Phenotypic and Genotypic Correlations in 218 Patients with VWD Including Platelet Function

Objectives: In the past, progress in the diagnosis of von Willebrand Disease (VWD) due to more advanced molecular techniques allowed the first phenotype-genotype correlations. In contrast, improvement of methods for phenotypic description has not developed comparably. Therefore, diagnosis of VWD remains difficult because the relationship between laboratory assays and clinical presentation remains uncertain. Here, we present genetic results combined with laboratory and clinical data of patients with various types of VWD.

Methods: Long-range PCRs combined with sequencing were used to completely sequence VWF exons and exon-intron flanking regions. Laboratory investigations included Factor VIII:C, VWF antigen, VWF GP Ib-binding, PFA-200 closure time, VWF collagen binding, multimeric analysis, a standardized bleeding score and blood group. In addition, CRP and BMI were analyzed to assess environmental confounders. Closure time measured by PFA-200 was correlated with the clinical type of VWD, VWF domains carrying mutations, e.g. A1 domain which is known to play a role in VWF-collagen-interaction (YEE, A. & KRETZ, C., 2014) and those missense mutations which occurred in our cohort, respectively.

Results: Molecular analysis of the VWF gene in 218 patients with VWD has led to the identification of 1350 exonic missense sequence variations, 936 were heterozygous and 414 homozygous. Furthermore, we found 1114 silent sequence variations (761 heterozygous and 353 homozygous). Numerous intronic variants were detected, in addition. High allele frequencies of sequence variation were detected in exon 13, 18, 20, 28, 35 and 42, respectively. We identified a Thr1381Ala mutation in 162 patients, Ser1378Phe mutation (n=15), Arg1379Lys mutation (n=12) and in 6 patients we detected Arg1399His mutation. These residues are located on VWF A1 domain coding for a collagen binding region.

In the total group, mean PFA-Coll/ADP was 111.9 ± 43.3 sec and mean PFA-Coll/Epi was 156.8 ± 53.0 sec. In VWD type 1-patients mean PFA-Coll/ADP was 115.6 ± 45.8 sec and mean PFA-Coll/Epi was 165.4 ± 56.8 sec and in patients with type 2N 99.9 ± 19.3 sec and 130.8 ± 22.9 sec. In patients with VWD type 3 mean PFA-Coll/ADP was 269.0 ± 43.8 sec and mean PFA-Coll/Epi was 292.5 ± 10.6 sec. The differences between these groups were highly significant (p≤ .001). By analysing the closure time according to the mutations in the collagen binding region the mean PFA-Coll/ADP in patients with Thr1381Ala was 112.3 ± 43.8 sec and the mean PFA-Coll/Epi was 163.5 ± 52.1 sec, in Ser1378Phe or Arg1379Lys mean PFA-Col/ADP was 114.1 ± 42.3 and mean PFA-Coll/Epi was 164.8 ± 46.8 sec. In Arg1399His mean PFA-Coll/ADP was 104.8 ± 25.7 sec and the mean PFA-Coll/Epi was 183.4 ± 63.1 sec showing the strongest prolongation. Further analyses with other frequent mutations will be presented, too.

Conclusion: Here, we present a large number of newly described mutations in various regions of the VWF gene including mutations in the collagen binding region of domain A1. Although some mutations are located in the collagen binding region which is believed to promote binding to collagens type IV and VI patients showed prolonged closure time using the PFA-200 Collagen/Epinephrine cartridge which works with collagen type I but not cross reacting to collagens type IV or VI. Furthermore, even when a comprehensive panel of assays including genetic analysis is used both diagnosis as well as classification of VWD remain uncertain in some cases. Thus, further investigation of phenotypic genotypic correlations in VWD is warranted to better understand the VWF molecule.