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Journal of Thrombosis and Thrombolysis 10,23±28,2000.
# Kluwer Academic Publishers. Printed in The Netherlands.
The Decanucleotide Polymorphism in the Factor VIIPromoter Predicts Factor VII Plasma Levels but not theRisk of Acute Coronary Syndromes
Ester Jimenez-Boj,1 JoÈ rg SchuÈ ttrumpf,1 EvaForberg,1 Herbert H. Watzke,1 and Kurt Huber2
1Division of Hematology and Hemostaseology, University of
Vienna, Austria; 2Division of Cardiology, University of Vienna,
Austria
Abstract. It is known from large epidemiologicalstudies that the elevation of coagulation factor VII inplasma is an independent risk factor for acute coron-ary syndromes. The level of factor VII is in¯uenced bypolymorphic sites in the factor VII gene. However, dataon the association of such polymorphisms with the riskof acute coronary syndromes are con¯icting. A deca-
nucleotide insertion=deletion polymorphic site hasbeen described in the promoter of the factor VII genethat leads to a dramatic change in the plasma factor VIIlevels. We therefore analyzed the association of thispolymorphism with the risk of acute coronarysyndromes in a case-control study. Included in thestudy were 111 patients with angiographically docu-mented acute coronary syndromes and 108 age- andsex-matched individuals from the same geographicarea without signs or symptoms of coronary heartdisease. The presence or absence of the decanucleotidestretch at position ÿ323 in the promoter of factor VIIwas monitored using a polymerase chain reaction(PCR)±based restriction technique. The prevalence ofthe genotype with the homozygous deletion was similarin the patients with acute coronary syndromes (79.2%)and in the control patients (79.6%). There was a non-signi®cant trend toward a higher prevalence of thehomozygote deletion in patients with prematureacute coronary syndromes (77.4%) compared with anage-matched subgroup of the control patients (67.5%)(odds ratio [OR] 1.6, con®dence interval [CI] 0.95, 0.61±3.93). Thus, we could not ®nd a signi®cant associationof the ocurrence of acute coronary events with theinsertion=deletion polymorphism in factor VII.
Key Words. factor VII polymorphism, acute coronarysyndromes, factor VII plasma level, cardiovascular riskfactors
Blood coagulation factors play an important rolein the development of acute coronary thrombosis.Elevated plasma levels of factor VII and ®brino-gen have been identi®ed in large epidemiologicalstudies as independent risk factors for the occur-rence of acute coronary syndromes or death frommyocardial infarction [1±5]. Many environmentalfactors such as body weight, smoking, and
hypercholesterolemia in¯uence the plasmalevels of these factors [6±9]. In addition, geneticfactors play an important role in the determina-tion of the plasma levels of various coagulationfactors [10±17]. It has been shown that a singlepolymorphic site in the 30 untranslated region ofprothrombin is associated with elevated plasmalevels of prothrombin and is also associated withan increased risk of acute coronary syndromes inyoung women [13]. The picture evolving fromstudies of the factor VII gene is much moreheterogeneous. A total of ®ve polymorphic siteshave been identi®ed in that gene that all are in amore or less strong linkage disequilibrium [18±22]. Three of them, a decanucleotide inser-tion=deletion polymorphism and two single basepair mutations at positions ÿ122, ÿ401, andÿ402, are clustered in the promoter region ofthe factor VII gene and are in complete linkagedisequilibrium [21]. A slightly less strong linkagedisequilibrium has been observed between thedecanucleotide insertion=deletion polymorphismand two other polymorphic sites in the factor VIIgene, a hypervariable region in intron 7 and asingle base pair substitution in exon VIII (R323Q)[20]. Both of these genetic variations also seem toin¯uence factor VII plasma levels. There arecon¯icting data on the association of these poly-morphisms with the occurrence of acute coronarysyndromes. Lane and coworkers [23] could not®nd any association of the exon VIII polymorph-ism with acute myocardial infarction despite itsclear impact on the factor VII plasma levels intheir patients. In contrast, Iacoviello et al. [24]reported a signi®cant association between famil-ial myocardial infarction and the polymorphismsin intron 7 and exon VIII, respectively. The
23
Address for correspondence: Herbert H. Watzke, MD, Divisionof Hematology and Hemostaseology, WaÈhringer GuÈrtel 18-20,A-1090, Vienna, Austria E-mail: [email protected]
association of acute coronary syndromes with thedecanucleotide insertion=deletion polymorphismin the factor VII promoter has only been studiedin a cohort from the Mediterranean area of Spainand no association with acute myocardial infarc-tion was found [25]. However, patients in thisstudy were selected for a low frequency ofacquired coronary risk factors. Consequently, asynergistic effect of metabolic and genetic factorsmight have been missed in this study. Studies indifferent ethnic groups suggest that the differentpolymorphic sites in factor VII exert a popula-tion-dependent effect on the factor VII levelwhereby the insertion=deletion polymorphismmay be the functional mutation having the stron-gest impact [26,27]. The prevalence of the inser-tion=deletion polymorphism in the promoterregion of Factor VII has so far only been studiedin patients from the southern parts of Europe[21,25]. We therefore investigated the associationof this polymorphism with factor VII plasmalevels and with the occurrence of acute coronarysyndromes in a case-control study of patientsfrom Austria with a typical coronary risk pro®le.
Patients and Methods
We conducted a single center case-control studyof acute coronary syndromes. The study groupincluded 111 consecutive patients from the urbanarea in Vienna, Austria, who underwent coronaryangiography for unstable angina or acutemyocardial infarction. Criteria were de®ned byevidence of symptoms associated with electrocar-diographic changes for unstable angina and addi-tional enzyme changes for myocardial infarction.The presence of coronary heart disease (CHD)was veri®ed by coronary angiography in allpatients. The age at ®rst event was recorded asage at event when a history of multiple coronaryevents was present. The control population(n� 108) consisted of hospital personnel andfriends of these individuals without a history ofcardiovascular disease. Cases and controls werematched for age and gender.
A history of diabetes or hypertension wasregarded as positive in cases and controls whensuch a diagnosis was ever mentioned to theindividual by a physician or when a lipid orblood pressure±lowering medication had everbeen prescribed. Hypercholesterolemia wasde®ned as plasma cholesterol levels> 200 mg=dLor medication with lipid-lowering drug. Smokerswere de®ned as individuals who were smokingregularly at entry into the study or had stoppedsmoking regularly no more than 10 y ago.
Blood was drawn from an antecubital veinafter informed consent and anticoagulated bythe addition of sodium citrate. Plasma was sepa-
rated by centrifugation at 4000 g for 10 minutesand stored at ÿ20�C in aliquots. Genomic DNAwas isolated from blood leukocytes using stan-dard methods.
A 307-bp fragment ranging from nucleotide(nt) ÿ380 to nt ÿ73 of the 50 untranslatedregion of human coagulation factor VII wasampli®ed by PCR (upstream oligo: 50-TGATTGC-TATGG GACAGG-30, downstream oligo: 50-CCCATCCCTATGTCAC-CCTT-30). The decanucleotide insertion withinthe PCR product results in the generation of aBlnI site. Digestion of the PCR product with BlnIresults in fragments of 252 bp and 65 bp in case ofthe insertion and in a fragment of 307 bp in thecase of the deletion (Figure 1A). The size of theresulting fragments were analyzed on a 4±20%NovexR gel (Novel Experimental Technology, SanDiego, CA).
Determination of factor VIIThe plasma level of factor VII was determined byenzyme-linked immunosorbent assay (ELISA)(Asserachrom VII:Ag. Stago Diagnostica,Ansiere, France) in 11 healthy individuals fromthe same geographic area with either a homozy-gous insertion (n� 3) or a homozygous deletion(n� 8).
Statistical analysisThe frequencies of the genotypes and risk factorsamong case patients and controls were deter-mined and compared by the chi-squared test.The factor VII levels were compared usingStudent's t-test. ORs were calculated togetherwith their 95 percent CI using standard methods.An analysis of maximum likelihood estimateswas performed to assess the extent to which thedecanucleotide insertion genotype, age, smoking,cholesterol levels, body mass index (BMI), anddiabetes mellitus (DM) in these populationsamples are associated with acute coronarysyndromes. Power calculation for the testbgen� 0 was performed using Query Advisor3.0. The assumption that the remaining covari-ates are not signi®cantly correlated with thevariable gen was made. All computations wereperformed with the SAS statistical package.
Results
Characteristics of the study populationTable 1 shows the demographic data of the studygroup and the control group. Patients in thestudy group had a signi®cantly higher prevalenceof hypertension (p< 0.05), of diabetes (p< 0.05),of a BMI> 26 (p< 0.05), and of cigarette smoking(p< 0.01) and had a borderline higher prevalence
24 Jimenez-Boj et al.
of hypercholesterolemia (p� 0.06) when comp-ared with the control group.
Genotype analysisGenotype frequencies are shown in Table 2.There was no statistical signi®cant difference inthe frequencies of the genotypes between thepatients with acute coronary syndromes and thecontrol population. The distribution of the geno-types was not signi®cantly different from Hardy-
Weinberg equilibrium (not shown). When asubgroup of patients with a ®rst cardiac eventat age< 45 y was analyzed (n� 53), the preva-lence of the homozygous deletion was slightly butnot signi®cantly higher in patients with acutecoronary syndromes when compared with thecontrol group (n� 43) (OR: 1.6; CI 0.95, 0.64±3.93).
A signi®cant association was found in ourlogistic regression analysis between the occur-rence of acute coronary syndromes and hyper-
Fig. 1. (a) Ampli®cation of a DNA stretch in the 50 untranslated region of the factor VII gene results in a fragment of 307 bp in caseof the allele without insertion (deletion) and a fragment of 317 bp in case of the decanucleotide insertion. This insertion within thepromoter region of the factor VII gene (the size of the insertion is exaggerated for better visibility) results in the generation of a BlnIsite. (b) Ampli®cation and subsequent digest with BlnI from an individual homozygous for the deletion (lane 1) results in a singleband of 307 pb. Ampli®cation and subsequent digest with BlnI from an individual homozygous for the insertion results in bands of252 pb and 65 bp (lane 2). Ampli®cation and subsequent digest with BlnI from an individual with a heterozygous genotype(insertion=deletion) results in bands at 307 bp, 252 bp, and 65 bp (lane 3). M�DNA size marker (PhiX174).
Factor VII Polymorphism 25
cholesterolemia (OR: 2.3, p� 0.002), elevatedBMI (OR: 10.6; p� 0.0001), or age (OR: 1.57,p� 0.0017).
Factor VII levelsFactor VII antigen levels (% of normal plasmapool) determined by ELISA, were signi®cantlyhigher in plasma of subjects with the homozygousdeletion (mean: 110� 24 standard deviation [SD])compared with subjects with the homozygousinsertion (mean: 69.1� 3.15 SD, p< 0.05).
Discussion
Our study suggests that the decanucleotide inser-tion=deletion polymorphism in the promoterregion of the factor VII gene is not associatedwith the risk of acute coronary syndromes. This isconsistent with the results from a similar studyby DellAcqua et al. [21]. In their study, patientsand controls were balanced for the presence ofacquired risk factors to eliminate their effect onthe coronary risk. The decanucleotide inser-tion=deletion was not associated with the occur-rence of myocardial infarction in this highlyselected group of low-risk patients. In contrast,we studied consecutive patients with acute coron-ary syndromes who had a typical pro®le of meta-bolic and environmental risk factors for coronaryheart disease (see Table 1) but could not ®nd anyassociation either. Our study was not poweredenough to detect small differences in the cardiacrisk between carriers and non-carriers of the
polymorphism but should have allowed us torule out any major or clinical important differ-ences. When we analyzed a subgroup of patientsfrom our study with acute coronary syndromesbefore age 45, a non-signi®cant trend toward anincreased prevalence of cardiac events was foundin homozygote carriers of the deletion with an ORof 1.6 (CI 0.95, 0.64±3.93). This phenomenon ofan identi®cation of genetic risk factors mainly inpatients at younger age is consistent with obser-vations in studies involving polymorphic sites inother coagulation factors [13]. Analyzing patientswith early onset of disease probably limits theimpact of metabolic and environmental riskfactors on the disease and helps genetic riskfactors to be recognized. Larger cohorts ofpatients with premature CHD need to be studiedto establish the true impact of the decanucleotidepolymorphism. Iacoviello and coworkers [24]studied a polymorphism in the coding region offactor VII (R353Q). This polymorphism has beenshown to be in a strong linkage disequilibriumwith the decanucleotide insertion=deletion poly-morphism and would therefore be expected togenerate a similar phenotype. In contrast to ourpatients with the homozygous deletion poly-morphism, patients with RR genotype had asigni®cantly increased risk of acute myocardialinfarction. Among others, different patient selec-tion might explain this different outcome;patients in the R353Q study were selected forthe presence of familial myocardial infarction orstroke, which potentially increases the impact ofgenetic factors.
It has been convincingly demonstrated by invitro techniques [28] that the insertion of thedecanucleotide stretch within the promoterregion of the factor VII gene leads to a dramaticdecrease in the factor VII expression. Thisdecrease in factor VII levels of approximately30% is also present in our healthy individualswith a homozygous insertion when comparedwith individuals with a homozygous deletion ofthe decanucleotide. This result from our study isin line with other studies [29,30], and representsa remarkably consistent ®nding with regard tothe amount of reduction of the factor VII levels.These data would suggest that other polymorphicsites in the factor VII gene play a less importantrole in the determination of the factor VII plasmalevels. This is particularly true for the otherpolymorphisms in the promoter region (ÿ401and ÿ122) that are in complete linkage disequili-brium with the decanucleotide polymorphism[21]. A small additional in¯uence on the factorVII plasma levels of the two other presentlyknown polymorphic sites in the factor VII genecannot be ruled out on the basis of our study, butit has been suggested by other authors [20].
Table 1. Prevalence (%) of metabolic and environmental riskfactors and age and gender of patients and controls
Patients Controls p
Sex (m=f) 87=24 80=28 ÐAge 48 51 ÐHypertension 30.6 9.3 < 0.05Diabetes 8.1 0.9 < 0.05Hypercholesterolemia 67.5 46.3 � 0.06Smoking 55 33 < 0.01BMI<26 53.7 33.9 < 0.05
Table 2. Prevalence (%) of the different genotypes of thedecanucleotide insertion (I)=deletion (D) polymorphism inpatients and controls
Patients Controls
n 111 108D=D 79.2 79.6D=I 19.8 20.4I=I 0.9 Ð
26 Jimenez-Boj et al.
The frequency of the genotype with the homo-zygote deletion in the Middle European controlpopulation of our study (77%) is similar to the onereported previously from Spain (73%) and fromItaly (87%). This is in contrast to polymorphismsin other coagulation factors, which are based onfounder effects and show a clear ethnic distribu-tion even among populations in Europe [31,32].
In conclusion, our data suggest that the deca-nucleotide insertion=deletion polymorphism inthe promoter region of the factor VII gene isassociated with a signi®cant change in factorVII plasma levels. Despite its impact on thefactor VII levels, we could not ®nd a signi®cantassociation between the occurrence of acutecoronary syndromes and the decanucleotideinsertion=deletion polymorphism.
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