Three Missense Mutations in the Protein C Heavy Chain Causing Type I and Type II Protein C Deficiency

Summary

We have studied the molecular basis of protein C deficiency in three families with a history of thromboembolic disease. An approximately 50% reduction in both functional and immunologic levels of protein C was detected in the plasma from two unrelated patients, designated protein C Osaka 1 and protein C Osaka 2. An approximately 50% reduction in functional level but normal immunologic level of protein C was detected in plasma from a third patient, designated protein C Osaka 3. DNA sequencing of the amplified DNA revealed one missense mutation in each case. Additional mutations in the coding sequence were excluded by DNA sequencing of all protein C exons. We identified a C-to-T change at nucleotide number 6,218 of the protein C gene in protein C Osaka 1. This results in the amino acid substitution of Arg-169 by Trp at the a-thrombin cleavage site. In protein C Osaka 2, a G-to-A change at nucleotide number 8,807 was identified leading to the amino acid substitution of Met-364 by lie in the protease domain. This substitution may impair the synthesis or stability of protein C Osaka 2. In protein C Osaka 3, a G-to-C change at nucleotide number 8,868 was identified. This results in substitution of Gly-385 by Arg in the protease domain. Based on these, it was concluded that Arg-169-to-Trp mutation and Met-364-to-Ile mutation cause type I protein C deficiency and Gly-385-to-Arg mutation causes type II deficiency.

• References

• 1 Stenflo J. A new vitamin K-dependent protein: Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251: 355-63
• 2 Kisiel W, Ericsson LH, Davie EW. Proteolytic activation of protein C from bovine plasma. Biochemistry 1976; 15: 4893-900
• 3 Esmon CT. Protein-C: Biochemistry, physiology, and clinical implications. Blood 1983; 62: 1155-8
• 4 Foster D, Davie EW. Characterization of a cDNA coding for human protein C. Proc Natl Acad Sci USA 1984; 81: 4766-70
• 5 Beckmann RJ, Schmidt RJ, Santerre RF, Plutzky J, Crabtree GR, Long GL. The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs. Nucl Acid Res 1985; 13: 5233-47
• 6 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-7
• 7 Plutzky J, Hoskins JA, Long GL, Crabtree GR. Evolution and organization of the human protein C gene. Proc Natl Acad Sci USA 1986; 83: 546-50
• 8 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-31
• 9 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-10
• 10 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-72
• 11 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-3
• 12 Pabinger-Fasching I, Bertina RM, Lechner K, Niessner H, Korninger Ch. Protein C deficiency in two Austrian families. Thromb Haemostas 1983; 50: 810-3
• 13 Brockmans AW, Veltkamp JJ, Bertina RM. Congenital protein C deficiency and venous thromboembolism: A study of three Dutch families. N Engl J Med 1983; 309: 340-4
• 14 Bovill EG, Bauer KA, Dickerman JD, Callas P, West B. The clinical spectrum of heterozygous protein C deficiency in a large New England kindred. Blood 1989; 73: 712-7
• 15 Horellou MH, Conard J, Bertina RM, Samama M. Congenital protein C deficiency and thrombotic disease in nine French families. Br Med J 1984; 289: 1285-7
• 16 Romeo G, Hassan HJ, Staempfli S, Roncuzzi L, Cianetti L, Leonardi A, Vicente V, Mannucci PM, Bertina R, Peschle C, Cortese R. Hereditary thrombophilia: Identification of nonsense and missense mutations in the protein C gene. Proc Natl Acad Sci USA 1987; 84: 2829-32
• 17 Matsuda M, Sugo T, Sakata Y, Murayama H, Mimuro J, Tanebe S, Yoshitake S. A thrombotic state due to an abnormal protein C. N Engl J Med 1988; 319: 1265-8
• 18 Reitsma PH, Poort SR, Allaart CF, Briet AE, 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: 890-4
• 19 Grundy C, Chitolie A, Talbot S, Bevan D, Kakkar V, Cooper DN. Protein C London 1: Recurrent mutation at Arg 169 (CGG →TGG) in the protein C gene causing thrombosis. Nucl Acid Res 1989; 17: 105-13
• 20 Bovill EG, Tomczak JA, Grant BN, Bhushan F, Pillemer E, Rainville IR, Long GL. Protein C_vermont: Symptomatic type II protein C deficiency associated with two Gla domain mutations. Blood 1992; 79: 1456-65
• 21 Sugahara Y, Miura O, Yuen P, Aoki N. Protein C deficiency Hong Kong 1 and 2: Hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation. Blood 1992; 80: 126-33
• 22 Yamamoto K, Tanimoto M, Emi N, Matsushita T, Takamatsu J, Saito H. Impaired secretion of the elongated mutant of protein C (Protein C-Nagoya); Molecular and cellular basis for hereditary protein C deficiency. J Clin Invest 1992; 90: 2439-46
• 23 Bernardi F, Patracchini P, Gemmati D, Boninsegna S, Guerra S, Legnani C, Ballerini G, Marchetti G. Rapid detection of a protein C gene mutation present in the asymptomatic and not in the thrombosisprone lineage. Br J Haematol 1992; 81: 277-82
• 24 Tokunaga F, Wakabayashi S, Sato H, Arakawa M, Tawaraya H, Koide T. Identification of one base deletion in exon IX of the protein C gene that causes a type I deficiency. Thromb Res 1992; 68: 417-23
• 25 Reitsma PH, Poort SR, Bernardi F, Gandrille S, Long GL, Sala N, Cooper DN. Protein C deficiency: A database of mutations. Thromb Haemost 1993; 69: 77-84
• 26 Sakata T, Tsushima N, Hatanaka K, Morishita T, Hatsuyama H, Uchida K, Katayama Y, Matsuyama T. Probable dysfunctional protein S in a patient with congenital heterozygous protein C deficiency: A family study. Jpn J Thromb Hemostas 1991; 2: 518-23
• 27 Bell GI, Karam JH, Rutter WJ. Polymorphic DNA region adjacent to the 5’ end of the human insulin gene. Proc Natl Acad Sci USA 1981; 78: 5759-63
• 28 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988; 239: 487-91
• 29 Orita M, Suzuki Y, Sekiya X, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5: 874-9
• 30 Yamamoto K, Tanimoto M, Matsushita T, Kagami K, Sugiura I, Hamaguchi M, Takamatsu J, Saito H. Genotype establishments for protein C deficiency by use of a DNA polymorphism in the gene. Blood 1991; 77: 2633-6
• 31 Barker D, Schafer M, White R. Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 1984; 36: 131-8
• 32 Cooper DN, Youssoufian H. The CpG dinucleotide and human disease. Hum Genet 1988; 78: 151-5
• 33 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: 5653-6
• 34 Tada N, Sato M, Tsujimura A, Iwase R, Hashimoto-Gotoh T. Isolation and characterization of a mouse protein C cDNA. J Biochem 1992; 111: 491-5
• 35 Okafuji T, Maekawa K, Nawa K, Marumoto Y. The cDNA cloning and mRNA expression of rat protein C. Biochim Biophys Acta 1992; 1131: 329-32
• 36 Giannelli F, Green PM, High KA, Sommer S, Lillicrap DP, Ludwig M, Olek K, Reitsma PH, Goossens M, Yoshioka A, Brownlee GG. Haemophilia B: database of point mutations and short additions and deletions – third edition, 1992. Nucl Acid Res 1992; 20: 2027-63
• 37 Degen SJ F, MacGillivray RT A, Davie EW. Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry 1983; 22: 2087-97
• 38 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: 5098-102
• 39 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: 3736-50
• 40 Hagen FS, Gray CL, O’Hara P, Grant FJ, Saari GC, Woodbury RG, Hart CE, Insley M, Kisiel W, Kurachi K, Davie EW. Characterization of a cDNA coding for human factor VII. Proc Natl Acad Sci USA 1986; 83: 2412-6
• 41 Walsh KA. Trypsinogens and trypsins of various species. Methods Enzymol. Perlmann GE, Lorand L. (eds) Academic Press; New York: 1970. (; 19). 41-63
• 42 Wilcox PE. Chymotrypsinogens – chymotrypsin. Methods Enzymol. Perlmann GE, Lorand L. (eds) Academic Press; New York: 1970. (; 19). 64-108
• 43 Sugimoto M, Miyata T, Kawabata S, Yoshioka A, Fukui H, Takahashi H, Iwanaga S. Blood clotting factor IX Niigata: Substitution of alanine-390 by valine in the catalytic domain. J Biochem 1988; 104: 878-80
• 44 Spitzer SG, Pendurthi UR, Kasper CK, Bajaj SP. Molecular defect in factor IX _{Bm Lake Elsinore}. Substitution of Ala³⁹⁰ by Val in the catalytic domain. J Biol Chem 1988; 263: 10545-8
• 45 Tarnower A, Gribble TJ, Smith KJ. Molecular basis of hemophilia B in New Mexico, U.S. A. Thromb Haemost 1991; 65: 967