Willebrand factor gene for diagnosing von Willebrand disease 2N

WOODS AI; KELLER, L; KEMPFER A C; FARIAS CE; CASAIS P; LAZZARI MA

Congreso; 19th International Congress on Thrombosis; 2005

Background: Conformation Sensitive Gel Electrophoresis (CSGE) is used as scanning for diagnosis of von Willebrand disease type 2N. Aims: In previous abstract we described its efficiency to scan exons 18-21 of VWF gene. Now, we evaluated its performance for exon 22 study. Methods: Exon 22 was amplified by PCR in 30 patients with low FVIII and FVIII binding to VWF and 20 normal controls. Heteroduplexes were generated using homozygous G for 2880G>A polymorphism. PCR-products and heteroduplexes were analyzed by CSGE. Amplicons were also analyzed by sequencing analysis (ABI PRISM 310). Results: By sequencing analysis, 14 heterozygotes for the 2880G>A polymorphism, 6 homozygotes A and 30 homozygotes G were detected (alelic frequency: 0,74/0,26). By CSGE, in all cases only one band was observed. Double bands in both homozygous A and heterozygous patients should be observed; then, 40% of false negatives were obtained. Characteristics among exons 20, 21 and 22 were: Exon 22: PCR-product size: 254 bp; 2880G>A position: 125 bp; GC content: 55,5%. Exon 20: PCR-product size: 202 bp; 2555G>A position: 66 bp; GC content: 58,9%. Exon 21: PCR-product size: 268 bp; 2771G>A position: 147 bp; GC content: 54,5%. Summary: Given that the mismatches were the same and their positions in the amplicons, the of PCR-product size and the GC content were similar among the 3 exons, we did not find out explanation for the absence of double bands in the CSGE scanning of exon 22. Conclusion: CSGE is not useful to scan exon 22 of VWF gene for us.