title: Characterisation and quantification of F8 transcripts of ten putative splice site mutations.
abstract: Mutations affecting splice sites comprise approximately 7.5 % of the known F8 gene mutations but only a few were verified at mRNA level. In the present study, 10 putative splice site mutations were characterised by mRNA analysis using reverse transcription PCR (RT-PCR). Quantitative real-time RT-PCR (RT-qPCR) and co-amplification fluorescent PCR were used in combination to quantify the amount of each of multiple F8 transcripts. All of the mutations resulted in aberrant splicing. One of them (c.6187+1del1) generated one form of F8 transcript with exon skipping, and the remaining nine mutations (c.602-6T>C, c.1752+5_1752+6insGTTAG, c.1903+5G>A, c.5219+3A>G, c.5586+3A>T, c.969A>T, c.265+4A>G, c.601+1_601+5del5 and c.1444-8_1444del9) produced multiple F8 transcripts with exon skipping, activation of cryptic splice site and/or normal splicing. Residual wild-type F8 transcripts were produced by the first six of the nine mutations with amounts of 3.9 %, 14.2 %, 5.2 %, 19.2 %, 1.8 % and 2.5 % of normal levels, respectively, which were basically consistent with coagulation phenotypes in the related patients. In comparison with the mRNA findings, software Alamut v2.3 had values in the prediction of pathogenic effects on native splice sites but was not reliable in the prediction of activation of cryptic splice sites. Our quantification of F8 transcripts may provide an alternative way to evaluate the low expression levels of residue wild-type F8 transcripts and help to explain the severity of haemophilia A caused by splicing site mutations.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liang
authors.forename: Mingjie
authors.initials: M
authors.lastname: Xiang
authors.forename: Yeling
authors.initials: Y
authors.lastname: Lu
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Ruan
authors.affiliation: Qiulan Ding, Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, No.197 Ruijin Second Road, Shanghai, 200025, China, Tel.: +86 21 54667770, Fax: +86 21 64333548, E-mail: qiulan_ding@hotmail.com.
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xi
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
mesh_terms: D001777:Blood Coagulation / Q000235:genetics*; D003198:Computer Simulation; D004252:DNA Mutational Analysis; D005091:Exons; D005169:Factor VIII / Q000235:genetics*; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D006467:Hemophilia A / Q000097:blood / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D022821:RNA Splice Sites; D012333:RNA, Messenger; C078147:F8 protein, human; D005169:Factor VIII
keywords: Factor VIII; gene expression; gene mutations