آنالیز مولکولی ژن فون ویلیبرانت در بیماران فون ویلیبرانت تیپ یک با استفاده از تکنیک ژل الکتروفورز حساس به ساختار (CSGE): مقایسه دو روش دستی و ماشینی (فلورسانت)

Background and purpose: Von Willebrand Disease (VWD) type 1, is the most common inherited bleeding disorder caused by defect in Von Willebrand Factor (VWF) gene with 178000 nucleotide length. Different methods are available to detect unknown mutations in a genetic study. The fluorescent conformation sensitive gel electrophoresis (F-CSGE) was designed for the VWF gene by using fluorescent dyes in this study and two methods of CSGE (manual and fluorescent) were compared to detect VWF gene mutations. Materials and methods :The VWF gene in 7 index cases with type 1 VWD and one normal control were amplified using 52 exons, exon-intron boundaries and 3’ region of the gene using 56 different PCR. The PCR fragments were analyzed using both manual and fluorescent CSGE and the fragments with bandshifts were sequenced. Results :In total, there were 125 PCR fragments with bandshift using F-CSGE and 101 fragments with bandshift using M-CSGE. Five different mutations were identified using both techniques including three new mutations, glycine 19 to argenine (G19R) in exon 2, Argenine 1315 to histidine (R1315H) in exon 28, cystin 2304 to lysine(C2304Y) in exon 40 and two known mutations,prolin 1266 to Lysine (P1266L) and Serine 1285 to Prolin (S1285P) in exon 28. In addition, 45 different polymorphisms were detected using F-CSGE and 39 were detected using M-CSGE. Also 192 different PCR fragments were analyzed in each run using F-CSGE and maximum 40 samples were analyzed using M-CSGE. Conclusion:Although F-CSGE is more time consuming and more expensive than M-CSGE, but it is a rapid, more sensitive and high-throughput method to identify unknown mutations in VWF gene compared with M-CSGE.