Clinical Chemistry 43:9 1678–1683 (1997) Oak Ridge Conference Factor V Leiden and other coagulation factor mutations affecting thrombotic risk Rogier M. Bertina Five genetic defects have been established as risk fac- dominant and recessive inheritances (fibrinogen Naples) tors for venous thrombosis. Three are protein C, protein have been reported [2]. The evidence that dysfibrinogen- S, and antithrombin deficiencies, defects in the antico- emia can be associated with thrombosis is scarce. The agulant pathways of blood coagulation. Together they most convincing support was published in 1995 in a can be found in ;15% of families with inherited throm- report from the SSC subcommittee on fibrinogen [2].So bophilia. Their laboratory diagnosis is hampered by the far at least 15 different mutations in the fibrinogen a, b, large genetic heterogeneity of these defects. The other and g genes have been reported in patients with throm- two genetic risk factors, resistance to activated protein C bosis that result in the phenotype of dysfibrinogenemia. associated with the factor V Leiden mutation and in- Antithrombin deficiency was first reported in 1965 by creased prothrombin associated with the prothrombin Egeberg [3]. It is inherited as an autosomal dominant trait, 20210 A allele, are much more prevalent and together with heterozygotes having an increased risk of venous can be found in 63% of the thrombophilia families. thrombosis [4]. Basically there are two types of antithrom- Because both defects are caused by a single mutation, bin deficiency: type 1 deficiency (reduction of both func- DNA analysis is the basis of their laboratory diagnosis. tional and immunological antithrombin) and type II defi- ciency (presence of an abnormal molecule). At present, Venous thrombosis is a common disease, with an esti- .79 different mutations in the antithrombin gene have mated annual incidence of 1 in 1000 persons. The devel- been reported that are associated with a type I or type II opment of a thrombotic event is the final result of multiple deficiency [5]. Heterozygosity for antithrombin deficiency interactions between different genetic and environmental can be found in ;4% of families with inherited thrombo- components. This is most clearly demonstrated in inher- philia, in 1% of consecutive patients with a first deep vein ited thrombophilia, a genetically determined tendency to thrombosis (DVT), and in 0.02% of healthy individuals venous thrombosis caused by the segregation of one [6].1 (monogenetic trait) or more (complex trait) disease alleles. Protein C deficiency was first reported in 1981 by Because a first thrombotic event is itself a very strong risk Griffin et al. [7]. In families with thrombophilia it is factor for thrombosis, it is important to identify individ- inherited as an autosomal dominant disorder: In these uals at risk and to offer them adequate treatment and (or) families heterozygosity for protein C deficiency is a prophylaxis. significant risk factor for venous thrombosis [8].Onthe other hand, autosomal recessive inheritance has been genetic risk factors for thrombophilia observed in families from newborns with severe throm- During the past 30 years, six genetic defects (listed in bosis resulting from homozygous or compound heterozy- Table 1) have been identified that are associated with an gous protein C deficiency [9]. Also here, type I and type II increased risk of venous thrombosis. Dysfibrinogenemia deficiencies have been reported. Protein C deficiency is was first described in 1965 by Beck et al. [1]. It is a rare genetically very heterogenous: .160 different mutations disorder (prevalence of 1% among selected thrombosis in the protein C gene have been reported to be associated patients), most commonly identified by an abnormal with a type I or II protein C deficiency [10]. Heterozygos- Reptilase® time and (or) thrombin time. Both autosomal ity for protein C deficiency is found in ;6% of families with inherited thrombophilia, in 3% of consecutive pa- Hemostasis and Thrombosis Research Center, Leiden University Medical Center, C2-R-143, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Fax 1 Nonstandard abbreviations: DVT, deep vein thrombosis; APC, activated 131-71-5266755; e-mail [email protected]. protein C; SR, sensitivity ratio; CI, confidence interval; and APTT, activated Received May 14, 1997; revised and accepted June 27, 1997. partial thromboplastin time. 1678 Clinical Chemistry 43, No. 9, 1997 1679 Table 1. Genetic defects in inherited thrombophilia. deficiency. Hopefully in the future other techniques will become available that will make sequencing of a particu- Prevalence, % Mutations Dysfibrinogenemia 1.0 .11 lar gene a feasible option in the routine hematology Antithrombin deficiency 4.3 .79 laboratory. Protein C deficiency 5.7 .160 The diagnostic procedures for identification of protein Protein S deficiency 5.7 .69 C and protein S deficiencies especially need further im- APC resistance 45 1 provement. Because of biological variation and gene– Increased prothrombin 18 1 environment interactions, there is a large overlap in, for Unknown 30 ? instance, protein C values between proven carriers of a protein C deficiency (heterozygotes) and non-protein C- deficient family members [8]. In practice this leads to the tients with a first DVT, and in 0.3% of healthy individuals definition of arbitrary cutoff points that never will pre- [6]. vent substantial percentages of false-positive and false- Protein S deficiency was first described in 1984 by negative diagnoses. Also, it has become clear that the Comp et al. [11]. In thrombophilic families it is inherited range of protein C values that can be measured in healthy as an autosomal dominant disorder. Heterozygotes in individuals depends on age and gender [17]. these families have an increased risk of venous thrombo- sis when compared with their unaffected family members A further complication in the diagnosis is that many [12]. Also, protein S deficiency is genetically heteroge- patients will be treated with oral anticoagulants. This nous. Almost 70 different mutations in the protein S gene treatment will result in a decrease both in the plasma (PROS-1) have been reported now [13]. The large majority concentration and in the degree of carboxylation of the of protein S-deficient patients have a type I deficiency. vitamin K-dependent proteins, including protein C and The prevalence of heterozygotes for a type I protein S protein S. This problem has been extensively discussed in deficiency is 6% in families with inherited thrombophilia the past and several solutions have been proposed but and 1–2% in consecutive patients with a first DVT [6]. later found to be not good enough (e.g., comparison of Interestingly, heterozygosity for protein S deficiency was protein C/protein S concentrations with those of factor not identified as a risk factor for venous thrombosis in a VII or factor II). recent large patient control study, whereas heterozygosity In the case of the diagnosis of protein S deficiency, an for protein C or antithrombin deficiency was [14]. additional complication is that in plasma part of the More recently, two other genetic risk factors have been protein S circulates in a complex with the C4b binding reported that differ from the previous ones in two re- protein, and that these complexes do not have APC spects. They are more common and they are always cofactor activity [18, 19]. Only very recently have some associated with the same genetic defect. The first concerns hard data been presented that in families with a known activated protein C (APC) resistance due to the factor V type I mutation in the protein S gene, heterozygotes can Leiden (FV R506Q) mutation [15], and the second is be identified better on the basis of their reduced free increased prothrombin, which is associated with the protein S concentrations than on the basis of their reduced 20210 A allele of the prothrombin gene [16]. In the next total protein S concentrations. Hopefully these new in- paragraphs these genetic defects will be discussed in more sights will result in the near future in new recommenda- detail. tions for the laboratory diagnosis of protein S deficiency. Interestingly, protein C, protein S, and antithrombin A technical problem with many of the protein C and deficiencies all involve defects in anticoagulant pathways, protein S clotting assays was discovered during the last 3 whereas the factor V Leiden mutation and the prothrom- years. Most of these assays apparently will give abnormal bin gene mutation involve procoagulant factors. In all results in plasmas of patients that are APC resistant but cases the expected result of the genetic defect is an not protein C or protein S deficient [20, 21]. Therefore new enhanced thrombin generation. functional tests are needed that are more specific for these laboratory diagnosis two anticoagulant proteins. Screening of patients for genetic defects associated with From the foregoing it is clear that laboratory diagnosis thrombophilia is done almost exclusively in specialized of protein C and protein S deficiencies is still far from hemostasis and thrombosis centers. For the detection of optimal, especially where the relatively low prevalence of heterozygotes of protein C, protein S, or antithrombin these defects among thrombosis patients further contrib- deficiency, we still rely on the results of specific functional utes to the rather unfavorable positive and negative and (or) immunological tests. These genetic defects