Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): comprehensive genetic analysis by next-generation sequencing of 480 patients

Abstract

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

Figure 1.

Flowchart depicting the molecular analysis and identification of potential mutations in individuals enrolled in the study. VWF from 48 patients were simultaneously amplified in a 48.48 Access-Array. Alignment, variant calling and annotation of each variant identified was performed by the CLC Genomic Workbench software. This analysis allowed selection of mutations/variants aligned against VWF (shown in gray) and elimination of those aligned against VWFP (shown in black). Variant filtering was performed by the Variant Studio software. *MAF<0.01 for all variants except three (p.Arg854Gln, p.Arg924Gln, p.Pro2063Ser). VWF: von Willebrand factor gene; VWFP: von Willebrand factor pseudogene; dbSNP: dbSNP database; 1000G: 1000 Genomes Project; ExAC: Exome Aggregation Consortium; MAF: minor allele frequency; NGS: next-generation sequencing.

Figure 2.

Classification of VWD patients based on central phenotypic diagnosis and final assignment according to the genotype-phenotype correlation. The dataset highlights the number of patients reincluded or reclassified after molecular study, from the initial classification based on central phenotypic results (shown in black) to a definite, refined classification based on molecular data analysis together with phenotypic results (shown in gray). *Patients diagnosed initially as type 1 VWD and reclassified to type 2M VWD due to the presence of collagen binding mutations.^† Patient with uncertain classification reclassified to type 3 VWD due to the presence of nonsense mutation in homozygous state. This is a discrepant case since laboratory levels do not correlate and it is pending a new analysis from a freshly obtained sample. Out: number of patients removed from this subtype; To: final definite classification; In: number of patients reclassified to this subtype; From: previous phenotypic classification; UC: uncertain classification; AVWS: acquired von Willebrand Syndrome.

Figure 3.

Summary of the final diagnosis of patients in terms VWD type. On the basis of phenotype data, 98 of the 556 recruited individuals did not meet any inclusion criteria and were initially excluded. Following the molecular analysis, 43 of these patients were reincluded due to the presence of a candidate mutation in VWF. The remaining 55 patients did not meet any inclusion criteria. Moreover, molecular analysis prompted the exclusion of 21 additional individuals: eight HA patients, five HA carriers, and eight patients finally diagnosed as AVWS and confirmed by the absence of mutations in VWF or GP1BA. Of note, 280 families were finally included (shown in black) and nine of them had members with different VWD types. In those particular cases, the family may be counted more than once; that is, within each VWD type where a family member was classified. VWD: von Willebrand disease.

Figure 4.

Schematic representation of phenotype/genotype correlations by VWD type. The correlation between genotype and phenotype in each patient was based on the concordance between the results of the phenotypic test panel and the results of the genetic analysis.

Figure 5.

Classification of the 280 families included according to VWD type. Numbers in parentheses refer to the total of families included in each VWD type. Of note, nine of the 280 families included in the PCM-EVW-ES presented a mixed phenotype (families in which some members had different VWD subtypes). UC: patients with an uncertain classification.