Novel genetic risk factors for venous thrombosis; a haplotype- based candidate gene approach Uitte de Willige, S.

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Novel genetic risk factors for venous thrombosis; a haplotype-

based candidate gene approach

Uitte de Willige, S.

Citation

Uitte de Willige, S. (2007, May 23). Novel genetic risk factors for venous

thrombosis; a haplotype-based candidate gene approach. Hemostasis and

Thrombosis Research Center, Department of Hematology, Faculty of Medicine,

Leiden University. Retrieved from https://hdl.handle.net/1887/11970

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis

in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/11970

Note: To cite this publication please use the final published version (if

applicable).

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Chapter 3.4

Haplotypes of the fibrinogen gamma gene do not

affect the risk of myocardial infarction

Shirley Uitte de Willige, Carine J.M. Doggen, Marieke C.H. de Visser, Rogier M. Bertina and Frits .R. Rosendaal

Journal of Thrombosis and Haemostasis 2006;4(2):474-6

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Gamma fibrinogen haplotypes and MI risk

93

Summary

Background Fibrinogen is an essential component of the haemostatic system and elevated plasma fibrinogen levels have consistently been shown to be associated with thrombotic disorders. We previously studied the association of haplotypes of the three fibrinogen genes (FGA, FGB, FGG) with the risk of deep venous thrombosis. We showed that haplotype 2 of the fibrinogen gamma gene (FGG-H2) was associated with decreased fibrinogen γ' levels and a 2.4-fold increased risk of venous thrombosis.

Objectives Here, we studied the association of the four common haplotypes of the FGG gene with the risk of myocardial infarction.

Patients and Methods We used the 'Study of Myocardial Infarctions Leiden' (SMILE), including 560 men with a first myocardial infarction and 646 healthy control subjects. All subjects were genotyped for three FGG haplotype-tagging SNPs by TaqMan. In addition, we studied the association between the FGG haplotypes and fibrinogen levels. Finally, we analysed the combined effect of the FGG haplotypes and known cardiovascular risk factors such as smoking and metabolic risk factors with regard to the risk of myocardial infarction.

Results None of the four FGG haplotypes was associated with the risk of myocardial infarction or with fibrinogen levels. Also the analyses of the combined effects did not show an association between the FGG haplotypes and the risk of myocardial infarction.

Conclusions We conclude that the four common haplotypes of the FGG gene do not affect the risk of myocardial infarction.

Introduction

As the precursor of fibrin, fibrinogen plays an important role in haemostasis.¹ Of all the components of the coagulation system, elevated plasma fibrinogen levels have most consistently been shown to be associated with occlusive vascular disorders.^2-6 Several polymorphisms in the fibrinogen genes (FGA, FGB, FGG) have been reported to be associated with fibrinogen levels. Most studies focussed on FGB polymorphisms. However, results are not consistent and none of these polymorphisms have been found to be associated with an increased risk of venous^7,8 or arterial thrombosis.⁸ Recently, we reported that a specific haplotype (H2) of the fibrinogen gamma gene (FGG), tagged by haplotype-tagging (ht) SNP 10034C>T [rs2066865], was associated with a 2.4-fold increased risk of deep venous thrombosis (95% confidence intervals (CI): 1.5-3.9).⁷ In the same study we found that another haplotype, FGG-H3, tagged by htSNP 9340T>C [rs1049636], was associated with a slight reduction in risk (OR=0.8, 95%CI: 0.6-1.0). None of these haplotypes was associated with total fibrinogen levels. However, the FGG-H2 haplotype was associated with reduced levels of fibrinogen γ', a product of alternative splicing of the FGG gene. In a recent report, Mannila et al⁹ studied the effect of haplotypes across the fibrinogen gene cluster on the risk of myocardial

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infarction. They determined the *216C>T polymorphism, which is identical to 10034C>T (FGG-H2). The FGG-H2 haplotype was not associated with the risk of myocardial infarction. However, they found a significant difference in the frequency distribution of the minor allele of the 1299+79T>C polymorphism between patients and controls (0.294 vs. 0.342, p=0.04), which suggests that this haplotype might be protective against the development of myocardial infarction. This polymorphism is identical to 9340T>C (FGG-H3).

The aim of the present study was to investigate the effect of the four most common haplotypes of the FGG gene on the risk of myocardial infarction. In addition, we studied the relation between the FGG haplotypes and fibrinogen levels. Finally, we analysed the combined effect of the FGG haplotypes and known cardiovascular risk factors with regard to the risk of myocardial infarction. For this study a large population-based case-control study, 'Study of Myocardial Infarctions Leiden' (SMILE) was used.

Study design Study population

Full details of the SMILE study have been described elsewhere.¹⁰ In short, patients were 560 men below the age of 70 years with a first myocardial infarction. The control group consisted of 646 men, frequency matched for age to the patients, who had undergone an orthopaedic intervention and had received prophylactic anticoagulants for a short period after this intervention. The mean age of the patients was 56.2 (range 32.1-70.0) years and of the control subjects 57.3 (range 27.2-74.8) years. DNA was available for all subjects.

Genotyping

Previously, we investigated the association between fibrinogen haplotypes and the risk of deep venous thrombosis.⁷ The subjects used in that study were all of Dutch origin. Four common haplotypes of the fibrinogen gamma gene were present in this population.⁷ Because the subjects of the SMILE study were also of Dutch origin, we assumed that in the SMILE population only these four common haplotypes would be present. To tag these four common haplotypes, all subjects were genotyped for three haplotype-tagging polymorphisms by the 5’ nuclease/TaqMan assay.¹¹ Polymorphism 10034C>T [rs2066865] tagged FGG-H2, polymorphism 9340T>C [rs1049636] tagged FGG-H3 and polymorphism 5836G>A [rs2066860] tagged FGG- H4.⁷ FGG-H1 consisted of the common alleles of the three mentioned polymorphisms. (Position numbering according to SeattleSNPs,¹² GenBank Accession number AF350254).

Fibrinogen measurement

In the control subjects, total fibrinogen levels were measured in plasma using the

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95 Clauss thrombin time method on a fully automated coagulometer STA (Diagnostica Stago, Boehringer Mannheim), as described previously.¹³ Results were expressed in g/L.

Statistical analysis

In the control subjects, Hardy-Weinberg equilibrium for each htSNP was tested by χ²-analysis. No haplotypes could be assigned to four patients and two control subjects, because of intragenic recombination. These samples were excluded from the analyses. To estimate the relative risk of myocardial infarction for a certain haplotype, odds ratios (OR) were calculated in the standard unmatched fashion.

Ninety-five percent confidence intervals (95% CI) were calculated according to the method of Woolf.¹⁴ Risks were also calculated after stratification for age younger than 50 years (150 patients and 158 control subjects) and age 50 years or older.

Furthermore, we investigated the combined effect of FGG haplotypes and smoking (345 patients and 214 control subjects) and FGG haplotypes and metabolic risk factors (203 patients and 197 control subjects), in which having a metabolic risk factor was defined as the presence of obesity, diabetes, hypertension or hypercholesterolemia.¹⁰

Results and Discussion

The distribution of the genotypes among control subjects was as expected for a population in Hardy-Weinberg equilibrium. Haplotypes could be assigned to 1200 of the 1206 subjects. As shown in Table 1, none of the haplotypes of the FGG gene was associated with the risk of myocardial infarction, including FGG-H2, which we found to be a risk factor for venous thrombosis.⁷ The allele frequency of FGG-H2 was 0.276 in cases and 0.277 in control subjects. This is in agreement with the data of Mannila et al,⁹ who did not find an association in 377 myocardial infarction patients and 387 control subjects aged less than 60 years in Sweden. However, we could not confirm the reduction in risk for the FGG-H3 tagging polymorphism 9340T>C. They reported a significant difference in the frequency distribution of the minor allele of the 1299+79T>C polymorphism between patients and control subjects (0.294 vs. 0.342, p=0.04), while we found allele frequencies of 0.298 and 0.284 for patients and control subjects, respectively (Table 1).

Most patients with myocardial infarction are middle-aged or elderly. Those suffering myocardial infarction at younger age usually have an excess of risk factors, including genetic ones. For this reason, we stratified the risk analyses for age of first myocardial infarction (<50 and ≥50 years) (Table 1). For FGG-H1, the risk of myocardial infarction was slightly decreased in the subgroup with age younger than 50 years, compared to the risk of myocardial infarction in the subgroup with age 50 years or older. This was also seen for FGG-H4. For FGG-H3, there was no difference in risk between the different age groups. Only for FGG-H2, the risk of myocardial

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infarction was increased in the subgroup with age younger than 50 years, whereas the risk in the subgroup with age 50 years or older was somewhat decreased. This might be an indication that FGG-H2, besides venous thrombosis also influences the risk on myocardial infarction, although the effect was weak (OR for FGG-H2H2 carriers is 1.5) with a wide confidence interval (0.7-3.5). Of course, the sample size of the subgroup with age below 50 years was relatively small (150 cases and 158 control subjects) and it would be useful to investigate the role of FGG-H2 in a larger study of patients with myocardial infarction at a young age.

Table 1 The risk of myocardial infarction in the presence of the four most common haplotypes of FGG

Haplotype Patients (%) Controls (%) < 50 ≥ 50

(htSNP) n =556 n = 644 OR 95% CI

OR 95% CI OR 95% CI H1 (all common)

HxHx 210 (37.8) 228 (35.4) 1* 1* 1*

H1Hx 265 (47.7) 318 (49.4) 0.9 0.7-1.2 0.7 0.4-1.2 1.0 0.7-1.3 H1H1 81 (14.6) 98 (15.2) 0.9 0.6-1.3 0.7 0.4-1.4 1.0 0.6-1.5 Frequency H1 0.384 0.399

H2 (10034C>T)

HxHx 290 (52.2) 336 (52.2) 1* 1* 1*

H3 (9340T>C)

HxHx 266 (47.8) 322 (50.0) 1* 1* 1*

H4 (5836G>A)

HxHx 509 (91.5) 593 (92.1) 1* 1* 1*

H4Hx 47 (8.5) 51 (7.9) 1.1 0.7-1.6 0.9 0.4-2.1 1.2 0.7-1.8

H4H4 - - - - - - - -

Frequency H4 0.042 0.040

*Reference category

We also analysed the combined effect of FGG haplotypes and cardiovascular risk factors such as smoking or metabolic risk factors. The risks of the four haplotypes in the different subgroups with or without the cardiovascular risk factors did not change compared to the risks in the overall population. In fact, the risks did not exceed that of the single effects of the cardiovascular risk factors, so we did not find evidence for synergistic effects between FGG haplotypes and cardiovascular risk factors.

In addition, we studied the relation between the four common FGG haplotypes and fibrinogen levels. In a previous study we did not find an association of these haplotypes with fibrinogen levels in the 473 control subjects of the Leiden Thrombophilia Study.⁷ Consistently, we also did not observe an association of FGG

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97 haplotypes with fibrinogen levels in the control subjects of the SMILE study (Table 2).

Previously we demonstrated in the SMILE study that the FGB polymorphisms Bcl I (11046C>T [rs209502]) and 1437G>A (-455G>A [rs1800790]) were not associated with fibrinogen levels (in the control subjects) and the risk of myocardial infarction.¹³ These two polymorphisms, which are both present in haplotypes FGB- H4 and FGB-H5,⁷ have been reported to be associated with increased plasma fibrinogen concentrations in several studies and this association seems to be more relevant under specific circumstances like inflammation.^8,15 Although it has been reported that genetic factors contribute to the variation in plasma fibrinogen levels (heritability, h²= 0.2-0.5),^16-18 so far no evidence has been obtained for linkage between the fibrinogen locus and the regulation of plasma fibrinogen levels.^19,20

Table 2 Association of FGG haplotypes with fibrinogen levels (g/L) in control subjects

Haplotype FGG N Mean (95%CI)

H1H1 98 3.31 (3.17-3.46)

H1H2 140 3.28 (3.14-3.42)

H1H3 157 3.22 (3.13-3.32)

H1H4 21 3.23 (2.98-3.48)

H2H2 49 3.25 (3.06-3.44)

H2H3 105 3.20 (3.09-3.31)

H2H4 14 3.41 (3.05-3.76)

H3H3 44 3.35 (3.15-3.55)

H3H4 16 2.98 (2.73-3.22)

H4H4 - -

We conclude that none of the four common haplotypes of the FGG gamma gene have a strong effect on the risk of myocardial infarction. Homozygosity for FGG-H2, which we found previously to be a risk factor for venous thrombosis, might slightly increase the risk of myocardial infarction in patients younger than 50 years. These findings are in line with studies on other prothrombotic factors that affect the risk of venous thrombosis but have small or no effects on arterial disease.²¹

Acknowledgments

This study was financially supported by grant 912-02-036 from the Netherlands Organization for Scientific Research (NWO). The SMILE study was supported by grant 92.345 from the Netherlands Heart Foundation.

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