Messenger RNA analysis and multiplex ligation probe amplification forhemophilia A patients without found mutation or suspected exon(s)deletion in genomic DNA

Introduction and Objectives:Hemophilia A (HA) is a congenital X-linked recessivebleeding disorder resulting from mutation of FVIII gene. There are around 5–7% ofHA patients without found any DNA encoded mutation despite using powerful andsensitive techniques for molecular diagnosis. The aims of this study were to identifythe genetic defect in three different genetic cohorts of hemophilia patients by analysisof FVIII mRNA and multiplex ligation amplication assay (MLPA).Patients and Methods:We have assessed 19 hemophilia A patients (5 mild, 2moderate, 6 severe and 6 inhibitor) from 19 unrelated families from Jan. 1 to Dec. 15,2015. With regard to the genetic defect, there were two patients with splice sitemutations, 8 patients with suspected deletion of exons and 9 patients withoutidentifiable DNA mutation by previous genetic testings. RNA was extracted from200ll buffy coat according to QIAamp RNA Blood Mini Kit. For reversetranscription FVIII cDNA, the method was modified from the report in the literature