title: Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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 termination codons, we investigated whether this transcript is subjected to nonsense-mediated mRNA decay degradation. The results confirmed the involvement of nonsense-mediated mRNA decay in the degradation of F5 PTC(+) mRNA. Moreover, a comprehensive analysis of the F5 splicing pattern led to the identification of two in-frame splicing variants resulting from skipping of exons 3 and 5-6.
  
  
  CONCLUSIONS
  The functional consequences of three splicing mutations leading to FV deficiency were elucidated. Furthermore, we report the identification of three alternatively spliced F5 transcripts.
authors: Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3/5, 20133 Milan, Italy.
authors: Claudia
authors: C
authors: Dall'Osso
authors: Ilaria
authors: I
authors: Guella
authors: Stefano
authors: S
authors: Duga
authors: Nadia
authors: N
authors: Locatelli
authors: Elvezia Maria
authors: EM
authors: Paraboschi
authors: Marta
authors: M
authors: Spreafico
authors: Abdolreza
authors: A
authors: Afrasiabi
authors: Christoph
authors: C
authors: Pechlaner
authors: Flora
authors: F
authors: Peyvandi
authors: Maria Luisa
authors: ML
authors: Tenchini
authors: Rosanna
authors: R
authors: Asselta
mesh_terms: D000328:Adult; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D005165:Factor V / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005166:Factor V Deficiency / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D005165:Factor V