Phenotypic characterization and genetic analysis found that 28 of 63 participants in a Spanish centralized cohort of patients with von Willebrand Disease (VWD) had at least one mutation for type 2N of the disease. Nine individuals had the diagnosis of type 2N VWD, and 19 individuals carried a heterozygous type 2N mutation.
Further testing allowed for the diagnosis of 8 of these 19 carriers with another type of VWD. Thirteen individuals had a type 2N mutation combined with other variations.
Next-generation sequencing (NGS) revealed 3 type 2N mutations: p.Arg854GIn on exon 20, p.Arg816Trp on exon 19, and p.Arg763Ser on exon 18 of the VWF (von Willebrand factor) gene. NGS demonstrated that 3 participants had p.His817GIn mutations, which the authors explained may “modulate the capacity of VWF to bind to FVIII, but [it] is not a disease-causing mutation.”
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Researchers performing phenotype analysis found abnormal ratios of von Willebrand factor (VWF) to factor VIIIB (FVIIIB) in 19 of the 27 participants who had it done. Nine of these individuals presented with severe VWD phenotypes, and 10 with moderate VWD phenotypes. The remaining 8 individuals did not demonstrate abnormal VWF:FVIIIB ratios; thus, they were diagnosed with other types of VWD.
The investigators observed that the 19 type 2N carriers who had borderline FVIII plasma levels may not have been detected based on clinical and laboratory tests alone.
The authors stressed the importance of genetic analysis, writing, “The high detection yield and affordability of next-generation sequencing support use of this technology as a first-line diagnostic tool” in clinical practice settings.
Study Methods
Researchers recruited participants from the multicenter, prospective, Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES) project. Out of all the participants in this project, 480 individuals had the diagnosis of VWD. The investigators selected 63 of them who met at least 1 of the following 4 inclusion criteria: a local diagnosis of type 2N VWD, a decreased FVIII:C/VWF:Ag ratio in the centralized phenotype study, type 2N mutations following in the genetic study, or the p.His817GIn mutation.
Only 9 of these participants had type 2N VWD and 19 were carriers of a type 2N missense mutation, comprising only 10% (28/280 index cases) of the entire Spanish cohort.
Phenotypic analysis included plasma FVIII:C, VWF:Ag, VWF collagen binding (VWF:CB), VWF ristocetin cofactor activity (VWF:RCo), and VWF:FVIIIB ratio. The investigators used an in-house ELISA method to analyze the VWF:FVIIIB ratio. This VWF:FVIIIB ratio determined the phenotypic disease severity (severe <.3, moderate .3-.78, or normal ≥.79).
Genetic analysis using NGS examined relevant regions of the VWF gene, including exons 1-52, intronic flanking regions, and 1300 base pairs of the promoter. Investigators performed Sanger F8 sequencing for participants in whom type 2N VWD had been ruled out, confirming F8 mutations in 13 of the 63 participants with 8 obtaining the diagnosis of hemophilia A and the remaining 5 being categorized as female carriers.
“Genetic analysis discriminates between [hemophilia A and VWD] by identifying the causative mutation in the genes coding for VWF (VWF) in type 2N VWD or FVIII (F8) in [hemophilia A],” the authors said.
Reference
Pérez-Rodríguez A, Batlle J, Pinto JC, et al. Type 2N VWD: conclusions from the Spanish PCM-EVW-ES project. Haemophilia. Published online September 7, 2021. doi:10.1111/hae.14405
