VWF levels were different in different plasma samples, even for those with the same variant. (C) VWF levels for nonsense variants. We identified a number of cases with nonsense variants who did not have low VWF levels. Even for those with the same variant, VWF levels varied widely from sample to sample. For cases with VWF:Rco of >200% and VWF:Ag of >220%, their values were entered as 200% and 220%, respectively.

Figure 3.

Location of each variant analyzed and its respective VWF:pp/VWF:Ag and VWF:CB/VWF:Ag ratios. (A) The VWF:pp/VWF:Ag ratio of the Gly1672Arg and Arg1943Cys variants was >1.6, suggesting abnormality with enhanced clearance. (B) The VWF:CB/VWF:Ag ratios of Arg1145Cys and Ala1461Val were below the threshold value of 0.7, suggesting molecular abnormalities.

Figure 4.

Correlation between age and plasma VWF levels. The correlation between age and VWF levels was examined in all cases and those with missense and nonsense variants.

Figure 5.

Correlation of blood group and sex with plasma levels of VWF. (A-D) Correlation between blood groups and VWF-related measurements in plasma samples obtained from the donors. The number of individuals with blood groups A, AB, B, and O were 14, 3, 9, and 17, respectively. (E) Correlation between sex and VWF-related measurements. Statistical analysis was performed using a 2-tailed t test. n.s., not significant.