Studies on the molecular mechanisms of large deletion in the F8 gene in a severe haemophilia A patient

Abstract

Objective To disclose the molecular mechanisms of large deletion in the F8 gene in a sever haemophilia A patient and to screen the potential carriers. Methods Coagulation assay was performed to establish the phenotype.LD-PCR and sequence-specific PCR were adopted for detecting intron 22 and intron 1 inversion of F8 gene.All the exons of F8 gene and its flanking sequences were amplified and sequenced directly.Deletion breakpoints were identified by primer walking strategy.Breakpoint-specific assays were performed for the identification of carriers.Multiplex fluorescent PCR was used to detect the polymorphism of six STR locus (F8Up226,F8Up146,F8Int13,F8Int25,F8Down48 and DXS1073) for gene linkage analysis. Results The proband had a significantly reduced activity of factorⅧ (<1%).Inversion of intron 22 and intron 1 showed negative results in the proband.Deletion of exons 4-7 in the F8 gene in the proband was suspected by the repeated failure to amplify the corresponding exons by PCR.Sequencing the products of prime walking showed that the patient had a deletion of 27 685 bp comprising exons 4-7,a 2-bp microhomology (AT) was observed at the breakpoint junction.The results of breakpoint-specific PCR showed that the fetal and the proband′s daughter were deletion carriers,consistent with the results of the genetic linkage analysis. Conclusions The deletion of exons 4-7 in the F8 gene results in a sever haemophilia A.Microhomology-mediated end-joining mediates the formation of detected deletion.The breakpoint-specific PCR provids a reliable diagnostic tool for identification of carrier status.(Chin J Lab Med,2013,36:534-537) Key words: Hemophilia A; Factor Ⅷ; Mutation; Exons; Multiplex polymerase chain reaction