In type 2N von Willebrand disease (VWD), von Willebrand factor (VWF) is characterized by a markedly decreased affinity for factor (F)VIII and a recessive inheritance pattern. The FVIII binding domain has been localized within a fragment corresponding to the first 272 amino acid residues of mature VWF (aa 764–1035) encoded by exons 18–23 . Most of type 2N VWD patients have been found to harbor missense mutations in the VWF D′ domain (aa 769–865) encoded by exons 18–20 [http://www.shej.ac.uk/vw/]. The R854Q mutation, in exon 20, is the most frequent mutation identified, on at least one allele, in 90% of type 2N VWD patients studied thanks to the French INSERM Network (unpublished data). Until now, only the Q1053H and C1060R mutations in exon 24  and the C1225G mutation in exon 27 of VWF gene  have been identified outside the N-terminal FVIII binding domain of VWF.
We report here on the identification of a new VWF gene defect in a 24-year-old French male patient with a history of postsurgical bleedings, normal bleeding times (6 and 7 min), normal VWF antigen levels (VWF:Ag = 67 and 91 IU dL−1) and VWF ristocetin cofactor activity (VWF:RCo = 72 and 90 IU dL−1), normal VWF multimeric profile but low FVIII coagulant activity (range 8–23 IU dL−1 in one-stage chronometric assay). Capacity of plasma VWF to bind FVIII (VWF:FVIIIB), measured with a solid-phase system , was nil (Fig. 1), establishing the type 2N VWD diagnosis after 5 years of misdiagnosis of ‘mild hemophilia A’ and one episode of inadequate treatment with recombinant FVIII for appendicectomy. During the procedure, despite a high baseline factor VIII:C level due to the inflammatory state, the dose of recombinant FVIII (Refacto®; Wyeth, Madison, New Jersey, USA) required to maintain a FVIII level above 50 IU dL−1 was unusually high (82 U kg−1 at day 1, 65 U kg−1 at day 2, 50 U kg−1 at days 3 and 4), suggesting a short half-life of the infused FVIII. After intranasal spray of desmopressin (Octim®; Ferring, Copenhagen, Denmark) dosing 300 µg, VWF:Ag increased from 91 to 216 IU dL−1 (x 2.37), while FVIII:C increased from 20 to 116 IU dL−1 (× 5.8) and subsequently decreased with a short half-life of 3 h.
Sequencing of exons 18–27 of the patient's VWF gene revealed the presence of two molecular abnormalities in the heterozygous state: the 2561G→A transition in exon 20 inducing the R854Q mutation and the not yet reported 3223G→A transition in exon 25, inducing substitution of lysine for glutamic acid 1078 (E1078K). The patient's mother and sister who displayed subnormal FVIII:C levels (55 IU dL−1), normal multimeric profile and moderately decreased VWF:FVIIIB (Fig. 1) harbored, respectively, only the R854Q or E1078K abnormality in the heterozygous state.
The E1078K candidate mutation was introduced in pSVvWFA with the Quick Change XL site directed mutagenesis kit (Stratagene, La Jolla, CA, USA) and the corresponding mutated vector was transiently transfected in Cos-7 cells . Cotransfection experiments [K1078 vector with either Q854 vector or wild-type (WT) vector] were performed to mimic the VWF synthesized in vivo in the patient and his sister, respectively. All these recombinant VWFs (rVWFs) showed normal secretion and multimerization. Both K1078 and K1078/Q854 rVWFs displayed dramatically reduced VWF:FVIIIB (Fig. 1). In contrast, K1078/WTrVWF and Q854/WTrVWF, expressed to mimic, respectively, the patient's sister and mother plasma VWF, showed only partially reduced function (Fig. 1). Taking all these data together, we demonstrated the relationship between patients' phenotype and genotype and confirmed the diagnosis of type 2N VWD, enabling establishment of optimal treatment for the patient. Furthermore, we showed again the implication of VWF D3 domain in the interaction with FVIII by identifying the first molecular abnormality in exon 25 of the VWF gene responsible for type 2N VWD.