Genetic Characterization of Protein C Deficiency in Japanese Subjects Using a Rapid and Nonradioactive Method for Single-Strand Conformational Polymorphism Analysis and a Model Building

Toshiyuki Miyata; Toshiyuki Sakata; Yan-Zhen Zheng; Hiroaki Tsukamoto; Hideaki Umeyama; Shinichiro Uchiyama; Masaomi Ikusaka; Akira Yoshioka; Yasufumi Imanaka; Hironobu Fujimura; Jun-ichi Kambayashi; Hisao Kato

doi:10.1055/s-0038-1650575

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 76(03): 302-311
DOI: 10.1055/s-0038-1650575

Original Article

Schattauer GmbH Stuttgart

Genetic Characterization of Protein C Deficiency in Japanese Subjects Using a Rapid and Nonradioactive Method for Single-Strand Conformational Polymorphism Analysis and a Model Building

Toshiyuki Miyata

¹The Research Institute, Suita, Japan

,

Toshiyuki Sakata

²The Department of Clinical Laboratory, National Cardiovascular Center, Suita, Japan

,

Yan-Zhen Zheng

¹The Research Institute, Suita, Japan

,

Hiroaki Tsukamoto

³The Department of Physical Chemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo

,

Hideaki Umeyama

³The Department of Physical Chemistry, School of Pharmaceutical Sciences, Kitasato University, Tokyo

,

Shinichiro Uchiyama

⁴The Department of Neurology, Tokyo Women’s Medical College, Tokyo

,

Masaomi Ikusaka

⁴The Department of Neurology, Tokyo Women’s Medical College, Tokyo

,

Akira Yoshioka

⁵The Department of Pediatric, Nara Medical University, Kashihara, Japan

,

Yasufumi Imanaka

⁵The Department of Pediatric, Nara Medical University, Kashihara, Japan

,

Hironobu Fujimura

⁶Department of Surgery II, Osaka University Medical School, Suita, Japan

,

Jun-ichi Kambayashi

⁶Department of Surgery II, Osaka University Medical School, Suita, Japan

,

Hisao Kato

¹The Research Institute, Suita, Japan

› Author Affiliations

Abstract

PDF Download

Summary

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

PDF (565 kb)

References

References
1 Stenflo J. A new vitamin K-dependent protein: Purification from bovine plasma and preliminary characterization. J Biol Chem 1976; 251: 355-363
2 Kisiel W, Ericsson LH, Davie EW. Proteolytic activation of protein C from bovine plasma. Biochemistry 1976; 15: 4893-4900
3 Esmon CT. Protein-C: Biochemistry, physiology, and clinical implications. Blood 1983; 62: 1155-1158
4 Foster D, Davie EW. Characterization of a cDNA coding for human protein C. Proc Natl Acad Sci USA 1984; 81: 4766-4770
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. Nucleic Acids Res 1985; 13: 5233-5247
6 Foster DC, Yoshitake S, Davie EW. The nucleotide sequence of the gene for human protein C. Proc Natl Acad Sci USA 1985; 82: 4673-4677
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: 546-550
8 Patracchini P, Aiello V, Palazzi P, Calzolari E, Bemardi F. Sublocalization of human protein C gene on chromosome 2ql3-ql4. Hum Genet 1989; 81: 191-192
9 Kisiel W, Canfield WM, Ericsson LH, Daviee EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
10 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII (antihemophilic factor). Biochemistry 1980; 19: 401-410
11 Marlar RA, Kleiss AJ, Griffin JH. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
12 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373
13 Reitsma PH, Bemardi F, Doig RG, Gandrille S, Greengard JS, Ireland H, Krawczak M, Lind B, Long GL, Poort SR, Saito H, Sala N, Witt I, Cooper DN. Protein C deficiency: A database of mutations, 1995 update. Thromb Haemost 1995; 73: 876-889
14 Miyata T, Zheng YZ, Sakata T, Tsushima N, Kato H. Three missense mutations in the protein C heavy chain causing type I and type II protein C deficiency. Thromb Haemost 1994; 71: 32-37
15 Zheng YZ, Sakata T, Matsusue T, Umeyama H, Kato H, Miyata T. Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling. Blood Coagul Fibrinolysis 1994; 5: 687-696
16 Miyata T, Zheng YZ, Sakata T, Kato H. Protein C Osaka 10 with aberrant propeptide processing: Loss of anticoagulant activity due to an amino acid substitution in the protein C precursor. Thromb Haemost 1995; 74: 1003-1008
17 Matsudaex M, Sugo T, Sakata Y, Murayama H, Mimuro J, Tanabe S, Yoshitake S. A thrombotic state due to an abnormal protein C. N Engl J Med 1988; 319: 1265-1268
18 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: 2439-2446
19 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: 417-423
20 Ohwada A, Takahashi H, Uchida K, Nukiwa T, Kira S. Gene analysis of heterozygous protein C deficiency in a patient with pulmonary arterial thromboembolism. Am Rev Respir Dis 1992; 145: 1491-1494
21 Yamamoto K, Matsushita T, Sugiura I, Takamatsu J, Iwasaki E, Wada H, Deguchi K, Shirakawa S, Saito H. Homozygous protein C deficiency: Identification of a novel missense mutation that causes impaired secretion of the mutant protein C. J Lab Clin Med 1992; 119: 682-689
22 Mimuro J, Muramatsu S, Kaneko M, Yoshitake S, Iijima K, Nakamura K, Sakata Y, Matsuda M. An abnormal protein C (protein C Yonago) with an amino acid substitution of Gly for Arg-15 caused by a single base mutation of C to G in codon 57 (CCG-GGG). Int J Hematol 1993; 57: 9-14
23 Ido M, Ohiwa M, Hayashi T, Nishioka J, Hatada T, Watanabe Y, Wada H, Shirakawa S, Suzuki K. A compound heterozygous protein C deficiency with a single nucleotide G deletion encoding Gly-381 and an amino acid substitution of Lys for Gla-26. Thromb Haemost 1993; 70: 636-641
24 Nakagawa K, Tsuji H, Masuda H, Kitamura H, Nakahara Y, Ogasahara Y, Okajima Y, Sawada S, Nakagawa M. Protein C deficiency found in a patient with acute myocardial infarction: a single base mutation 157 Arg (CGA) to stop codon (TGA). Int J Hematol 1994; 60: 273-280
25 Sugahara Y, Miura O, Hirosawa S, Aoki N. Compound heterozygous protein C deficiency caused by two mutations, Arg-178 to Gin and Cys-331 to Arg, leading to impaired secretion of mutant protein C. Thromb Haemost 1994; 72: 814-818
26 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: 487-489
27 Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989; 5: 874-9
28 Padmanabhan K, Padmanabhan KP, Tulinsky A, Park CH, Bode W, Huber R, Blankenship DT, Cardin AD, Kisiel W. Structure of human des (1-45) factor Xa at 2.2 A resolution. J Mol Biol 1993; 232: 947-966
29 Kajihara A, Komooka H, Kamiya K, Umeyama H. Protein modelling using a chimera reference protein derived from exons. Protein Engineering 1993; 6: 615-620
30 Rezaie AR, Mather T, Sussman F, Esmon CT. Mutation of Glu-80 → Lys results in a protein C mutant that no longer requires Ca²⁺ for rapid activation by the thrombin-thrombomodulin complex. J Biol Chem 1994; 269: 3151-3154
31 Bartunik HD, Summers LJ, Bartsch HH. Crystal structure of bovine (3-trypsin at 1.5 A resolution in a crystal form with low molecular packing density. J Mol Biol 1989; 210: 813-828
32 Bernstein FC, Koetzle TF, Williams GJB, Meyer Jr EF, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The protein data bank: A computer-based archival file for macromolecular structures. J Mol Biol 1977; 112: 535-542
33 Long GL, Belagaje RM, MacGillivray RT. Cloning and sequencing of liver cDNA coding for bovine protein C. Proc Natl Acad Sci USA 1984; 81: 5653-5656
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: 491-495
35 Okafuji T, Maekawa K, Nawa K, Marumoto Y. The cDNA cloning and mRNA expression of rat protein C. Biochim Biophys Acta 1992; 1131: 329-332
36 Murakawa M, Okamur T, Kamura T, Kuroiwa M, Harada M, Niho Y. A comparative study of partial primary structures of the catalytic region of mammalian protein C. Br J Haematol 1994; 86: 590-600
37 Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Ann Rev Biochem 1981; 50: 349-383
38 Reitsma PH, Poort SR, Allaart CF, Briet 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
39 Tsay W, Greengard JS, Griffin JH. Exonic polymorphisms in the protein C gene: Interethnic comparison between Caucasian and Asians. Hum Genet 1994; 94: 177-178
40 Yamamoto K, Tanimoto M, Matsushita T, Kagami K, Sugiura I, Hama-guchi M, Takamatsu J, Saito H. Genotype establishments for protein C deficiency by use of a DNA polymorphism in the gene. Blood 1991; 77: 2633-2636
41 Gandrille S, Alach M. Polymorphism in the protein C gene detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 1991; 19: 6982
42 Barker D, Schafer M, White R. Restriction sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 1984; 36: 131-138
43 Fisher CL, Greengard JS, Griffin JH. Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen. Protein Science 1994; 3: 588-599
44 Wacey AI, Pemberton S, Cooper DN, Kakkar VV, Tuddenham EGD. A molecular model of the serine protease domain of activated protein C: Application to the study of missense mutations causing protein C deficiency. Br J Haematol 1993; 84: 290-300
45 Greengard JS, Griffin JH, Fisher CL. Possible structural implications of 20 mutations in the protein C protease domain. Thromb Haemost 1994; 72: 869-873
46 Terukina S, Matsuda M, Hirata H, Takeda Y, Miyata T, Takao T, Shimonishi Y. Substitution of gArg-275 by Cys in an abnormal fibrinogen, fibrinogen Osaka II. J Biol Chem 1988; 263: 13579-13587