Type 2N von Willebrand Disease: Rapid Genetic Diagnosis of G2811A (R854Q), C2696T (R816W), T2701A (H817Q) and G2823T (C858F) – Detection of a Novel Candidate Type 2N Mutation: C2810T (R854W)

Summary

The majority of patients with type 2N von Willebrand disease (VWD type 2N) have mutations in the region of the von Willebrand factor (VWF) gene encoding the factor VIII binding domain of VWF. Two mutations predominate among VWD type 2N patients: G2811A and C2696T, which respectively bring about the amino acid substitutions R854Q and R816W in VWF. Several other mutations have been found in VWD type 2N, including T2701A (H817Q) and G2823T (C858F). We have developed a genetic test which permits rapid screening for these four mutations in a single polymerase chain reaction (PCR). The test employs induced heteroduplex formation using two universal heteroduplex generators, one of which detects G2811A (R854Q) and G2823T (C858F), the other detects C2696T (R816W) and T2701A (H817Q). The allele frequency of the common G2811A (R854Q) mutation was investigated in the local (S. Wales) population by examination of 216 VWF genes (108 individuals) and was found to be 0.01. The heteroduplex-based test additionally detected a novel candidate type 2N mutation, C2810T (R854W) and a previously described polymorphism, G2805A (R852Q). The polymorphism showed allele frequencies of 0.92 (G nucleotide) and 0.08 (A nucleotide) in the population study.

• References

• 1 Nishino M, Girma JP, Rothschild C, Fressinaud E, Meyer D. New variant of von Willebrand disease with defective binding to factor VIII.. Blood 1989; 74: 1591-9.
• 2 Mazurier C, Dieval J, Jorieux S, Delobel J, Goudemand M. A new von Willebrand factor (vWF) defect in a patient with factor VIII deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterisation of abnormal vWF/FVIII interaction.. Blood 1990; 75: 20-6.
• 3 Takahashi Y, Kalafatis M, Girma JP, Sewerin K, Andersson LD, Meyer D. Localisation of a factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor.. Blood 1987; 70: 1679.
• 4 Foster PA, Fulcher CA, Marti T, Titani K, Zimmerman TS. A major factor VIII binding domain resides within the amino terminal 272 amino acid residues of von Willebrand factor.. J Biol Chem 1987; 262: 8443.
• 5 Mazurier C, Meyer D. Factor VIII binding assay of von Willebrand factor and the diagnosis of type 2N von Willebrand disease – results of an international survey.. Thromb Haemost 1996; 76: 720-4.
• 6 Kroner PA, Friedman KD, Fahs S, Scott JP, Montgomery RR. Abnormal binding of factor VIII is linked with the substitution of glutamine for argi-nine in von Willebrand factor in a variant form of von Willebrand disease.. J Biol Chem 1991; 266: 19146-9.
• 7 Jorieux S, Tuley EA, Gaucher C, Mazurier C, Sadler JE. The mutation Arg(53)-Trp causes von Willebrand disease Normandy by abolishing binding to factor VIII. Studies with recombinant von Willebrand factor.. Blood 1992; 79: 563-7.
• 8 Sadler JE. A revised classification of von Willebrand disease.. Thromb Haemost 1994; 71: 502-5.
• 9 Ginsburg D, Sadler JE. Von Willebrand factor (vWF) database.. 1997 http//mmg2.im.med.umich.edu/vWF.
• 10 Eikenboom JCJ, Reitsma PH, Peerlinck KMJ, Briët E. Recessive inheritance of von Willebrand’s disease type 1.. Lancet 1993; 341: 982-6.
• 11 Culpan D, Standen G, Wood N, Mazurier C, Gaucher C, Bidwell J. Rapid mutation screening in type 2A von Willebrand’s disease using universal heteroduplex generators.. Br J Haematol 1997; 96: 464-9.
• 12 Wood N, Standen GR, Murray EW, Lillicrap D, Holmberg L, Peake IR, Bidwell J. Rapid genotype analysis of type 2B von Willebrand’s disease using a universal heteroduplex generator.. Br J Haematol 1995; 89: 152-6.
• 13 Wood N, Bidwell J. Genetic screening and testing by induced heteroduplex formation.. Electrophoresis 1996; 17: 247-54.
• 14 Cacheris PM, Nichols WC, Ginsburg D. Molecular characterisation of a unique von Willebrand disease variant. A novel mutation affecting von Willebrand factor/factor VIII interaction.. J Biol Chem 1991; 266: 13499-502.
• 15 Lahiri DK, Nurnberger Jr. JI. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies.. Nucleic Acids Res 1991; 19: 5444.
• 16 Bowen DJ, Standen GR, Granville S, Bowley S, Wood NAP, Bidwell J. Genetic diagnosis of factor V Leiden using heteodupelx technology.. Thromb Haemost 1997; 77: 119-22.
• 17 Mancuso DJ, Tuley EA, Westfield LA, Worral NK, Shelton-Inlies BB, Sorace JM, Alevy YG, Sadler JE. Structure of the gene for human von Willebrand factor.. J Biol Chem 1989; 264: 19514-27.
• 18 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia.. Science 1985; 230: 1350-4.
• 19 Maniatis T, Fritsch EF, Sambrook J. Analysis of DNA clones.. In: Molecular cloning, a laboratory manual.. Maniatis T. ed. New York: Cold Spring Harbour Laboratory; 1982. pp. 363-73.
• 20 Budowle B, Allen RC. Discontinuous polyacrylamide gel electrophoresis of DNA fragments.. In: Protocols in human molecular genetics, Methods in Molecular Biology 9.. Mathew CG. ed. New Jersey: Humana Press; 1991. p. 128.