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

Claudia Dall'Osso, Ilaria Guella, Stefano Duga, Nadia Locatelli, Elvezia Maria Paraboschi, Marta Spreafico, Abdolreza Afrasiabi, Christoph Pechlaner, Flora Peyvandi, Maria Luisa Tenchini, Rosanna Asselta

Research output: Contribution to journalArticle

16 Citations (Scopus)

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. Design and 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-D8-mRNA). Interestingly, F5-D8-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-D8-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.

Original languageEnglish
Pages (from-to)1505-1513
Number of pages9
JournalHaematologica
Volume93
Issue number10
DOIs
Publication statusPublished - Oct 2008

Fingerprint

Factor V Deficiency
Statistical Factor Analysis
Messenger RNA
Mutation
Nonsense Mediated mRNA Decay
Exons
Nonsense Codon
Genes
COS Cells
RNA Stability
Reverse Transcriptase Polymerase Chain Reaction
Hemorrhagic Disorders
RNA Splice Sites
Factor IX
Factor V
Hep G2 Cells
DNA Sequence Analysis
Transfection
Real-Time Polymerase Chain Reaction
Blood Platelets

Keywords

  • Exon skipping
  • F5
  • Factor V deficiency
  • Nonsense-mediated mRNA decay
  • Premature termination codons
  • Splicing mutations

ASJC Scopus subject areas

  • Hematology

Cite this

Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. / Dall'Osso, Claudia; Guella, Ilaria; Duga, Stefano; Locatelli, Nadia; Paraboschi, Elvezia Maria; Spreafico, Marta; Afrasiabi, Abdolreza; Pechlaner, Christoph; Peyvandi, Flora; Tenchini, Maria Luisa; Asselta, Rosanna.

In: Haematologica, Vol. 93, No. 10, 10.2008, p. 1505-1513.

Research output: Contribution to journalArticle

Dall'Osso, C, Guella, I, Duga, S, Locatelli, N, Paraboschi, EM, Spreafico, M, Afrasiabi, A, Pechlaner, C, Peyvandi, F, Tenchini, ML & Asselta, R 2008, 'Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern', Haematologica, vol. 93, no. 10, pp. 1505-1513. https://doi.org/10.3324/haematol.12934
Dall'Osso, Claudia ; Guella, Ilaria ; Duga, Stefano ; Locatelli, Nadia ; Paraboschi, Elvezia Maria ; Spreafico, Marta ; Afrasiabi, Abdolreza ; Pechlaner, Christoph ; Peyvandi, Flora ; Tenchini, Maria Luisa ; Asselta, Rosanna. / Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. In: Haematologica. 2008 ; Vol. 93, No. 10. pp. 1505-1513.
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T1 - Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern

AU - Dall'Osso, Claudia

AU - Guella, Ilaria

AU - Duga, Stefano

AU - Locatelli, Nadia

AU - Paraboschi, Elvezia Maria

AU - Spreafico, Marta

AU - Afrasiabi, Abdolreza

AU - Pechlaner, Christoph

AU - Peyvandi, Flora

AU - Tenchini, Maria Luisa

AU - Asselta, Rosanna

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N2 - 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. Design and 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-D8-mRNA). Interestingly, F5-D8-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-D8-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.

AB - 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. Design and 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-D8-mRNA). Interestingly, F5-D8-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-D8-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.

KW - Exon skipping

KW - F5

KW - Factor V deficiency

KW - Nonsense-mediated mRNA decay

KW - Premature termination codons

KW - Splicing mutations

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