PATIENT WITH TYPE 2N VON-WILLEBRAND-DISEASE IS HETEROZYGOUS FOR A NEWMUTATION - GLY22GLU - DEMONSTRATION OF A DEFECTIVE EXPRESSION OF THE 2ND ALLELE BY THE USE OF MONOCLONAL-ANTIBODIES

We report the case of a Chinese patient who has subnormal von Willebra nd factor (VWF) level and normal VWF multimeric pattern, but a lack of vWF capacity to bind factor VIII (FVIII). Exons 18 to 20 of the patie nt's VWF gene were analyzed by DGGE and a G2354 --> A substitution whi ch changes the encoded amino acid sequence from Gly22 to Glu was ident ified. The patient is heterozygous for this substitution, creating a u nique Sac I restriction site. Recombinant vWF (rvWF) containing the ca ndidate mutation was transiently expressed in COS-7 cells, It was proc essed and secreted normally but failed to bind FVIII. FVIII binding ab ility of hybrid rvWF, obtained by cotransfection of normal and mutated expression vectors and corresponding to a heterozygous genotype, was moderately decreased. To explain this functional discrepancy between p atient's plasma vWF and hybrid rvWF, we used anti-vWF monoclonal antib odies (MoAbs) as capture in an enzyme-linked immunosorbent assay test. MoAb 32B12 recognized both wild-type and mutated rvWFs whereas MoAb 4 18 did not recognize mutated rvWF. Because MoAb 418 also failed to cap ture the plasma vWF from propositus, it means that his second nonmutat ed allele is not expressed or expressed at a very low level. (C) 1997 by The American Society of Hematology.