Gene Mutations in 21 Unrelated Cases of Phenotypic Heterozygous Protein C Deficiency and Thrombosis

Summary

Mutations have been identified in the protein C gene in 21 patients with venous thromboembolism and phenotypic heterozygous protein C deficiency. In 20 probands, single mutations were the only abnormalities identified by sequencing all coding regions, intron exon boundaries and the promoter region back to -1540. In one proband 2 mutations were identified and in another family 2 mutations were identified (but not both in the proband). Of the 23 mutations, 18 resulted in predicted amino acid substitutions, 3 were mutations resulting in stop codons, one was a mutation within a consensus splice sequence and another a 9 base pair insertion within exon 5 (this region within exon 5 is proposed as a deletion/insertion hot spot). A novel polymorphism was also, uniquely, identified in the propeptide region of the molecule (Pro-21 Pro; CCT to CCC) in a kindred from Hong Kong. Cosegregation of the protein C gene mutation with protein C deficiency could be determined in 13 families. In a further family, phenotypic protein C deficiency and the genetic mutation cosegregated in only 4/5 members.

The first thrombotic incident occurred in the probands between the ages of 11 and 59 years and 12 individuals suffered recurrent thrombosis. Thrombosis occurred in at least one other family member in 9/21 families, but in 2 of these it was inconsistently associated with protein C deficiency. An independent genetic risk factor, factor V Arg506Gln (FV Leiden) was identified in 2 probands (and 3 family members) and in 4 protein C deficient members of a third family but not in the proband. The results suggest that in the majority of probands with thrombosis and phenotypic protein C deficiency, a single protein C gene mutation is associated with thrombosis. However, it is also possible that additional unknown genetic risk factors contribute to the thrombotic risk. An added, acquired, risk factor leads to thrombosis at an early age (< 25 years).

• References

• 1 Stenflo J. A new vitamin K dependent protein. Purification from bovine plasma and preliminary characterisation. J Biol Chem 1976; 251: 355-363
• 2 Kisiel W, Canfield W, Eriksson L, Davie E. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
• 3 Marlar RA, Kliess AJ, Griffin HJ. Mechanism of action of human activated protein C, a thrombin-dependent anticoagulant enzyme. Blood 1982; 59: 1067-1072
• 4 Esmon TS. Protein C: Biochemistry, physiology, and clinical implications. Blood 1983; 62: 1155-1158
• 5 Walker FG. Regulation of activated protein C by protein S. The role of phospholipids in factor Va inactivation. J Biol Chem 1981; 256: 11128-11131
• 6 Susuki K, Stenflo J, Dahlbäck B, Theodorsson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983; 258: 1914-1920
• 7 Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984; 63: 486-489
• 8 Branson HE, Marble R, Katz J, Griffin JH. Inherited protein C deficiency and coumarin-responsive chronic relapsing purpura fulminans in a newborn infant. Lancet 1983; ii: 1165-1168
• 9 Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI. Homozygous protein C deficiency manifested by massive venous thrombosis in the newborn. N Engl J Med 1984; 310: 559-562
• 10 Marciniak E, Wilson HD, Marlar RA. Neonatal purpura fulminans: a genetic disorder related to the absence of protein C in the blood. Blood 1985; 65: 15-20
• 11 Marlar RA, Montgomery R, Broekmans AW. Diagnosis and treatment of homozygous protein C-deficient children. J Pediatrics 1989; 114: 528-535
• 12 Bertina RM, Broekmans AW, Van der Linden IK, Mertens K. Protein C deficiency in a Dutch family with thrombotic disease. Thromb Haemost 1982; 48: 1-5
• 13 Horellou MH, Conrad J, Bertina RM, Samama M. Congenital protein C deficiency and thrombotic disease in nine French families. Brit Med J 1984; 289: 1285-1287
• 14 Heijboer H, Brandjes DPM, Büller HR, Sturk A, ten Cate JW. Deficiencies of coagulation-inhibiting and fibrinolytic proteins in outpatients with deep-vein thrombosis. N Engl J Med 1990; 323: 1512-1515
• 15 Allaart CF, Poort SR, Rosendaal F, Reitsma PH, Bertina R, Briët E. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993; 341: 134-138
• 16 Miletich J, Sherman L, Broze G. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987; 317: 991-996
• 17 Dahlbäck B. Familial thrombophilia due to a previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-1008
• 18 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden P, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67
• 19 Catto A, Carter A, Ireland H, Bayston TA, Philippou H, Barrett J, Lane DA, Grant PJ. Factor V Leiden gene mutation and thrombin generation in relation to the development of acute stroke. Arterioscler Thromb Vase Biol 1995; 15: 783-785
• 20 Emmerich J, Poirier O, Evans A, Marques-Vidal P, Arveiler D, Luc G, Aiach M, Cambien F. Myocardial infarction, Arg506 to Gin factor V mutation, and activated protein C resistance. Lancet 1995; 345: 321
• 21 Ireland H, Bayston T, Thompson E, Adami A, Concalves C, Lane DA, Finazzi G, Barbui T. Apparent heterozygous Type II protein C deficiency caused by the factor V 506 Arg to Gin mutation. Thromb Haemost 1995; 73: 727-738
• 22 Ireland H, Boisclair MD, Taylor J, Thompson E, Thein SL, Girolami A, De Caterina M, Scopacasa F, De Stefano V, Leone G, Finazzi G, Cohen H, Lane DA. Two novel (R[-l1]C; T394D) and two repeat missense mutations in the protein C gene associated with venous thrombosis in six kindreds. Hum Mutat 1996; 7: 176-179
• 23 Kogan SC, Doherty M, Gitchier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. New Engl. J Med 1987; 317: 985-990
• 24 Reitsma PH, Poort SR, Alaart 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
• 25 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
• 26 Gandrille S, Jude B, Alhenc-Gelas M, Millaire A, Aiach M. Compound heterozygosity in a family with protein C deficiency illustrating the complexity of the underlying molecular mechanism. Thromb Haemost 1993; 70: 747-752
• 27 Gandrille S, Aiach M. and the French INSERM Network on Molecular Abnormalities Responsible for Protein C and ProteinS deficiencies. Identification of mutations in 90 of 121 consecutive symptomatic French patients with a Type I protein C deficiency. Blood 1995; 86: 2598-2605
• 28 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
• 29 Spek CA, Koster T, Rosendaal FR, Bertina RM, Reitsma PH. Genotypic variation in the promoter region of the protein C gene is associated with plasma protein C levels and with thrombotic risk. Thromb Haemost 1995; 73: 937
• 30 Erdjument H, Lane DA, Ireland H, Panico M, Di Marzo V, Blench I, Morris HR. Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin “Northwick Park”. J Biol Chem 1987; 262: 13381-13384.
• 31 Cooper DN, Krawczak M. Mechanisms of insertional mutagenesis in human genes causing genetic disease. Hum Genet 1991; 87: 409-415
• 32 Efstradiadis A, Posakony JW, Maniatis T, Lawn RM, O’Connell C, Spritz RA, De Riel JK, Forget BG, Weissman SM, Slightom JL, Blechl AE, Smithies O, Baralle FE, Shoulders CC, Proudfoot NJ. The structure and evolution of the B-globin gene family. Cell 1980; 21: 653-668
• 33 Streisinger G, Okada Y, Emrich J, Newton J, Tsugita A, Terzaghi E, Inouye M. Frameshift mutations and the genetic code. Cold Spring Harbor Symp Quant Biol 1966; 31: 77-84
• 34 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, 2nd edn. Nucleic Acids Res 1991; 19: 2193-2219
• 35 Fearon ER, Cho KR, Nigro JM, Kern SE, Simmons JW, Ruppert JM, Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 1990; 247: 49-56
• 36 Koeleman BPC, Reitsma PH, Allart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C deficient families. Blood 1994; 84: 1031-1035
• 37 Pabinger I, Mustafa S, Rintelen C, Kyrle PA, Lechner K, Mannhalter C. Factor V (FV) Leiden mutation (APC-resistance) increases the risk for venous thromboembolism in patients with a gene defect of the protein C (PC) or protein S (PS) gene. Thromb Haemost 1995; 73: 1361
• 38 Gandrille S, Greengard JS, Alhenc-Gelas M, Juhan-Vague I, Abgrall JF, Jude B, Griffin JH, Aiach M. French Network on behalf of INSERM. Incidence of activated protein C resistance caused by the Arg506 Gin mutation in factor V in 113 unrelated symptomatic protein C deficient patients. Blood 1995; 86: 219-224
• 39 Soria JM, Fontcuberta J, Borrell M, Estivill X, Sala N. Two novel mutations in exon 5 of the protein C gene in two Spanish families with thrombophilia due to protein C deficiency. Hum Mol Genet 1994; 3: 1205-1206
• 40 Tait RC, Walker ID, Reitsma PH, Islam SIAM, McCall F, Poort SR, Conkie JA, Bertina RM. Prevalence of protein C deficiency in the healthy population. Thromb Haemost 1995; 73: 87-93
• 41 Doig RG, Begley CG, McGrath KM. Hereditary protein C deficiency associated with mutations in exon IX of the protein C gene. Thromb Haemost 1994; 72: 203-208