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Open Access

Peer-reviewed

Research Article

Evaluation of single nucleotide polymorphisms in 6 candidate genes and carotid intima-media thickness in community-dwelling residents

  • Fang-Yang Wu ,

    Contributed equally to this work with: Fang-Yang Wu, Chia-Ing Li, Li-Na Liao

    Roles Methodology, Writing – original draft

    Affiliation Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan

    ⨯
  • Chia-Ing Li ,

    Contributed equally to this work with: Fang-Yang Wu, Chia-Ing Li, Li-Na Liao

    Roles Data curation, Formal analysis, Project administration, Writing – review & editing

    Affiliations School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan

    ⨯
  • Li-Na Liao ,

    Contributed equally to this work with: Fang-Yang Wu, Chia-Ing Li, Li-Na Liao

    Roles Data curation, Formal analysis

    Affiliation Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan

    ⨯
  • Chiu-Shong Liu,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliations School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan, Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan

    ⨯
  • Wen-Yuan Lin,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliations School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan

    ⨯
  • Chih-Hsueh Lin,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliations School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan

    ⨯
  • Chuan-Wei Yang,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Medical Research, China Medical University Hospital, Taichung, Taiwan

    ⨯
  • Tsai-Chung Li ,

    Roles Data curation, Formal analysis, Methodology, Writing – original draft

    cclin@mail.cmuh.org.tw (CCL); tcli@mail.cmu.edu.tw (TCL)

    ‡ These authors also contributed equally to this work.

    Affiliations Department of Public Health, College of Public Health, China Medical University, Taichung, Taiwan, Department of Healthcare Administration, College of Medical and Health Sciences, Asia University, Taichung, Taiwan

    ⨯
  • Cheng-Chieh Lin

    Roles Data curation, Methodology, Project administration, Writing – review & editing

    cclin@mail.cmuh.org.tw (CCL); tcli@mail.cmu.edu.tw (TCL)

    ‡ These authors also contributed equally to this work.

    Affiliations School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan, Department of Family Medicine, China Medical University Hospital, Taichung, Taiwan

    ⨯

Abstract

Evidence suggests the existence of association between a large panel of modifiable biomarkers representing inflammation, coagulation, paraoxonase, and endothelial activation pathways and carotid atherosclerosis. Thus, this study investigated whether CRP, FGA, FGB, FGG, PON1, and EDNRA gene variants affected plasma hs-CRP, fibrinogen levels, and thickness of carotid intima media thickness (IMT). Nineteen single-nucleotide polymorphisms of CRP, FGA, FGB, FGG, PON1, and EDNRA genes were examined in 480 participants from 160 families. Carotid IMT was measured by ultrasound. Generalized linear models with generalized estimating equation were utilized to consider the dependence of subjects within families. In the recessive model, homozygotes for the minor alleles of rs1800789, rs1800790 and rs4220 SNPs in FGB gene indicated a reduced risk of IMT (Exp. β = 0.89, 0.89, 0.88), which remained significant after adjustment for confounding factors. Significant interaction effects between CRP SNP rs1130864 and rs3093059 and gender for IMT were observed with a significant association in men only. Men carrying minor-minor genotype of CRP SNP rs1130864 and rs3093059 had 0.70- and 0.78-fold lower IMT than men carrying minor-major/major-major genotype. We also observed that the interaction of CRP SNP rs1130864 and rs3093059 with obesity on IMT, hs-CRP and fibrinogen levels. These results support the hypothesis that inflammatory genes are involved in atherosclerosis, most likely via complex gene-gender and gene-obesity interactions.

Citation: Wu F-Y, Li C-I, Liao L-N, Liu C-S, Lin W-Y, Lin C-H, et al. (2020) Evaluation of single nucleotide polymorphisms in 6 candidate genes and carotid intima-media thickness in community-dwelling residents. PLoS ONE 15(3): e0230715. https://doi.org/10.1371/journal.pone.0230715

Editor: Cassandra Nichole Spracklen, University of North Carolina at Chapel Hill, UNITED STATES

Received: July 26, 2019; Accepted: March 6, 2020; Published: March 26, 2020

Copyright: © 2020 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript.

Funding: Ministry of Science and Technology, Taiwan - MOST 104-2314-B-039-016, MOST 105-2314-B-039-021-MY3, MOST 105-2314-B-039-025-MY3, MOST 107-2314-B-039-049, MOST 108-2314-B-039-039, MOST 108-2314-B-039-035-MY3 and MOST 108-2314-B-039-031-MY2 China Medical University Hospital - DMR-107-200 The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Carotid intima-media thickness (IMT) is one of the best established and most commonly used early surrogate markers of atherosclerosis [1]. Carotid IMT has recently emerged as an important predictor for cardiovascular events in the general population [2] and in asymptomatic subjects with multiple cardiovascular disease (CVD) risk factors [3]. The Atherosclerosis Risk in Communities (ARIC) study show an increase of 0.19 mm in the carotid IMT, an increase of 36%-69% in the risk of coronary disease, and an increase in the risk of stroke by 3.6–8.5 times [4, 5].

Atherosclerosis is one kind of chronic inflammatory disease. Inflammation and endothelial injury play pivotal roles in the pathogenesis of atherosclerosis [6, 7]. Endothelial injury affects the balance between vasodilation and vasoconstriction [8]. In order to repair the endothelial tissue, fibrinogen was cleaved by thrombin to form fibrin. Low-density lipoprotein (LDL) accumulates at the site of endothelial injury and may become oxidized [9], and further induce inflammation, although paraoxonase 1 (PON 1) stimulate the antioxidant activity of high-density lipoprotein (HDL) to fight the oxidized- LDL [10]. Accumulation of macrophages at the site of endothelial injury regulate the endothelin A receptors shift to endothelin B receptors and enhance endothelin system, and further express C-reactive protein (CRP) mRNA contribute to the progression of atherosclerosis [11, 12]. These six genes are involved in the development of atherosclerosis, including CRP, endothelin receptor type A (EDNRA), PON 1, fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), and fibrinogen gamma chain (FGG).

Many prior studies have demonstrated that the increase in IMT is associated with most of the known CVD and atherosclerotic risk factors, such as CRP, fibrinogen, PON 1, and EDNRA levels or gene variants [13–19], although those associations have not been confirmed in some studies [20, 21]. Some studies indicated a modest graded relation between CRP and carotid plaque and carotid IMT in different populations [13, 19, 22]. The studies of CRP and IMT confirmed that increased CRP is a risk factor for ischemic stroke, and atherosclerosis is measured by carotid IMT degree, i.e. the increase in IMT also increases the risk of atherosclerosis [13, 19].

Fibrinogen plays an important role in blood coagulation and is also a key regulator of inflammation processes [23]. Elevated fibrinogen level is a well-characterized risk factor for CVD [24]. Prior studies indicated that increased fibrinogen was correlated with carotid IMT in adults without clinically overt atherosclerotic disease [17, 18]. Studies on genetic determinants of fibrinogen typically focused on the FGA, FGB, and FGG genes. However, polymorphisms of these genes denoted only a small part of the estimated heritable effects on fibrinogen [23, 25, 26]. Reports show inconsistent evidence of the association between CRP and fibrinogen gene variants and carotid IMT [27, 28].

PON1 degrades oxidized lipids. Previous studies revealed the association between the low plasma PON1 activity and the increase CVD risk regardless of age and ethnicity [29, 30]. Thus, PON1 activity is genetically adjusted and differs widely among populations. Cece et al. study showed that decreased PON1 activity and increased IMT suggest significantly relationship in subjects with atherosclerosis and ankylosing spondylitis [14]. Similarly, a negative relationship was observed between PON1 activity and IMT in individuals with coronary heart disease [31]. As for EDNRA protein, its existence is found in the membrane of vascular smooth muscle cells [32]. The proliferation and migration of smooth muscle cells from the media into the intima mediated by EDNRA is significant with atherosclerosis development and progression [33, 34]. Little is known regarding the relationship between EDNRA gene and carotid IMT [15, 16]. Li’s study indicated the interaction of smoking with EDNRA gene variation on left carotid IMT in Africa Americans [15], whereas Lopez-Mejias’s study revealed no significant differences in carotid IMT according to EDNRA gene polymorphisms in patients with rheumatoid arthritis [16]. In our previous analyses of this study population, genetic polymorphisms of EDNRA revealed a significant interaction with regular exercise or obesity on IMT [35].

Carotid IMT is related to a family history of pre-clinical carotid artery cardiovascular disease [36]. The measurements of carotid IMT is a good quantitative phenotype for the assessment of its genetic determinants. Prior studies estimated the heritability of carotid IMT, thereby demonstrating great variation depending on the population. Most family studies revealed the heritability between 0.30 and 0.60, thereby indicating that genetic factors play a key role in carotid IMT [37–40]. This study was designed to investigate the association between a large panel of modifiable biomarkers representing inflammation, coagulation, paraoxonase, and endothelial activation pathways and carotid IMT. In any complex polygenic event, the overall influence of each gene is probably small and may be masked by the effect of gene-environment interactions. Therefore, we hypothesized that the tag single nucleotide polymorphisms (SNPs) of CRP, FGA, FGB, FGG, PON1, and EDNRA genes are related to higher high-sensitivity CRP (hs-CRP) and fibrinogen levels and to increased thickness of IMT. We also hypothesized that their effects would vary according to environmental factors, such as gender, obesity, and regular exercise.

Methods

Study subjects

This study was conducted after obtaining approval from the Institutional Review Board of China Medical University Hospital and all methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from each participant. The study conforms to the recognized standards (Declaration of Helsinki). A community-based family study was conducted to explore the relationship between genetic variants and subclinical atherosclerosis [35]. All proband cases and proband controls were identified from our cohort of Taichung Community Health Study (TCHS). The TCHS was conducted in 2004 and it consisted of 2,359 participants who were randomly selected from among Taichung’s general population aged ≥40 years old. A total of 1,666 subjects were followed up, and the over-all follow-up rate was 73.3%. Our analyses are based on carotid IMT measurements collected at the second wave of the follow-up evaluations, which were conducted between 2006 and 2009. We invited family members of study subjects from TCHS individually by letter and by phone to participate in our genetic study. This family study recruited 484 TCHS participants and 1080 family members to estimate the familiar aggregation and heritability of specific cardiovascular risk phenotypes in our prior study. In this genetic study, we included 80 proband cases and 80 proband controls with their spouses and one first-degree blood relative aged 20 years old and older for this genetic study (a total of 480 people). Proband cases were those who had their maximum IMT (IMTmax) in the area of the common carotid arteries (CCAs), carotid bulb, and internal carotid artery (ICA) ≥ 1.5 mm, and IMTmax< 1.5 mm as proband controls, whereas proband control had their IMT ranges from 0.25 to 1.5 mm [41]. The inclusion criterion for this genetic study was that study subjects must have more than one first-degree relatives.

Measurements

A standardized questionnaire was designed to collect data on sociodemographic characteristics, including gender, age, education, smoking, drinking, regular exercise, betel nut chewing, family and personal history of cardiovascular-related diseases, and medication history. The questionnaire used in this study was shown in Supplemental Information. Hypertension, diabetes, and hyperlipidemia were defined as the use of medications or self-reported. Anthropometric measurements were obtained from physical examination. Weight and height were measured on an autoanthropometer (super-view, HW-666), with no shoe and light clothing. Body mass index (BMI) was defined as weight (kg) ÷ (height)2 (m) 2. Obesity was defined as BMI > = 27 kg/m2. Study subjects who had regular exercise were those who participated in regular leisure-time activities for at least 30 min per week for at least 6 months when they were interviewed. Venous blood samples were obtained after 12-hour overnight fasting. Biochemical blood tests such as total cholesterol (TC), high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, triglyceride (TG), urine albumin, creatinine, uric acid, blood urea nitrogen (BUN), fasting plasma glucose (FPG), and insulin levels were measured by using a biochemical autoanalyzer (Beckman Coluter, Lx-20, USA) at the Clinical Laboratory Department of China Medical University Hospital. Hs-CRP and fibrinogen levels was measured using nephelometry, a latex particle-enhanced immunoassay (TBA-200FR, Tokyo, Japan). The inter-assay and intra-assay coefficient of variation (CV) for hs-CRP are <2.0% and 1.9%, respectively. The lower detection limit of the assay is 0.1 mg/L. The inter-assay and intra-assay CVs for fibrinogen are 3.6% and 6.1%, respectively.

All study subjects were measured by the same machine (GE L7000, GE, Milwaukee, Wis., USA). After having a rest for at least 10 min in the supine position with the neck in slight hyperextension, all study subjects underwent carotid ultrasound examination using 7.5-MHz probe to scan the near and far wall of arterial segments bilaterally in order to get the longitudinal (anterior oblique, lateral, and posterior oblique) and transverse views. Each ultrasound image was recorded on a computer with an online digital filing system, and the intima-media complex thickness and atherosclerotic plaques were measured. The 12 arterial segments involves 1) 1 to 2 cm proximal to the tip of the flow divider into the common carotid arteries (CCAs); 2) the carotid bifurcations beginning at the tip of the flow divider and extending 1 cm proximal to the flow divider tip; and 3) the proximal (first 1 cm) of the internal carotid arteries (ICAs). Maximal IMT of each segment at the CCA was determined from a minimum of 3 frames as the maximum IMT (M-IMT) of each segment by averaging these frames. The total carotid IMT was calculated as a composite measure (mean of the 8 carotid sites) that combine the near and the far wall of the CCA IMT, the bifurcation IMT, and the ICA IMT of both sides of the neck. Total IMT were expressed in a mean of the maximums of the 8 carotid sites.

Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood leucocytes by using QIAamp Blood Kit (Qiagen, Chatsworth, CA, USA). DNA concentration was measured by using a NanoDrop ND-2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The purity of genomic DNA was verified by evaluation of OD260/OD280 ratio. The criteria used for SNPs selection were as follows: 1) A minimum of 60 bases is required for upstream or downstream flanking region of each SNP with another SNP; 2) Minor allele frequency of each SNP must be 5% or greater in the HapMap dataset (Han Chinese in Beijing (CHB) population); and 3) The final score of each SNP must be 0.6 or greater by using the Illumina Assay Design Tool. We selected the CRP (SNP rs12093699, rs876537, rs1205, rs1130864, and rs3093059), FGA (SNP rs2070016 and rs2070011), FGB (SNP rs1800789, rs1800790, rs1800788, and rs4220), FGG (SNP rs7659024, rs1049636, and rs7681423), PON1 (SNP rs854555 and rs662), and EDNRA (SNP rs1395821, rs1878406 and rs5333) genes based on the findings on the CHB population in the HapMap dataset (CHB population). These polymorphisms were detected by using Illumina GoldenGate Genotyping Assay (Illumina, San Diego, CA, USA). Each of the genomic DNA samples was amplified and the polymerase chain reaction products were hybridized to VeraCode microbeads and detected on a VeraCode BeadXpress Reader. SNP genotypes were typed by GenomeStudio Data Analysis software. The quality controls of GoldenGate Genotyping Assay were verified in each steps, such as allele-specific extension, first hybridization, PCR uniformity, extension gap, and second hybridization.

Statistical analysis

The values of mean and standard deviation (SD) for continuous variables and number and proportion for categorical variables were presented stratified by proband type (case and control) and type of family member (proband, spouse, and offspring). Nineteen SNPs were assessed for deviation from Hardy-Weinberg equilibrium (HWE) using PLINK software. Covariates for IMT were age, gender, BMI, waist-hip ratio (WHR), hs-CRP, fibrinogen, lifestyle behaviors, biochemical markers (including FPG, TC, HDL, and TG), and disease history (hypertension, diabetes, hyperlipidemia, cardiovascular disease, and cancer). The average values of IMT, hs-CRP, and fibrinogen were compared among genotypes by using generalized linear model(s) (GLM) with generalized estimating equation(s) (GEE) to account for the dependence of subjects within families. Because distributions of Max IMT, hs-CRP, ACR (albumin to creatinine ratio), and TG skewed to the right, natural log-transformation for these markers was used to normalize data and geometric means of these markers values and SDs were reported. Estimated coefficient of regression and standard error (SE) were calculated. Genotypic models were used for the single-SNP association analysis. Because most SNPs exerted recessive effects, recessive model was adopted for multivariate models examining gene-environment interactions. Gene-environment interactions containing gene-gender, gene-obesity, and gene-regular exercise interactions on carotid IMT, hs-CRP, and fibrinogen were evaluated by GLM with GEE adjusted for confounders. Then, interaction plots were presented for those analyses with significant interactions. A significant gene-environment interaction was interpreted as “a different effect of a genotype on disease risk in persons with different environmental exposures” [42]. The p-values corrected for false discovery rate method were reported to consider multiple testing problem. All analyses used Statistical Analysis System software (v9.4, SAS Institute Inc., Cary, NC, USA) and PLINK (v1.07) (http://pngu.mgh.harvard.edu/purcell/plink) [43].

Results

Basic characteristics of probands, spouses, and offspring

The characteristics of study subjects, consisting of 80 proband cases and 80 proband controls along with their spouses and offsprings, are shown in Table 1. Mean ages were 63.0 and 59.8 years old for proband cases and controls, 61.2 and 59.5 years old for their spouses, and 33.9 and 30.9 years old for their offspring. Males accounted for 70% and 50% of proband cases and controls, 30% and 50% of their spouses, and 60% and 53.8% of their offspring. The number of individuals with max IMT ≥1.5 mm for proband cases and controls were 80 and 0, whereas those for their spouses were 30 and 28, and those for their offspring were 5 and 2, respectively. Mean values of BMI, IMT, ACR, and proportions of smoking, hypertension, heart disease, and cerebral vascular accident (CVA) were higher in proband cases than in the other five groups.

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Table 1. Characteristics of study subjects.

https://doi.org/10.1371/journal.pone.0230715.t001

Allele and genotype frequencies in CRP, FGA, FGB, FGG, PON1, and EDNRA genes and their relation with IMT, hs-CRP, and fibrinogen

All 19 SNPs studied in the six genes were tested to be in the HWE (P > 0.05). The allele and genotype distributions for all polymorphisms are shown in Table 2. The associations of CRP, FGA, FGB, FGG, PON1, and EDNRA polymorphisms with IMT, hs-CRP, and fibrinogen were examined. The mean of IMT in individuals carrying the AA variant of SNPs (rs12093699 and rs1130864) of CRP gene and SNPs (rs1800789, rs1800790 and rs4220) of FGB gene were lower compared with genotype GG. Significant differences in hs-CRP level were found among genotype groups for SNPs rs876537, rs1205 and rs3093059 of CRP gene (p< 0.05). Higher hs-CRP concentration was linked with a greater number of G allele of rs876537, rs1205 and rs3093059 polymorphisms. Increasing levels of fibrinogen were also linked with higher numbers of minor alleles of rs1800789, rs1800790, rs1800788, and rs4220 SNPs of FGB gene.

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Table 2. Genotype and allele distributions of study subjects as well as IMT, hs-CRP and fibrinogen distributions according to genotype status†.

https://doi.org/10.1371/journal.pone.0230715.t002

Comparison of IMT, serum CRP and fibrinogen level in the recessive models

Linear regression with GEE model was performed for CRP, FGA, FGB, FGG, PON1, and EDNRA gene SNPs separately, under the assumption of recessive models. Regression coefficients (β) and their SE were calculated for minor-minor genotype before and after adjustment for age, sex, BMI, physical activity, smoking, alcohol drinking, and betel nut chewing (Table 3) with minor-major/major-major genotype as the reference genotype. CRP gene bearing two minor alleles of SNP rs12093699, rs876537, rs1205, and rs1130864 was associated with IMT in the unadjusted analysis, but their effects were not statistically significant after adjustment for confounding variables. Subjects carrying GG genotype of SNP rs876537 and rs1205 were associated with an increased hs-CRP levels and had an average 1.55- and 1.53-fold higher than subjects carrying GA/GG genotype in the adjusted analysis (S1 Table). In the FGB gene, A allele of SNPs rs1800789, rs1800790, and rs4220 were correlated with IMT, and the correlation remained significant after adjustment (Exp. β = 0.89, 0.89, 0.88), but were no significant association in serum FBG concentration and in IMT, hs-CRP, and fibrinogen levels for the SNPs of FGA, FGG, PON1, and EDNRA genes with and without adjustment.

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Table 3. The association between SNPs and IMT, hs-CRP, and fibrinogen in the recessive model†.

https://doi.org/10.1371/journal.pone.0230715.t003

The findings from significant interactions of CRP, FGA, PON1, and EDNRA gene SNPs with gender and obesity are summarized in Table 4. Significant gene-gender interactions were found for CRP gene SNPs rs1130864 and rs3093059 on IMT (P<0.001 and 0.036, respectively), as well as rs1130864 on serum hs-CRP level (P = 0.004) and fibrinogen level (P<0.001) with multivariate adjustment. The interactions of CRP gene SNP rs1130864 and rs3093059 with obesity were significant on IMT (P<0.001), serum hs-CRP (P <0.01) and fibrinogen level (P<0.001). In addition, obesity significantly interacted with FGA rs2070016, PON1 rs854555 and EDNRA rs5333 on fibrinogen (P = 0.024, 0.036, and <0.001, respectively). Significant gene-regular exercise interactions were not observed.

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Table 4. Significant gene-gender and obesity interaction on ln IMT, ln hs-CRP, and fibrinogen in recessive model#.

https://doi.org/10.1371/journal.pone.0230715.t004

To further evaluate the significant interactions identified above, we estimated the adjusted mean differences in fibrinogen and the adjusted mean ratios in carotid IMT and hs-CRP level for CRP, FGA, PON1, and EDNRA minor-minor genotype in comparison with minor-major/major-major genotype stratified by gender (Fig 1) and obesity (Fig 2). For gene-gender interaction, the adjusted mean IMT for men carrying minor-minor genotype of CRP SNP rs1130864 and rs3093059 were 0.70 and 0.78-fold lower than that for men carrying minor-major/major-major genotype (95% confidence interval (CI): 0.64, 0.76; 0.71, 0.86) (Fig 1A and S1 Table). Men carrying AA genotype of rs1130864 had 1.87-fold higher serum hs-CRP level than men carrying AG/GG genotype (95% CI: 1.45, 2.41) (Fig 1B). On the contrary, we did not observe significant effects for women (Fig 1A and 1B). Among individual with the AA genotype of the CRP SNP rs1130864, the adjusted mean difference for fibrinogen levels increased by 33.01 mg/dL (95% CI: 16.57, 49.45) in men and decreased by -74.09 mg/dL (95% CI:-91.53, -56.65) in women, compared with those carrying AG/GG genotype (Fig 1C).

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Fig 1.

(a) Adjusted mean ratios of IMT, (b) adjusted mean ratios of hs-CRP, and (c) adjusted mean differences of fibrinogen were from generalized linear regression with GEE model considering SNP, age, gender, obesity, regular exercise, smoking status, alcohol drinking, betel nut chewing, and interaction between gender and SNP. *P < 0.05; **P < 0.01; ***P < 0.001.

https://doi.org/10.1371/journal.pone.0230715.g001

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Fig 2.

(a) Adjusted mean ratios of IMT, (b) adjusted mean ratios of hs-CRP, and (c) adjusted mean differences of fibrinogen were from generalized linear regression with GEE model considering SNP, age, gender, obesity, regular exercise, smoking status, alcohol drinking, betel nut chewing, and interaction between obesity and SNP. *P < 0.05; **P < 0.01; ***P < 0.001.

https://doi.org/10.1371/journal.pone.0230715.g002

For gene-obesity interaction, obese individuals carrying the minor-minor genotype of CRP SNP rs1130864 and rs3093059 had a significantly lower adjusted mean IMT value than those carrying minor-major/major-major genotype (0.70 [0.64, 0.76] and 0.64 [0.56, 0.75], respectively) (Fig 2A and S1 Table). The adjusted mean hs-CRP levels for obese individuals carrying minor-minor genotype of CRP SNP rs1130864 and rs3093059 were 1.87-fold higher and 0.31-fold lower than that for obese individuals carrying minor-major/major-major genotype (95% CI:1.45, 2.41; 0.21, 0.45) (Fig 2B). As for fibrinogen levels, the adjusted mean differences in obese individuals carrying the minor-minor genotype of CRP rs1130864, FGA rs2070016 and PON 1 rs854555 was higher by 33.01, 55.19 and 64.77 mg/dL, respectively, compared with those carrying minor-major/major-major genotype (Fig 2C). Whereas individuals carrying the GG genotype of CRP SNP rs3093059 and EDNRA SNP rs5333 had lower mean values of fibrinogen levels than those carrying the GA/AA genotype among the subjects who obese (Fig 2C). In non-obese individuals, we observed significant effects for CRP SNP rs3093059 in hs-CRP levels (Fig 2B) and CRP SNP rs1130864 in fibrinogen levels (Fig 2C).

Discussion

The important role of hs-CRP and fibrinogen concentration and carotid IMT in the occurrence of atherosclerosis and associated complications is well recognized; it is implied that measurements of hs-CRP and fibrinogen levels and carotid IMT may aid in risk stratification and serve as targets for effective prevention of CVD [13, 24]. In this study, we assessed hs-CRP, fibrinogen levels, and 19 SNPs of 6 genes and their correlation with carotid IMT, which is a marker of subclinical cardiovascular disease. Conventional risk factors in this study such as BMI, IMT, smoking, hypertension, heart disease, and CVA were greatest in mean values or prevalence for proband cases than for the other five groups.

Previous studies suggested that inflammation, coagulation, paraoxonase, and endothelial activation pathways may play an important role in carotid atherosclerosis [13–19]. CRP, a major inflammatory biomarker, is associated with higher IMT and ischemic stroke, carotid atherosclerosis, or coronary heart disease [13, 19], although this association has not been confirmed in some studies [25, 44]. CRP tag SNP genotype has been associated with increased serum CRP concentration, and a few have been related with decreased hs-CRP levels [45]. Our prior studies indicated that CRP gene SNPs rs2794520, rs1205, and rs3093059 were associated with a higher serum CRP level and low handgrip strength in a random sample of elders [46]. This current study analyzed whether five tag SNPs in CRP gene were correlated with plasma CRP levels and IMT. Our present results demonstrated the significant relationship between serum CRP concentration and G alleles of SNPs rs876537 and rs1205 with family study design. Higher hs-CRP concentration was linked with a greater number of G alleles of rs876537 and rs1205 polymorphisms. Although SNP rs1130864 was correlated with increased plasma CRP levels in Caucasians [47], we found no correlation between SNPs rs1130864 with CRP levels.

In the recessive model of this study, individuals carrying the GG genotype of CRP gene SNP rs876537 and rs1205 were associated with an increased hs-CRP level, and separately had an average 1.55- and 1.53-fold higher levels in the adjusted analyses. Four tag SNP (rs12093699, rs876537, rs1205, and rs1130864) genotypes in CRP gene exhibited influence on carotid IMT in the unadjusted analysis, but their effects were not significant in further regression analyses. No evidence of CRP variants’ relation to carotid IMT was found in previous studies [27]. Thus, gene-gene and gene-environment interactions may provide useful assistance to the understanding of complexities of genetic determinants of carotid IMT. In this study, significant interaction effects of CRP gene SNP rs1130864 and rs3093059 and gender for IMT, hs-CRP and fibrinogen were observed. Men carrying AA genotype of CRP rs1130864 and GG genotype of rs3093059 have a 30% and 22% decrease in IMT, (adjusted mean ratios of 0.70 and 0.78, respectively), but carrying AA genotype of CRP rs1130864 was associated with a 87% increased serum hs-CRP level. We did not observe significant effects in women. Our findings on the gender difference in the association of IMT and serum hs-CRP level with two SNPs of CRP gene suggested that hormones may play an etiologic role on IMT-related cardiovascular disease.

Fibrinogen plays an important role in the pathways of blood coagulation [23]. Many studies have indicated that variation in the fibrinogen structural genes (FGA, FGB and FGG) are related with plasma fibrinogen concentration [23, 25, 26]. Some SNPs of these genetic variants are located in non-coding regions of FGB. Most studied fibrinogen polymorphism is SNP rs1800790 (-455G/A) change in the promoter region of FGB gene [48–50]. FGB rs1800790 variant elevated plasma fibrinogen levels [26, 51]. Boekholdt et al. suggested that rs1800790 SNP in FGB gene was correlated with decreased myocardial infarction risk using the recessive model in a meta-analysis [23]. This SNP exerted protective activity against premature myocardial infarction [52]. Theodoraki et al. proposed that FGB rs1800787 and rs1800789 SNPs offered protection for coronary artery disease onset by diminishing about 50% of the risk in homozygotes for the minor alleles [53]. By contrast, Zeng et al. revealed that three SNPs (rs1800790, rs1800787, and rs6050) of the fibrinogen gene were not related with sporadic cerebral hemorrhage when analyzed separately [28]. In our study, minor alleles of rs1800789, rs1800790 and rs4220 SNPs on FGB gene were associated with increased plasma fibrinogen levels. However, the effects of these alleles on IMT were the opposite. Homozygotes for the minor alleles of rs1800789, rs1800790 and rs4220 SNPs in FGB gene demonstrated significantly lower mean values of IMT after adjustment for confounding factors in the recessive model. These SNPs result in conflicting findings for functionally distinct fibrinogen and IMT, thereby suggesting that the causal pathway for variation in IMT may not be through the alteration of serum fibrinogen levels.

However, the correlation of subclinical atherosclerosis with PON1 activity has been reported [54, 55]. PON1 level and activity are reportedly predictors of cardiovascular diseases, whereas PON1 genotype was not [56]. Several negative relationships were observed between serum PON-1 activity and carotid IMT in subjects with coronary heart disease [31], hypertension, and ankylosing spondylitis. Previous studies showed inconsistent results regarding the associations between PON1 SNP rs662 (Q192R) and IMT [21]. A significant difference in mean IMT in female subjects was observed for the PON1 re662 variants [57]. Another relationship was also observed based on ethnicity for PON1 rs662 polymorphism in young subjects from Bogalusa Heart Study [57], whereas Karvonen et al. did not observe a significant association between PON1 rs662 and carotid IMT in the general Caucasian population [58]. Another study also showed a lack of association of PON1 rs662 with carotid IMT and carotid atherosclerotic plaques [59]. Our study’s findings support the conclusion that PON1 SNPs were not correlated with carotid IMT.

The EDNRA protein plays a central role in the endothelin-1 pathway and is its function with regard to vasoconstriction [34]. EDNRA gene is of great importance in different cardiovascular pathologies [33]. Yasuda et al. shown that EDNRA SNP rs5333 was positively associated with mean IMT, and this association was only apparent in male hypertensive patients [60]. Li et al. indicated the interaction of smoking with EDNRA rs6841473 on left carotid IMT in Africa Americans [15]. Lopez-Mejias et al. did not confirm the association between the EDNRA rs1878406 polymorphisms and carotid IMT in rheumatoid arthritis with subclinical atherosclerosis and cardiovascular problem [16]. Our previous study showed that EDNRA gene rs1395821 significantly interacted with regular exercise and rs5333 with obesity on IMT [35]. This study’s results identified a significant interaction of obesity with SNP rs5333 in the EDNRA gene on fibrinogen. Our findings provided the knowledge of how life-style associated risk factors may interact with EDNRA gene variants in IMT risk, which would be useful for research on carotid IMT risk prediction and prevention in the general population.

Conclusion

Our research was a community-based family study with detailed systematic examination of our quantitative phenotype. One limitation that must be explained is the relatively small sample size. The number individuals who underwent carotid IMT measurements (480 individuals) may be too small to provide sufficient statistical power for a stratified analysis. Our results provide evidence for the main effects of the FGB genes on IMT, for the interaction of CRP SNP rs1130864 and rs3093059 with gender on IMT, and with obesity on IMT, hs-CRP and fibrinogen levels. These results support the hypothesis that inflammatory genes are involved in atherosclerosis, most likely via complex gene-gender and gene-obesity interactions.

Supporting information

S1 Data.

https://doi.org/10.1371/journal.pone.0230715.s001

(DOCX)

S1 Table. A summary table of describing significant regression analysis and interaction results simply.

https://doi.org/10.1371/journal.pone.0230715.s002

(DOCX)

References

  1. 1. O'Leary DH, Polak JF. Intima-media thickness: a tool for atherosclerosis imaging and event prediction. The American journal of cardiology. 2002;90(10C):18L–21L. Epub 2002/12/03. pmid:12459422.
  2. 2. Nambi V, Chambless L, Folsom AR, He M, Hu Y, Mosley T, et al. Carotid intima-media thickness and presence or absence of plaque improves prediction of coronary heart disease risk: the ARIC (Atherosclerosis Risk In Communities) study. Journal of the American College of Cardiology. 2010;55(15):1600–7. Epub 2010/04/10. pmid:20378078; PubMed Central PMCID: PMC2862308.
  3. 3. Baldassarre D, Hamsten A, Veglia F, de Faire U, Humphries SE, Smit AJ, et al. Measurements of carotid intima-media thickness and of interadventitia common carotid diameter improve prediction of cardiovascular events: results of the IMPROVE (Carotid Intima Media Thickness [IMT] and IMT-Progression as Predictors of Vascular Events in a High Risk European Population) study. Journal of the American College of Cardiology. 2012;60(16):1489–99. Epub 2012/09/25. pmid:22999719.
  4. 4. Chambless LE, Heiss G, Folsom AR, Rosamond W, Szklo M, Sharrett AR, et al. Association of coronary heart disease incidence with carotid arterial wall thickness and major risk factors: the Atherosclerosis Risk in Communities (ARIC) Study, 1987–1993. American journal of epidemiology. 1997;146(6):483–94. Epub 1997/09/18. pmid:9290509.
  5. 5. Howard G, Manolio TA, Burke GL, Wolfson SK, O'Leary DH. Does the association of risk factors and atherosclerosis change with age? An analysis of the combined ARIC and CHS cohorts. The Atherosclerosis Risk in Communities (ARIC) and Cardiovascular Health Study (CHS) investigators. Stroke. 1997;28(9):1693–701. Epub 1997/09/26. pmid:9303011.
  6. 6. Lefkowitz RJ, Willerson JT. Prospects for cardiovascular research. Jama. 2001;285(5):581–7. Epub 2001/02/15. pmid:11176863.
  7. 7. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473(7347):317–25. Epub 2011/05/20. pmid:21593864.
  8. 8. Davignon J, Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation. 2004;109(23 Suppl 1):III27–32. Epub 2004/06/17. pmid:15198963.
  9. 9. Vasankari T, Ahotupa M, Toikka J, Mikkola J, Irjala K, Pasanen P, et al. Oxidized LDL and thickness of carotid intima-media are associated with coronary atherosclerosis in middle-aged men: lower levels of oxidized LDL with statin therapy. Atherosclerosis. 2001;155(2):403–12. Epub 2001/03/20. pmid:11254911.
  10. 10. Brites F, Martin M, Guillas I, Kontush A. Antioxidative activity of high-density lipoprotein (HDL): Mechanistic insights into potential clinical benefit. BBA Clin. 2017;8:66–77. Epub 2017/09/25. pmid:28936395; PubMed Central PMCID: PMC5597817.
  11. 11. Fan J, Unoki H, Iwasa S, Watanabe T. Role of endothelin-1 in atherosclerosis. Ann N Y Acad Sci. 2000;902:84–93; discussion -4. Epub 2000/06/24. pmid:10865828.
  12. 12. Kaplan M, Hamoud S, Tendler Y, Meilin E, Lazarovitch A, Nitecki S, et al. A significant correlation between C—reactive protein levels in blood monocytes derived macrophages versus content in carotid atherosclerotic lesions. J Inflamm (Lond). 2014;11(1):7. Epub 2014/03/05. pmid:24588988; PubMed Central PMCID: PMC3944991.
  13. 13. Cao JJ, Thach C, Manolio TA, Psaty BM, Kuller LH, Chaves PH, et al. C-reactive protein, carotid intima-media thickness, and incidence of ischemic stroke in the elderly: the Cardiovascular Health Study. Circulation. 2003;108(2):166–70. Epub 2003/06/25. pmid:12821545.
  14. 14. Cece H, Yazgan P, Karakas E, Karakas O, Demirkol A, Toru I, et al. Carotid intima-media thickness and paraoxonase activity in patients with ankylosing spondylitis. Clinical and investigative medicine Medecine clinique et experimentale. 2011;34(4):E225. Epub 2011/08/04. pmid:21810380.
  15. 15. Li C, Chen W, Jiang F, Simino J, Srinivasan SR, Berenson GS, et al. Genetic association and gene-smoking interaction study of carotid intima-media thickness at five GWAS-indicated genes: the Bogalusa Heart Study. Gene. 2015;562(2):226–31. Epub 2015/03/10. pmid:25746325.
  16. 16. Lopez-Mejias R, Genre F, Garcia-Bermudez M, Ubilla B, Castaneda S, Llorca J, et al. Lack of association between ABO, PPAP2B, ADAMST7, PIK3CG, and EDNRA and carotid intima-media thickness, carotid plaques, and cardiovascular disease in patients with rheumatoid arthritis. Mediators of inflammation. 2014;2014:756279. Epub 2014/05/06. pmid:24795506; PubMed Central PMCID: PMC3984817.
  17. 17. Martinez-Vila E, Paramo JA, Beloqui O, Orbe J, Irimia P, Colina I, et al. Independent association of fibrinogen with carotid intima-media thickness in asymptomatic subjects. Cerebrovascular diseases (Basel, Switzerland). 2003;16(4):356–62. Epub 2003/09/18. pmid:13130176.
  18. 18. Paramo JA, Beloqui O, Roncal C, Benito A, Orbe J. Validation of plasma fibrinogen as a marker of carotid atherosclerosis in subjects free of clinical cardiovascular disease. Haematologica. 2004;89(10):1226–31. Epub 2004/10/13. pmid:15477208.
  19. 19. Wang TJ, Nam BH, Wilson PW, Wolf PA, Levy D, Polak JF, et al. Association of C-reactive protein with carotid atherosclerosis in men and women: the Framingham Heart Study. Arteriosclerosis, thrombosis, and vascular biology. 2002;22(10):1662–7. Epub 2002/10/16. pmid:12377746.
  20. 20. Folsom AR, Pankow JS, Tracy RP, Arnett DK, Peacock JM, Hong Y, et al. Association of C-reactive protein with markers of prevalent atherosclerotic disease. The American journal of cardiology. 2001;88(2):112–7. Epub 2001/07/13. pmid:11448405.
  21. 21. Leus FR, Wittekoek ME, Prins J, Kastelein JJ, Voorbij HA. Paraoxonase gene polymorphisms are associated with carotid arterial wall thickness in subjects with familial hypercholesterolemia. Atherosclerosis. 2000;149(2):371–7. Epub 2000/03/24. pmid:10729387.
  22. 22. Jarvisalo MJ, Harmoinen A, Hakanen M, Paakkunainen U, Viikari J, Hartiala J, et al. Elevated serum C-reactive protein levels and early arterial changes in healthy children. Arteriosclerosis, thrombosis, and vascular biology. 2002;22(8):1323–8. Epub 2002/08/13. pmid:12171795.
  23. 23. Boekholdt SM, Bijsterveld NR, Moons AH, Levi M, Buller HR, Peters RJ. Genetic variation in coagulation and fibrinolytic proteins and their relation with acute myocardial infarction: a systematic review. Circulation. 2001;104(25):3063–8. Epub 2001/12/19. pmid:11748101.
  24. 24. Danesh J, Lewington S, Thompson SG, Lowe GD, Collins R, Kostis JB, et al. Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis. Jama. 2005;294(14):1799–809. Epub 2005/10/13. pmid:16219884.
  25. 25. Folsom AR, Aleksic N, Ahn C, Boerwinkle E, Wu KK. Beta-fibrinogen gene -455G/A polymorphism and coronary heart disease incidence: the Atherosclerosis Risk in Communities (ARIC) Study. Annals of epidemiology. 2001;11(3):166–70. Epub 2001/04/11. pmid:11293402.
  26. 26. Siegerink B, Rosendaal FR, Algra A. Genetic variation in fibrinogen; its relationship to fibrinogen levels and the risk of myocardial infarction and ischemic stroke. Journal of thrombosis and haemostasis: JTH. 2009;7(3):385–90. Epub 2009/01/16. pmid:19143925.
  27. 27. Lange LA, Carlson CS, Hindorff LA, Lange EM, Walston J, Durda JP, et al. Association of polymorphisms in the CRP gene with circulating C-reactive protein levels and cardiovascular events. Jama. 2006;296(22):2703–11. Epub 2006/12/14. pmid:17164456.
  28. 28. Zeng Y, Zhang L, Hu Z, Yang Q, Ma M, Liu B, et al. Fibrinogen polymorphisms associated with sporadic cerebral hemorrhage in a Chinese population. Journal of clinical neuroscience: official journal of the Neurosurgical Society of Australasia. 2012;19(5):753–6. Epub 2012/03/06. pmid:22386478.
  29. 29. Wang M, Lang X, Cui S, Zou L, Cao J, Wang S, et al. Quantitative assessment of the influence of paraoxonase 1 activity and coronary heart disease risk. DNA and cell biology. 2012;31(6):975–82. pmid:22320866.
  30. 30. Zhao Y, Ma Y, Fang Y, Liu L, Wu S, Fu D, et al. Association between PON1 activity and coronary heart disease risk: a meta-analysis based on 43 studies. Molecular genetics and metabolism. 2012;105(1):141–8. pmid:22030099.
  31. 31. Harangi M, Seres I, Magyar MT, Csipo I, Sipka S, Valikovics A, et al. Association between human paraoxonase 1 activity and intima-media thickness in subjects under 55 years of age with carotid artery disease. Cerebrovascular diseases (Basel, Switzerland). 2008;25(1–2):122–8. Epub 2007/12/13. pmid:18073465.
  32. 32. Yu JC, Pickard JD, Davenport AP. Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells. British journal of pharmacology. 1995;116(5):2441–6. Epub 1995/11/01. pmid:8581282; PubMed Central PMCID: PMC1909054.
  33. 33. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. The New England journal of medicine. 2004;351(14):1425–36. Epub 2004/10/02. pmid:15459304.
  34. 34. Hafizi S, Allen SP, Goodwin AT, Chester AH, Yacoub MH. Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ET(A) receptor and is co-mitogenic with growth factors. Atherosclerosis. 1999;146(2):351–9. pmid:10532691.
  35. 35. Li TC, Li CI, Liao LN, Liu CS, Yang CW, Lin CH, et al. Associations of EDNRA and EDN1 polymorphisms with carotid intima media thickness through interactions with gender, regular exercise, and obesity in subjects in Taiwan: Taichung Community Health Study (TCHS). BioMedicine. 2015;5(2):8. Epub 2015/06/05. pmid:26040574; PubMed Central PMCID: PMC4502041.
  36. 36. Bensen JT, Li R, Hutchinson RG, Province MA, Tyroler HA. Family history of coronary heart disease and pre-clinical carotid artery atherosclerosis in African-Americans and whites: the ARIC study: Atherosclerosis Risk in Communities. Genetic epidemiology. 1999;16(2):165–78. Epub 1999/02/25. pmid:10030399.
  37. 37. Fox CS, Polak JF, Chazaro I, Cupples A, Wolf PA, D'Agostino RA, et al. Genetic and environmental contributions to atherosclerosis phenotypes in men and women: heritability of carotid intima-media thickness in the Framingham Heart Study. Stroke. 2003;34(2):397–401. Epub 2003/02/08. pmid:12574549.
  38. 38. Juo SH, Lin HF, Rundek T, Sabala EA, Boden-Albala B, Park N, et al. Genetic and environmental contributions to carotid intima-media thickness and obesity phenotypes in the Northern Manhattan Family Study. Stroke. 2004;35(10):2243–7. Epub 2004/08/28. pmid:15331789; PubMed Central PMCID: PMC1325223.
  39. 39. Moskau S, Golla A, Grothe C, Boes M, Pohl C, Klockgether T. Heritability of carotid artery atherosclerotic lesions: an ultrasound study in 154 families. Stroke. 2005;36(1):5–8. Epub 2004/12/01. pmid:15569868.
  40. 40. Xiang AH, Azen SP, Buchanan TA, Raffel LJ, Tan S, Cheng LS, et al. Heritability of subclinical atherosclerosis in Latino families ascertained through a hypertensive parent. Arteriosclerosis, thrombosis, and vascular biology. 2002;22(5):843–8. Epub 2002/05/15. pmid:12006400.
  41. 41. Veller MG, Fisher CM, Nicolaides AN, Renton S, Geroulakos G, Stafford NJ, et al. Measurement of the ultrasonic intima-media complex thickness in normal subjects. Journal of vascular surgery. 1993;17(4):719–25. Epub 1993/04/01. pmid:8464091.
  42. 42. Ottman R. Gene-environment interaction: definitions and study designs. Prev Med. 1996;25(6):764–70. Epub 1996/11/01. pmid:8936580; PubMed Central PMCID: PMC2823480.
  43. 43. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. American journal of human genetics. 2007;81(3):559–75. Epub 2007/08/19. pmid:17701901; PubMed Central PMCID: PMC1950838.
  44. 44. Tracy RP, Psaty BM, Macy E, Bovill EG, Cushman M, Cornell ES, et al. Lifetime smoking exposure affects the association of C-reactive protein with cardiovascular disease risk factors and subclinical disease in healthy elderly subjects. Arteriosclerosis, thrombosis, and vascular biology. 1997;17(10):2167–76. Epub 1997/11/14. pmid:9351386.
  45. 45. Suk HJ, Ridker PM, Cook NR, Zee RY. Relation of polymorphism within the C-reactive protein gene and plasma CRP levels. Atherosclerosis. 2005;178(1):139–45. Epub 2004/12/09. pmid:15585211.
  46. 46. Lin CC, Wu FY, Liao LN, Li CI, Lin CH, Yang CW, et al. Association of CRP gene polymorphisms with serum CRP level and handgrip strength in community-dwelling elders in Taiwan: Taichung Community Health Study for Elders (TCHS-E). Experimental gerontology. 2014;57:141–8. Epub 2014/05/28. pmid:24862635.
  47. 47. Brull DJ, Serrano N, Zito F, Jones L, Montgomery HE, Rumley A, et al. Human CRP gene polymorphism influences CRP levels: implications for the prediction and pathogenesis of coronary heart disease. Arteriosclerosis, thrombosis, and vascular biology. 2003;23(11):2063–9. Epub 2003/07/05. pmid:12842840.
  48. 48. de Maat MP, Kastelein JJ, Jukema JW, Zwinderman AH, Jansen H, Groenemeier B, et al. -455G/A polymorphism of the beta-fibrinogen gene is associated with the progression of coronary atherosclerosis in symptomatic men: proposed role for an acute-phase reaction pattern of fibrinogen. REGRESS group. Arteriosclerosis, thrombosis, and vascular biology. 1998;18(2):265–71. Epub 1998/03/04. pmid:9484992.
  49. 49. Simmonds RE, Hermida J, Rezende SM, Lane DA. Haemostatic genetic risk factors in arterial thrombosis. Thrombosis and haemostasis. 2001;86(1):374–85. Epub 2001/08/07. pmid:11487027.
  50. 50. Voetsch B, Loscalzo J. Genetic determinants of arterial thrombosis. Arteriosclerosis, thrombosis, and vascular biology. 2004;24(2):216–29. Epub 2003/11/15. pmid:14615395.
  51. 51. van der Bom JG, de Maat MP, Bots ML, Haverkate F, de Jong PT, Hofman A, et al. Elevated plasma fibrinogen: cause or consequence of cardiovascular disease? Arteriosclerosis, thrombosis, and vascular biology. 1998;18(4):621–5. Epub 1998/04/29. pmid:9555868.
  52. 52. Rallidis LS, Gialeraki A, Fountoulaki K, Politou M, Sourides V, Travlou A, et al. G-455A polymorphism of beta-fibrinogen gene and the risk of premature myocardial infarction in Greece. Thrombosis research. 2010;125(1):34–7. Epub 2009/05/05. pmid:19409601.
  53. 53. Theodoraki EV, Nikopensius T, Suhorutsenko J, Peppes V, Fili P, Kolovou G, et al. Fibrinogen beta variants confer protection against coronary artery disease in a Greek case-control study. BMC medical genetics. 2010;11:28. Epub 2010/02/20. pmid:20167083; PubMed Central PMCID: PMC2834581.
  54. 54. Dursun B, Dursun E, Suleymanlar G, Ozben B, Capraz I, Apaydin A, et al. Carotid artery intima-media thickness correlates with oxidative stress in chronic haemodialysis patients with accelerated atherosclerosis. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association—European Renal Association. 2008;23(5):1697–703. Epub 2008/01/05. pmid:18174265.
  55. 55. Labib HA, Etewa RL, Gaber OA, Atfy M, Mostafa TM, Barsoum I. Paraoxonase-1 and oxidative status in common Mediterranean beta-thalassaemia mutations trait, and their relations to atherosclerosis. Journal of clinical pathology. 2011;64(5):437–42. Epub 2011/03/24. pmid:21427447.
  56. 56. Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E, et al. Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype? Arteriosclerosis, thrombosis, and vascular biology. 2001;21(9):1451–7. Epub 2001/09/15. pmid:11557671.
  57. 57. Srinivasan SR, Li S, Chen W, Tang R, Bond MG, Boerwinkle E, et al. Q192R polymorphism of the paraoxanase 1 gene and its association with serum lipoprotein variables and carotid artery intima-media thickness in young adults from a biracial community. The Bogalusa Heart Study. Atherosclerosis. 2004;177(1):167–74. Epub 2004/10/19. pmid:15488880.
  58. 58. Karvonen J, Kauma H, Paivansalo M, Kesaniemi YA. Paraoxonase-1 gene Leu-Met55 and Gln-Arg192 polymorphisms are not associated with carotid artery atherosclerosis in a population-based cohort. European journal of cardiovascular prevention and rehabilitation: official journal of the European Society of Cardiology, Working Groups on Epidemiology & Prevention and Cardiac Rehabilitation and Exercise Physiology. 2004;11(6):511–2. Epub 2004/12/08. pmid:15580063.
  59. 59. Scherrer DZ, Zago VH, Vieira IC, Parra ES, Panzoldo NB, Alexandre F, et al. p.Q192R SNP of PON1 seems not to be Associated with Carotid Atherosclerosis Risk Factors in an Asymptomatic and Normolipidemic Brazilian Population Sample. Arquivos brasileiros de cardiologia. 2015;105(1):45–52. Epub 2015/06/04. pmid:26039660; PubMed Central PMCID: PMC4523287.
  60. 60. Yasuda H, Kamide K, Takiuchi S, Matayoshi T, Hanada H, Kada A, et al. Association of single nucleotide polymorphisms in endothelin family genes with the progression of atherosclerosis in patients with essential hypertension. Journal of human hypertension. 2007;21(11):883–92. Epub 2007/05/26. pmid:17525706.