Six Different Point Mutations in Seven Danish Families with Symptomatic Protein C Deficiency

Summary

Six different point mutations of the protein C gene are described in seven Danish families with protein C deficiency associated with an increased risk of venous thromboembolism. All affected family members are heterozygotes for the mutated protein C genotype. One mutation is a G²⁹⁹²→A transition at position +5 in the 5’ splice site of intron D. The other five mutations affect the protein coding region. One is a C^l432→T transition in exon III converting the highly conserved Arg¹⁵ to Trp in the Gla-domain. Another mutation is a G³¹⁵⁷→C transversion in exon V converting the non-conserved Gly⁷² to Arg in the epidermal growth factor domain. The remaining three mutations are located in non-conserved amino acid positions in exon IX and affect the serine proteinase domain. The first is a G⁸⁵⁵⁹→C transversion converting Gly²⁸² to Arg. The second is a C⁸⁵⁷¹→T transition (present in two families) converting Arg²⁸⁶ to Cys. The third is a C⁸⁶⁹⁵→T transition converting Pro³²⁷ to Leu. In each family the protein C deficiency cosegregates or probably cosegregates (one family, G⁸⁵⁵⁹→C) with the mutation. All affected family members exhibit a reduction of both the antigen and the functional plasma concentration of protein C to approximately 50% of normal indicating that the mutated protein C is not present (type 1 deficiency) or only present in low amounts in plasma. Agarose gel electrophoresis followed by Western blotting shows that the Arg¹⁵→Trp substitution is associated with a normal as well as an abnormal migrating plasma protein C band. This provides positive evidence for that both the normal and mutated alleles are expressed (type 2 deficiency). The five other mutations are associated with only one band with the mobility of normal protein C. In one family a novel G¹³⁹⁰→A transition converting the normal Ala¹ to Thr was demonstrated. This mutation is not linked to the patient specific G⁸⁵⁵⁹→C transversion. In conclusion one splice site mutation and five different missense mutations of the protein C gene are described.

• References

• 1 Esmon CT. The protein C anticoagulant pathway. Arterioscler Thromb 1992; 12: 135-145
• 2 Kiesel W. Human plasma protein C. Isolation, characterization, and mechanism of activation by α-thrombin J Clin Invest 1979; 64: 761-769
• 3 Stenflo J. The biochemistry of protein C. In: Protein C and related proteins. Biochemical and clinical aspects. Bertina RM. ed. Edinburgh: Churchill Livingstone; 1988. pp 21-54
• 4 Foster D, Davie EW. Characterization of a cDNA coding for human protein C. Proc Natl Acad Sci USA 1994; 81: 4766-4770
• 5 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-4677
• 6 Plutzky J, Hoskins JA, Long GL, Crabtree GR. Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 1986; 83: 546-550
• 7 Foster DC, Sprecher CA, Holly RD, Gambee JE, Walker KM, Kumar AA. Endoproteolytic processing of the dibasic cleavage site in the human protein C precursor in transfected mammalian cells: Effects of sequence alterations on efficiency of cleavage. Biochemistry 1990; 29: 347-354
• 8 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 6: 2561-2567
• 9 Broekmans AW, Conard J. Hereditary protein C deficiency. In: Protein C and related proteins. Biochemical and clinical aspects. Bertina RM. ed. Edinburgh: Churchill Livingstone; 1988. pp 160-181
• 10 Reitsma PH, Poort SR, Allaart CF, Briët E, Bertina RM. The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: Heterogeneity and founder effects. Blood 1991; 78: 890-894
• 11 Allaart CF, Poort SR, Rosendaal FR, Reitsma PH, Bertina RM, Briët E. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993; 341: 134-138
• 12 Reitsma PH, Poort SR, Gandrille S, Long GL, Sala N, Cooper DN. Protein C Deficiency: A Database of Mutations. Thromb Haemost 1993; 69: 77-84
• 13 Lind B, Van Solinge WW, Schwartz M, Thorsen S. Splice site mutation in the human protein C gene associated with venous thrombosis: Demonstration of exon skipping by ectopic transcript analysis. Blood 1993; 82: 2423-2432
• 14 Philips M, Juul A-G, Selmer J, Lind B, Thorsen S. A specific immunologic assay for functional plasminogen activator inhibitor 1 in plasma-standardized measurements of the inhibitor and related parameters in patients with venous thromboembolic disease. Thromb Haemost 1992; 68: 486-494
• 15 Schwartz M, Rosenberg T, Niebuhr E, Lundsteen C, Sardemann H, Andersen O, Yang H, Lamm LU. Choroideremia: further evidence for assignment of the locus to Xql3-Xq21. Hum Genet 1986; 74: 449-452
• 16 De Stefano V, Leone G, Micalizzi P, Teofili L, Falappa PG, Pollari G, Bizzi B. Arterial thrombosis as clinical manifestation of congenital protein C deficiency. Blut 1991; 62: 180-183
• 17 Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base- pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54
• 18 Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA. Splicing of messenger RNA precursors. Annu Rev Biochem 1986; 55: 1119-1150
• 19 Green MR. Biochemical mechanisms of constitutive and regulated pre- mRNA splicing. Annu Rev Cell Biol 1991; 7: 559-599
• 20 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
• 21 Tokunaga F, Tsukamoto T, Wakabayashi S, Koide T. Analysis of protein C deficiency by expression and characterization of five recombinant Arg-15 mutants. Thromb Haemost 1993; 69: 979 (abstract)
• 22 Long GL, Belagaje RM, MacGillivray RT A. Cloning and sequencing of liver cDNA coding for bovine protein C. Proc Natl Acad Sci USA 1984; 81: 5653-5656
• 23 Schmidel DK, Tatro AV, Phelps LG, Tomczak JA, Long GL. Organization of the human protein S genes. Biochemistry 1990; 29: 7845-7852
• 24 Degen SJ F, Davie EW. Nucleotide sequence of the gene for human prothrombin. Biochemistry 1987; 26: 6165-6177
• 25 MacGillivray RT A, Davie EW. Characterization of Bovine Prothrombin mRNA and Its Translation Product. Biochemistry 1984; 23: 1626-1634
• 26 O’Hara PJ, Grant FJ, Haldeman BA, Gray CL, Insley MY, Hagen FS, Murray MJ. Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation. Proc Natl Acad Sci USA 1987; 84: 5158-5162
• 27 Yoshitake S, Schach BG, Foster DC, Davie EW, Kurachi K. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985; 24: 3736-3750
• 28 Katayama K, Ericsson LH, Enfield DL, Walsh KA, Neurath H, Davie EW, Titani K. Comparison of amino acid sequence of bovine coagulation Factor IX (Christmas Factor) with that of other vitamin K-dependent plasma proteins. Proc Natl Acad Sci USA 1979; 76: 4990-4994
• 29 Leytus SP, Foster DC, Kurachi K, Davie EW. Gene for human factor X: A blood coagulation factor whose gene organization is essentially identical with that of factor IX and protein C. Biochemistry 1986; 25: 5098-5102
• 30 Fung MR, Campbell RM, MacGillivray RT A. Blood coagulation factor X mRNA encodes a single polypeptide chain containing a prepro leader sequence. Nucleic Acids Res 1984; 12: 4481-4492
• 31 Zhang L, Jhingan A, Castellino FJ. Role of individual gamma-carboxy-glutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity. Blood 1992; 80: 942-952
• 32 Zhang L, Castellino FJ. The contributions of individual gamma-carboxy-glutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles. J Biol Chem 1993; 268: 12040-12045
• 33 Zhang L, Castellino FJ. Influence of Specific gamma-Carboxyglutamic Acid Residues on the Integrity of the Calcium-dependent Conformation of Human Protein C. J Biol Chem 1992; 267: 26078-26084