Expression of wild-type and recombinant D4-CK domains of VWF gene with novel variants in Pakistan

Inherited quantitative or qualitative abnormalities in the von Willebrand factor (VWF) gene underlie the pathophysiology of von Willebrand disease (VWD). This study aimed to evaluate the secretion profiles and structural analysis of two novel missense variants found in Pakistani patients located within the C1 domain of VWF.

Variants were generated by site-directed mutagenesis of the D4-CK domain cloned into the pcDNA3.1 vector. Constructs were then expressed in HEK293T cells, and protein expression and secretion were subsequently assessed using both qualitative and quantitative analyses. Alphafold was used to predict the structure of domain with the presence of variants.

Both variants, Arg2311Cys and Asp2328His, showed markedly reduced secretion in the conditioned medium, whereas intracellular protein levels remained comparable to the wild-type D4-CK domain, demonstrated by Western blot analysis. Quantitative ELISA showed that both variants, in the homozygous state, had protein concentrations less than half of the wild type. One-way ANOVA validated that the results were statistically significant. Finally, the C1 domain modelling predicted structural alterations in the presence of both variants.

The novel missense variants characterized in the current study, did not impair protein synthesis, however these interfered with post-translational processing, folding, and/or intracellular trafficking. Overall, the findings suggest that the variants are likely pathogenic and highlight their value of interpretative analysis.