Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern

Haematologica
Claudia Dall'OssoR Asselta

Abstract

Factor V deficiency is a rare autosomal recessive hemorrhagic disorder, associated with bleeding manifestations of variable severity. In the present study, we investigated the molecular basis of factor V deficiency in three patients, and performed a comprehensive analysis of the factor V gene (F5) splicing pattern. Mutational screening was performed by DNA sequencing. Wild-type and mutant F5 mRNA were expressed by transient transfection in COS-1 cells, followed by reverse-transcriptase polymerase chain reaction and sequencing. Real-time reverse-transcriptase polymerase chain reaction was used to evaluate degradation of mRNA carrying premature termination codons. Mutational screening identified three hitherto unknown splicing mutations (IVS8+6T>C, IVS21+1G>A, and IVS24+1_+4delGTAG). Production of mutant transcripts in COS-1 cells demonstrated that both IVS21+1G>A and IVS24+1_+4delGTAG cause the activation of cryptic donor splice sites, whereas IVS8+6T>C causes exon-8 skipping (F5-Delta 8-mRNA). Interestingly, F5-Delta 8-mRNA was also detected in wild-type transfected samples, human liver, platelets, and HepG2 cells, demonstrating that F5 exon-8 skipping takes place physiologically. Since F5-Delta 8-mRNA bears a premature termina...Continue Reading

Citations

Jun 16, 2010·Haemophilia : the Official Journal of the World Federation of Hemophilia·J A CutlerM J Mitchell
Jul 23, 2011·Haemophilia : the Official Journal of the World Federation of Hemophilia·E M ParaboschiR Asselta
Aug 10, 2011·Journal of Thrombosis and Haemostasis : JTH·S Duga, R Asselta
Nov 30, 2016·Hematology·Akbar DorgalalehMorteza Shamsizadeh
Jun 29, 2016·British Journal of Haematology·Francesca NuzzoElisabetta Castoldi
Feb 23, 2019·International Journal of Molecular Sciences·Elvezia Maria ParaboschiRosanna Asselta
Mar 27, 2021·Blood Coagulation & Fibrinolysis : an International Journal in Haemostasis and Thrombosis·Satish MaharajRajiv Pruthi

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