Atypical presentation of VWD leading to discovery of novel VWF mutation
The diagnosis of VWD and its subtypes depends on markers like Von Willebrand antigen, activity, multimer patterns and binding capacities to FVIII, collagen and platelets. Still some cases present diagnostic challenges. The extensive analysis of an atypical VWD patient leading to the discovery of a novel VWD mutation is discussed.
The diagnostic work-up included: VWF:AG, VWF:Activity, VWF fibrinogen binding, VWF FVIII binding, fibrinogen, FVIII activity and multimer assays. Additional measurement of VWF:AG, VWF:ACT and FVIII activity over time were carried out to detect potential signs of protein degradation. Genetic screening was performed by whole exome sequencing of the VWF gene. VWF mRNA analysis was also carried out by RT-PCR and Sanger sequencing.
Routine analysis showed PFA-ADP and PFA EPI >300 seconds, VWF:ACT of 37% with a VWF:AG of 36%. Collagen binding and FVIII-binding were 46% (ref: 64-155%) and 28% (ref: 80-120%) respectively.
The first multimer assays (handmade gels) showed no multimer pattern at all. Eventually an automated assay was able to show a normal multimer distribution pattern. Genetic analysis of the VWF gene disclosed 2 heterozygous variants of unknown significance of which one (c.2771 G>A in exon 21, p.Arg924.Gln) has a 1-2.5% population prevalence and has been previously described in type 1 and 2N VWD. The other VUS (c.2278 C>A in exon 17) is a novel mutation predicting a major amino acid substitution (p.Arg760Ser) in the D2 domain of VWF. Sequencing of exons 17 and 21 in the patient’s VWF mRNA revealed homozygosity for the mutated allele at both mutation sites, indicating that the two variants are in cis and that the ‘normal’ allele is not expressed at mRNA level. Moreover, an aberrantly spliced mRNA was identified which lacks exon 17, leading to a frameshift and a premature stop codon in exon 18.
The diagnosis of VWD can be challenging. In this case, routine diagnostic work-up would initially suggest a type 1 VWD. However the reduced FVIII-binding and collagen binding would suggest a type 2-like VWD. The patient carried two variants of unknown significance on the only VWF allele that was expressed at the mRNA level. The cause of the silencing of the other allele, as well as the phenotypic impact of the exon 17 variant (via amino acid substitution and/or aberrant splicing) are currently under investigation. Better understanding of the pathophysiology might allow for a better diagnosis in this kind of atypical VWD patients.