Multiple Assessment Of Genetic Risk Factors For Thrombophilia In The Chinese Population

Thrombophilia refers to a pathological condition where the blood has an increasedtendency to form clots and induce thromboembolism. Both genetic factors （for example,inherited natural anticoagulant deficiency） and environmental factors （for example,advancing age, smoking, hyperlipidemia, pregnancy, puerperium, cancer, antiphospholipidantibodies, oral contraceptives, hormone replacement therapy） are implicated inthrombophilia. The main clinical manifestation of thrombophilia is venousthromboembolism. Some types of thrombophilia, such as congenital protein S deficiencyand antiphospholipid syndrome, are also associated with an increased risk of arterialthrombotic disorders, especially early-onset arterial occlusive incidents. Arterial thromboticdiseases includes coronary heart disease, myocardial infarction, and ischemic stroke; whilevenous thrombotic diseases includes deep vein thrombosis and pulmonary embolism.Arterial and venous thromboembolism are vascular diseases that, although affectingdifferent vascular beds, share many traditional risk factors and major pathologicalprocesses such as atherosclerosis and thrombosis.In China, according to the World Health Organization, more than700,000people diefrom coronary heart disease each year; ischemic stroke is the leading cause of long-termadult disability and the second leading cause of death. Venous thrombosis, affecting1in1000individuals annually in Western countries, is also one of the leading causes ofmorbidity and mortality. Its overall mortality rate was estimated to be22.7per1,000person-years. Although there is no epidemiological data from non-Western countries formany years, venous thrombosis is thought to be as prevalent, if not more so, in the Chinesepopulation. Therefore, thrombophilia causes a predisposition to thrombosis and is a majormedical problem.Thrombosis is a complex multifactorial disease, involving both genetic andcircumstantial risk factors that affect a delicate balance between procoagulant andanticoagulant forces. Genetic factors can explain the lifelong susceptibility to thrombosis of an individual, and thrombotic events tend to occur when one or more of the acquired riskfactors come into play. Family and twin studies indicate that genetic factors contributeabout60%to venous thrombosis. Multiple reports based on twin studies and family studieshave also shown that genetics contribute to risk of coronary heart disease and ischemicstroke independently of traditional risk factors. Because treatments for thromboembolismare limited, the best approach to reducing the burden of disease is thrombosis riskprediction followed by primary prevention. In this context, definition of the geneticarchitecture of thrombophilia will offer an unequivocal diagnosis of affected andunaffected individuals and as a result, may help in risk prediction, secondary prevention,and development of novel therapies.It is known that a poor response of plasma to activated protein C caused by the factorV Leiden mutation in the coding sequence of F5gene, a common prothrombin G20210Amutation in the3’-untranslated region of F2gene are common genetic risk factors forthrombophilia in White populations. The prevalence of heterozygous factor V Leiden inWhites is5¹0%. It confers a5-fold increased risk of venous thrombosis in heterozygotesand a50-fold increased risk in homozygotes. Recent studies show that the factor V Leidenis also associated with a1.66-fold and1.33-fold increased risk of myocardial infarction andischemic stroke, respectively. As for the prothrombin G20210A mutation, its prevalence isestimated to be2⁴%in the general population. It confers a3-fold increased risk of venousthrombosis in heterozygotes. In addition, this polymorphism is also associated with a1.32-fold and1.44-fold increased risk of myocardial infarction and ischemic stroke,respectively.However, duo to ethnic differences, the two variants are rare in Eastern countriesincluding the Chinese population. Little is known about the genetic background ofthrombophilia, and no common genetic risk factors have been identified in the Chinesepopulation. Therefore, in this study, we set out to determine whether there were anyprevalent genetic variants associated with thrombosis in the Chinese population and investigate the genetic characteristics of thrombophilia in China. Since most of the knownrisk factors for thrombosis are linked to blood hypercoagulable state, hypoanticoagulablestate, or hypoactive fibrinolysis, we mainly focused on plasma factors that are involved inthe blood coagulation in the screening stage. In addition, because the main clinicalmanifestation of thrombophilia is venous thrombosis, we mainly selected participantsdiagnosed with venous thrombosis. We named these series of studies evaluating the risk ofthrombophilia and venous thrombosis as MAGIC: Multiple Assessment of Genetic riskfactors for thrombophilia In the Chinese population.Part1Sample bank establishment and abnormal plasma factors screeningObjectivesThrombophilia is a typical multifactorial disease, the pathogenesis involving bothcircumstantial and genetic mechanisms. As a result, we aimed to establish a large samplebank involving plasma samples from unselected consecutive venous thrombosis patientsand matched controls. Then, we carried out a series of coagulation screening tests toidentify possible common abnormal plasma factors that could contribute to thrombosissusceptibility.MethodsUnselected consecutive venous thrombosis patients （n=1304） were recruited between1March2008and30June2012at the Hubei Clinical and Research Center of Thrombosisand Hemostasis. The criteria for diagnosis of thrombosis was based on clinicalmanifestations, D-dimer assay, and objective techniques （compression ultrasound orvenography for deep vein thrombosis, and ventilation/perfusion lung scanning, computedtomography angiography, or pulmonary angiography for pulmonary thromboembolism）.Age-and sex-matched control participants （n=1334）, without a history of an individual orfamily history of thrombosis （arterial thrombosis and venous thrombosis）, were enrolledfrom a community screening program during the same period in the same area. Case group and control group had similar median age （52years versus51years）, mean age （51.5±14.4years versus50.3±14.5years）,5th-95thage （25.0⁷4.0years versus25.6⁷3.0years）,and similar sex distribution （53.0%male）. The demographic data and acquired risk factorswere recorded, including age, gender, smoking, malignancy, sedentariness or immobility,pregnancy or puerperium, oral contraceptives, hormone-replacement therapy, and lupusanticoagulant.Plasma samples from310patients were randomly selected for abnormal factor screening.Procoagulant activities of factor II, factor V, factor VII, factor VIII, factor IX, factor X,factor XI, factor XII, and activated protein C cofactor activity of protein S were determinedusing a clotting assay. Antithrombin activity, protein C amidolytic activity, plasminogenlevel, and plasminogen activator inhibitor-1level were measured using a chromogenicmethod. Fibrinogen level was tested using the Clauss assay. Activated protein C sensitivityof factor V was determined using a modified activated protein C resistance assay.ResultsFactor V procoagulant activities were significantly higher （>152U/dL, mean+2SD） in32cases. Eleven of the32cases were also accompanied with low protein C or protein Sactivities, indicating protein C or protein S deficiency. However, no other abnormalitieswere observed in the other21of the32cases. Modified activated protein C resistance testsshowed that factor V of the32subjects were all sensitive to activated protein C inactivation.These data suggested the abnormal factor might be factor V inactivator （protein C） or itsco-factor （protein S） rather than factor V itself. In addition,34,43, and6of the total310individuals were identified to be protein C deficiency, protein S deficiency, andantithrombin deficiency, respectively. Twenty-six of the310participants were found tohave lupus anticoagulant in plasma.ConclusionThe plasma screening tests indicated that the common abnormal factors associated withthrombophilia in the Chinese population are anticoagulants in the protein C system （protein C, protein S, and thrombomodulin）, antithrombin, and lupus anticoagulant. Therefore, thecorresponding candidate genes selected for further analysis are PROC, PROS1, THBD,SERPINC1, and APOH.Part2Study on the association between PROC variants and thrombophiliaObjectivesProtein C, the key component of the protein C anticoagulant system, is an importantvitamin K-dependent protein that regulates the physiological coagulation cascade byinactivating factors Va and VIIIa upon activation by thrombin. Hereditary protein Cdeficiency causes a predisposition to venous thrombosis. The genetic characteristics ofprotein C deficiency in the Chinese population remain unknown.MethodsThirty-four unrelated probands diagnosed with hereditary protein C deficiency wereinvestigated. Protein C activity and antigen levels were measured. Mutation analysis wasperformed by sequencing the PROC gene. In silico analyses, including PolyPhen-2, SIFT,multiple sequence alignment, splicing prediction, and protein molecular modeling wereperformed to predict the consequences of each variant identified. One recurrent mutationand its relative risk for thrombosis in relatives were analyzed in11families. Onepolymorphism and its functional impacts were analyzed by in vitro expression studies. Thetwo common PROC variants （the recurrent mutation and the polymorphism） weresubsequently detected in the case-control study （1304versus1334）, and the adjusted oddsratios for venous thrombosis risk were calculated by logistic regression analysis.ResultsA total of18different mutations, including12novel variants, were identified. One commonmutation, PROC c.565C>T, was found in17of the34probands. The family study showedthat first-degree relatives bearing this variant had an8.80-fold increased risk of venousthrombosis. The case-control study identified this mutation in68patients and in12controls, respectively. The mutant allele conferred a high predisposition to venous thrombosis（adjusted odds ratio=6.44）. The plasma protein C activity and antigen levels inheterozygotes were51.736.92U/dL and75.174.84U/dL, respectively. One commonpolymorphism, PROC c.574₅76del was identified in85of the patients and32of thecontrols, respectively. It had an adjusted odds ratio of2.80for thrombosis. Protein Camidolytic activities of most variant carriers were similar to those of non-carriers. But themean anticoagulant activity of them was only72.7U/dL. Expression studies in vitroshowed that the anticoagulant activity of the mutant was43.6%of the wild-type protein C.ConclusionThe PROC c.565C>T mutation is the most frequent cause of PC deficiency as well as aprevalent risk factor for venous thrombosis in the Chinese individuals. While the PROCc.574₅76del polymorphism is a common genetic risk factor for thrombophilia in China.Part3Study on the association between THBD variants and thrombophiliaObjectivesThrombomodulin, a critical component of the protein C pathway, is expressed mainly onthe endothelial surface of blood vessels. The anticoagulant property of thrombomodulin ismediated by its interaction with thrombin and protein C. The high affinity binding ofthrombin to thrombomodulin results in a greater than1000-fold amplification of the rate ofthrombin-dependent protein C activation. Based on the crucial antithrombotic role ofthrombomodulin, we investigated the THBD gene as a candidate gene to identify possiblevariants that could contribute to thrombosis susceptibility.MethodsThe putative promoter,5’-untranslated region, coding region, and3’-untranslated region ofthe THBD gene were screened using PCR-sequencing method in60cases randomlyselected from the venous thrombosis patients who were free from antithrombin/proteinC/protein S deficiency, the PROC c.565C>T mutation, and the PROC c.574₅76del variant. One recurrent mutation and its relative risk for thrombosis in relatives were analyzed in38index families involving176first-degree relatives. The recurrent mutation wassubsequently detected in the case-control study （1304versus1334）, and the adjusted oddsratio for venous thrombosis risk were calculated by logistic regression analysis. Itsfunctional impacts were analyzed by ex vivo plasma thrombomodulin measurements and invitro luciferase assays.ResultsFive rare mutations and a novel recurrent mutation, c.151G>T in the5’-untranslatedregion, were identified by re-sequencing of the THBD gene in the selected thrombophiliapatients and could cause a predisposition to venous thrombosis. Family analysis indicatedthat first-degree relatives with the c.151G>T had a3.42-fold increased risk of venousthrombosis and their probability of remaining thrombosis-free was significantly lower thanthat of relatives without the variant. Case-control study showed that this variant waspresent in35venous thrombosis individuals and13controls. The homozygous genotypewas absent in all the participants. Thus, carriers of the variant allele in the heterozygousstate had a2.55-fold risk of developing venous thrombosis. The mean values of solublethrombomodulin of variant carriers for the c.151G>T variant were significantly lowerthan that of non-carriers. Luciferase assay further confirmed that the151T variantsignificantly reduced the reporter gene expression level compared with the151Gwild-type allele.ConclusionThe common THBD c.151G>T variant was associated with both an impaired5’-untranslated region function and an increased risk of venous thrombosis. Inheritedthrombomodulin defect might be more common in Chinese thrombosis patients. Furthersystemic research on the genetics of thrombomodulin defects is thus warranted.Part4Study on the association between PROS1variants and thrombophilia ObjectivesProtein S, another important component of the protein C pathway, acts as a non-enzymaticcofactor for activated protein C in the inactivation of the procoagulant factor Va and factorVIIIa on phospholipid membranes. Protein S deficiency is a major risk factor for venousthrombosis. The objective of this study is to investigate the molecular basis of hereditaryprotein S deficiency in the Chinese population.MethodsA total of40probands diagnosed with hereditary protein S deficiency were available andwere investigated. The cofactor activity of protein S was assayed by using a clottingmethod. Total protein S antigen and free protein S antigen were further tested by an ELISAassay. The PROS1gene was analyzed in each proband by PCR-sequencing. Large deletionswere identified by multiplex ligation-dependent probe amplification analysis. To evaluatethe functional consequences of two novel missense variants, bioinformatics tools, ex vivothrombin-generation assays, and in vitro expression studies were employed.ResultsA total of20different mutations, including15novel mutations, were identified in21of the40index probands. Small mutations were detected in18（45.0%） probands, and largedeletions were found in3（7.5%） probands, leaving19（47.5%） patients without causativevariants. All of the novel mutations identified in probands cosegregated with a reduced PSactivity in the corresponding family. The endogenous thrombin potential, the peak, and thestart tail values for plasma from the proband with the p.Asn365Lys or the p.Pro410Hiswere much higher than the values from relatives without the mutations. The p.Asn365LysPS was found to have moderately impaired secretion and reduced activated protein Ccofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impairedsecretion but full anticoagulant activity.ConclusionThe molecular heterogeneity of protein S deficiency was observed in Chinese patients, and null mutations were relatively common. Further genetic analysis is warranted to understandthe causes of protein S deficiency in patients without a genetic explanation.Part5Study on the association between SERPINC1variants and thrombophiliaObjectivesAntithrombin is a major physiological inhibitor of hemostasis with an important function inthe inhibition of procoagulant serine proteases such as thrombin or factor Xa. Inheritedantithrombin deficiency is a major genetic factor for thrombosis that causes a20-foldincreased risk of venous thrombosis. The objective of this study is to investigate themolecular basis of hereditary antithrombin deficiency in the Chinese population.MethodsSix probands diagnosed with hereditary antithrombin deficiency in the coagulationscreening stage were enrolled. The antithrombin activity and antigen were assayed by aclotting and an ELISA assay, respectively. The SERPINC1gene was analyzed in eachproband by PCR-sequencing.ResultsSix different mutations, including four novel mutations, were identified in all of the sixindex probands. The mutation profile consisted of3nonsense mutations,2small deletions,and1splicing site mutation. These mutations caused type I antithrombin deficiency whichwas characterized by the reduction of both functional and antigenic antithrombin in thethrombosis individuals.ConclusionThere was not a predominant mutation in Chinese patients with antithrombin deficiency.The molecular heterogeneity of antithrombin deficiency was observed and null mutationswere relatively common in Chinese patients.Part6Study on the association between APOH variants and thrombophilia ObjectivesThe presence of anti-phospholipid antibodies is considered as an important risk factor thatdisturbs normal hemostasis. The so-called lupus anticoagulant is thought to be the mostcommon and the most important anti-phospholipid antibody which can confer high risk forthrombosis. Recent studies suggest that lupus anticoagulant may actually be anti-β2GPIantibody and that a conformational change in β2-Glycoprotein I is sufficient to induceautoantibodies. Therefore, we investigated the APOH gene encoding β2-Glycoprotein I as acandidate gene to identify possible variants that could contribute to lupus anticoagulantformation and thrombosis susceptibility.MethodsThe putative promoter,8exons, and3’-untranslated region of the APOH gene werescreened using the PCR-sequencing method in the26cases who had lupus anticoagulant intheir plasma in the coagulation stage. The haploid genotype of the APOH gene wassubsequently determined in the case-control study （1304versus1334）, and the adjustedodds ratios for venous thrombosis risk were calculated by logistic regression analysis. Theinfluence of different haploid genotypes on the global level of coagulation function wasevaluated by using ex vivo thrombin-generation assays.ResultsFour common polymorphisms g.5028C>A, c.422T>C （p.Ile141Thr）, c.461G>A（p.Arg154His）, and c.1004G>C （p.Trp335Ser） were identified. These variants were incomplete linkage disequilibrium with one another and produced3haploid genotypes: wildtype H1，CTGG/CTGG；heterozygous type H2，CTGG/ACAC；homozygous type H3，ACAC/ACAC. Case-control study showed that the H2type was present in157venousthrombosis individuals and135controls; while the H3type was identified in12of the casegroup and2of the control group. Thus, carriers of the H2type and the H3type had a1.29-fold and6.04-fold increased risk of venous thrombosis, respectively, after adjusted forother risk factors. The mean endogenous thrombin potential values for plasma from individuals with the H3type were much higher than those with the H2type and H1type.ConclusionThe common APOH haploid genotypes were associated with both a susceptibility to lupusanticoagulant formation and an increased risk of thrombosis.