Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.
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.
Dall'Osso
Claudia
C
Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3/5, 20133 Milan, Italy.
Guella
Ilaria
I
Duga
Stefano
S
Locatelli
Nadia
N
Paraboschi
Elvezia Maria
EM
Spreafico
Marta
M
Afrasiabi
Abdolreza
A
Pechlaner
Christoph
C
Peyvandi
Flora
F
Tenchini
Maria Luisa
ML
Asselta
Rosanna
R
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
D012333:RNA, Messenger; D005165:Factor V