Molecular characterisation of Tyr530Ser and IVS16–1G>T mutations causing severe factor V deficiency

Summary

Our previous study reported a missense mutation (Tyr530Ser) and a splicing site mutation (IVS16–1G>T) in blood coagulation factor V (FV) gene in a two-year-old Chinese boy. However, the linkage between the mutations and severe FV deficiency and the underlying mechanism has not been elucidated. The present study was designed to investigate the effect of the two mutations and the possible pathogenetic mechanism. FV procoagulant activity showed tremendous decrease in the patient with two mutations. The bioinformatics analyses predicted that IVS16–1G>T mutation may cause the entire exon 17 of FV to be skipped in transcription and thereby result in a deletion mutant. To confirm the predicted results, the fragment of exon 16 to exon 18 containing IVS16–1G>T mutation was obtained by PCR and site-directed mutagenesis. IVS16–1G>T mutant and wild-type constructs were transfected into COS-7 cells. Sequence analysis showed that mutant transcript lacked the entire 180-bp length of exon 17. Moreover, compared to wild-type, the expression of the two mutant proteins was decreased and the procoagulant activity was also reduced when the deletion mutant cDNA and Tyr530Ser site mutant cDNA were transfected into COS-7 cells, respectively. Our results indicate that Tyr530Ser and IVS16–1G>T could be separately responsible for severe FV deficiency, while the phenotype in the proband could be caused by the combination effect of the two defects.

Keywords

Factor V deficiency - splicing mutation - missense mutation - pathogenetic mechanism

• References

• 1 Jenny RJ, Pittman DD, Toole JJ. et al. Complete cDNA and derived amino acid sequence of human factor V.. Proc Natl Acad Sci USA 1987; 84: 4846-4850.
• 2 Kane WH, Ichinose A, Hagen FS. et al. Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats.. Biochemistry 1987; 26: 6508-6514.
• 3 Adams TE, Hockin MF, Mann KG. et al. The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function.. Proc Natl Acad Sci USA 2004; 101: 8918-8923.
• 4 Villoutreix BO, Dahlback B. Structural investigation of the A domains of human blood coagulation factor V by molecular modeling.. Protein Sci 1998; 7: 1317-1325.
• 5 Orban T, Kalafatis M, Gogonea V. Completed three dimensional model of human coagulation factor va. Molecular dynamics simulations and structural analyses.. Biochemistry 2005; 44: 13082-13090.
• 6 Pellequer JL, Gale AJ, Getzoff ED. et al. Three-dimensional model of coagulation factor Va bound to activated protein C.. Thromb Haemost 2000; 84: 849-857.
• 7 Autin L, Steen M, Dahlback B. et al. Proposed structural models of the prothrombinase (FXa-FVa) complex.. Proteins 2006; 63: 440-450.
• 8 Stoilova-McPhie S, Parmenter CDJ, Segers K. et al. Defining the structure of membrane-bound human blood coagulation factor Va.. J Thromb Haemost 2008; 6: 76-82.
• 9 Macedo-Ribeiro S, Bode W, Huber R. et al. Crystal structures of the membrane-binding C2 domain of human coagulation factor V.. Nature 1999; 402: 434-439.
• 10 Ngo JC, Huang M, Roth DA. et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex.. Structure 2008; 16: 597-606.
• 11 Koeleman BP, Reitsma PH, Bakker E. et al. Location on the human genetic linkage map of 26 genes involved in blood coagulation.. Thromb Haemost 1997; 77: 873-878.
• 12 Cripe LD, Moore KD, Kane WH. Structure of the gene for human coagulation factor V.. Biochemistry 1992; 31: 3777-3785.
• 13 Kane W, Davie E. Blood coagulation factors V and VIII: structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders.. Blood 1988; 71: 539-555.
• 14 Vos HL. An online database of mutations and polymorphisms in and around the coagulation factor V gene.. J Thromb Haemost 2007; 5: 185-188.
• 15 Duga S, Montefusco MC, Asselta R. et al. R2074C missense mutation in the C2-domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein.. Blood 2003; 101: 173-177.
• 16 Schrijver I, Koerper MA, Jones CD. et al. Homozygous factor V splice site mutation associated with severe factor V deficiency.. Blood 2002; 99: 3063-3065.
• 17 Yamakage N, Ikejiri M, Okumura K. et al. A case of coagulation factor V deficiency caused by compound heterozygous mutations in the factor V gene.. Haemophilia 2006; 12: 172-178.
• 18 Jones CD, Yeung C, Negro F. et al. Molecular characterization and subcellular localization of Tyr478del: a pathogenic in-frame deletion in coagulation factor V.. J Thromb Haemost 2007; 5: 431-433.
• 19 Steen M, Miteva M, Villoutreix BO. et al. Factor V New Brunswick: Ala221Val associated with FV deficiency reproduced in vitro and functionally characterized.. Blood 2003; 102: 1316-1322.
• 20 Delev D, Pavlova A, Heinz S. et al. Modelling and expression studies of two novel mutations causing factor V deficiency.. Thromb Haemost 2008; 100: 766-772.
• 21 Lin TM, Chen HY, Wu CL. et al. Gly392Cys missense mutation in the A2 domain of factor V causing severe factor V deficiency: molecular characterization by expression of the recombinant protein.. Thromb Haemost 2005; 93: 614-615.
• 22 Shinozawa K, Amano K, Suzuki T. et al. Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency.. Int J Hematol 2007; 86: 407-413.
• 23 Dall’Osso C, Guella I, Duga S. et al. Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.. Haematologica 2008; 93: 1505-1513.
• 24 Cai XH, Wang XF, Ding QL. et al. Factor V C1149G and 5609–10INSCGTGGTT causing factor V deficiency: molecular characterization by in-vitro expression.. Thromb Haemost 2007; 98: 683-685.
• 25 Asselta R, Montefusco MC, Duga S. et al. Severe factor deficiency :exon skipping in the factor V gene causing a partial deletion of the C1 domain.. J Thromb Haemost 2003; 1: 1237-1244.
• 26 Fu QH, Zhou RF, Liu LG. et al. Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees.. Haemophilia 2004; 10: 264-270.
• 27 Duga S, Asselta R, Malcovati M. et al. From Owren’s first parahemophilia case to present, characterization of missense mutations in the factor V gene by in vitro expression.. Blood 2003; 102: 306a.
• 28 Zheng WD, Liu YH, Liu HF. et al. Identification of two novel mutations of human blood coagulation factor V gene in a Chinese family with congenital factor V deficiency.. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2006; 5: 515-518.
• 29 Kaufman RJ. Vectors used for expression in mammalian cells.. Meth Enzymol 1990; 185: 487-511.
• 30 Shapiro MB, Senapathy P. RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression.. Nucleic Acids Res 1987; 15: 7155-7174.
• 31 Bento I, Peixoto C, Zaitsev VN. et al. Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites.. Acta Crystallogr D Biol Crystallogr 2007; 63: 240-248.
• 32 Mann KG, Lawler CM, Vehar GA. et al. Coagulation factor V contains copper ion.. J Biol Chem 1984; 259: 12949-12951.
• 33 Tagliavacca L, Moon N, Dunham WR. et al. Identification and functional requirement of Cu(I) and its ligands within coagulation factor VIII.. J Biol Chem 1997; 272: 27428-27434.
• 34 Bihoreau N, Pin S, de Kersabiec AM. et al. Copper-atom identification in the active and inactive forms of plasma derived FVIII and recombinant FVIII-delta II. Eur. J.. Biochem 1994; 222: 41-48.
• 35 Wakabayashi H, Koszelak ME, Mastri M. et al. Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity.. Biochemistry 2001; 40: 10293-10300.
• 36 Nakamura M, Shishido N, Nunomura A. et al. Three histidine residues of amyloid-beta peptide control the redox activity of copper and iron.. Biochemistry 2007; 46: 12737-12743.
• 37 Venkateswarlu D. Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study.. BMC Struct Biol 2010; 10: 7.
• 38 Shen BW, Spiegel PC, Chang CH. et al. The tertiary structure and domain organization of coagulation factor VIII.. Blood 2008; 111: 1240-1247.
• 39 Xie F, Cheng F, Zhu X. Studies on hereditary deficiency of coagulation factor V.. Zhonghua Xue Ye Xue Za Zhi 2001; 22: 453-456.
• 40 Shen MC, Wu YF, Lin BD. et al. Novel mutations in the factor V gene of Taiwaiese factor V deficiency patients.. J Thromb Haemost 2007; 5 (Suppl. 02) P-M-204.
• 41 Guinto ER, Esmon CT, Mann KG. et al. The Complete cDNA Sequence of Bovine Coagulation Factor V.. J Biol Chem 1992; 267: 2971-2978.
• 42 Yang TL, Cui J, Rehumtulla A. et al. The Structure and Function of Murine Factor V and Its Inactivation by Protein C.. Blood 1998; 91: 4593-4599.
• 43 Grimm DR, Colter MB, Braunschweig M. et al. Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains.. Cell Mol Life Sci 2001; 58: 148-159.
• 44 Lin SW, Lin SR, Shen MC. Characterization of genetic defects of hemophilia A in patients of Chinese origin.. Genomics 1993; 18: 496-504.
• 45 Kemball-Cook G, Tuddenham EGD, Wacey AI. The Factor VIII Structure and Mutation Resource Site: HAMSTeRS Version 4.. Nucleic Acids Res 1998; 26: 216-219.