Experimental and Molecular Pathology 96 (2014) 405–410

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Experimental and Molecular Pathology

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TheHIF1A rs2057482 polymorphism is associatedwith risk of developingpremature coronary artery disease and with some metabolic andcardiovascular risk factors. The Genetics of Atherosclerotic Disease (GEA)Mexican Study

Alberto López-Reyes a, José Manuel Rodríguez-Pérez b, Javier Fernández-Torres a, Nancy Martínez-Rodríguez b,Nonanzit Pérez-Hernández b, Arturo Javier Fuentes-Gómez a, Carlos Alberto Aguilar-González a,Edith Álvarez-León b, Carlos Posadas-Romero c, Teresa Villarreal-Molina d,Carlos Pineda a, Gilberto Vargas-Alarcón b,⁎a Molecular Synovioanalisis Laboratory, Instituto Nacional de Rehabilitación, Mexico City, Mexicob Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexicoc Department of Endocrinology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexicod Genomic Laboratory, Instituto Nacional de Medicina Genómica, Mexico City, Mexico

⁎ Corresponding author at: Department of Moleculde Cardiología Ignacio Chávez, Juan Badiano 1, SeccióCity, Mexico.

E-mail address: gvargas63@yahoo.com (G. Vargas-Ala

http://dx.doi.org/10.1016/j.yexmp.2014.04.0100014-4800/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 April 2014Available online 24 April 2014

Keywords:Cardiovascular risk factorsCoronary artery diseaseHypoxiaHypoxia-inducible factor 1-alphaIschemiaPolymorphisms

The aim of the present studywas to establish the role ofHIF1A gene polymorphisms in the risk of developing pre-mature coronary artery disease (CAD) in a well-characterized clinical cohort. Three polymorphisms in HIF1A(rs11549465, rs11549467, rs2057482) gene were genotyped in 949 patients with premature CAD, and 676healthy controls (with negative calcium score by computed tomography). Under a dominant model adjustedfor age, visceral to subcutaneous adipose tissue (VAT/SAT) ratio, hypertension, type 2 diabetes mellitus(T2DM), HDL-C levels, hypercholesterolemia and hypertriglyceridemia, the rs2057482 T allele was associatedwith decreased risk of premature CAD when compared to healthy controls (OR = 0.616, Pdom = 0.020). The ef-fect of the studied polymorphisms on variousmetabolic parameters and cardiovascular risk factorswas explored.In this analysis, the rs2057482 T allele was associated with decreased risk of obesity, central obesity, hyperten-sion, hypercholesterolemia, hypertriglyceridemia and increased risk of T2DM. Under a dominantmodel adjustedby age, the HIF1A rs2057482 T polymorphism was associated with high VAT/SAT ratio (P = 0.009) and HDL-Clevels (P = 0.04) in healthy controls. The results suggest that HIF1A rs2057482 polymorphism is involved inthe risk of developing CAD and is associated with some metabolic parameters and cardiovascular risk factors.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Coronary artery disease (CAD) is a complex, multifactorial, andpolygenic disorder caused by an excessive inflammatory responseto various forms of injury, resulting in endothelial dysfunction inthe arterial walls leading to accelerated atherosclerosis (Ross,2009). Multiple genetic factors may work in conjunction with envi-ronmental factors to confer susceptibility to CAD. It has been sug-gested that acute ischemic injury due to loss of oxygen homeostasisis present in this pathology (Garcia-Moll, 2005; Kumar and Cannon,2009; Sami andWilerson, 2010). An imbalance between oxygen supply

ar Biology, Instituto Nacionaln XVI, 14080, Tlalpan, Mexico

rcón).

and demand occurs as a result of reduced blood flow brought about byatherosclerotic plaque formation and inflammatory processes takingplace within the vascular endothelium (Lusis, 2000; Sluimer andDaemen, 2009).

Hypoxia regulates different processes in the cardiovascular sys-tem, such as angiogenesis, vascular smooth muscle contraction, ven-tricular remodeling, erythropoiesis, and cellular oxidative stressgenerated by free radicals derived from macrophages and endothelialcells (Dewhirst, 2009). As a consequence of oxidative stress in hypoxicconditions, cells respond through adaptive and repair mechanismsme-diated by the hypoxia-inducible factor-1 alpha (HIF-1α) (Hlatky et al.,2007).

HIF-1α is a transcriptional factor encoded by theHIF1A gene locatedon chromosome 14 (14q23.2) and plays a critical role in the regulationof different cellular processes involved in the preservation of oxygenhomeostasis. HIF-1α modulates the expression of approximately 100

406 A. López-Reyes et al. / Experimental and Molecular Pathology 96 (2014) 405–410

target genes, among which vascular endothelial growth factor (VEGF),nitric oxide synthase (NOS2), and genes codifying several antioxidantenzymes stand out (Görlach, 2009; Jong and Goo, 2011; Kolyada et al.,2009; Vogiatzi et al., 2009). Furthermore, HIF-1α promotes cellularadaptation responses in hypoxic environments through mechanismslike angiogenesis, proliferation, differentiation, and tissue remodeling(Semenza, 2000). A previous study found that the HIF-1α expressionwas significantly stronger in patients with CAD than controls, and thelevel of HIF-1α was associated with severity of atherosclerosis andhigher level of coronary collaterals (Chen et al., 2008, 2009). HIF-1αtriggers VEGF expression, which increases plaque capillary density andextends the depth of neo-vascularization to the intima (Celletti et al.,2001), resulting in mural hemorrhage, plaque rupture, and inducesacute coronary syndrome (Shah, 2003). Recently, two single nucleotidepolymorphisms (SNPs) of the HIF1A gene, HIFA1 C1772T (rs11549465)and G1790A (rs11549467) were identified (Zhao et al., 2009). TheseSNPs are thought to be important for protein stability because theyare located within the oxygen-dependent degradation domain, veryclose to proline 564 (P564) and proline 402(P402), key residues foroxygen-dependent degradation of the HIF.

The role of genetic predisposition in neoplasic, degenerative, andinflammatory disorders has been well described (Sartori-Cintra et al.,2012; Tang and Yu, 2013; Tzouvelekis et al., 2012; Wu et al., 2012).Moreover, several polymorphisms of the genes that encode HIF1Ahave been studied in cardiovascular diseases. Results however havebeen inconsistent and controversial, revealing both positive and nega-tive associations (Alidoosti et al., 2010; Bahadori et al., 2010; Torreset al., 2010).

The aim of the present study was to establish the role of the HIF1Agene polymorphisms in the risk of developing premature CAD in awell-characterized clinical cohort of Mexican Mestizo patients.

Materials and methods

Subjects

All participants provided written informed consent. The study com-plies with the Declaration of Helsinki and was approved by the EthicsCommittee of the Instituto Nacional de Cardiología Ignacio Chávez(INCICH). The primary aim of the Genetics of Atherosclerotic Disease(GEA) Study is to investigate genetic factors associated with prematureCAD, and other coronary risk factors in theMexican population. All GEAparticipants are unrelated and of self-reported Mexican-Mestizo ances-try (three generations). A Mexican Mestizo is defined as someone bornin Mexico who is a descendant of the original autochthonous inhabi-tants of the region, and of individuals of Caucasian (predominantlySpaniards) and/or African origin, who came to the American continentduring the sixteenth century. A total of 1625 individuals were includedin the study, 949 diagnosed with premature CAD and 676 apparentlyhealthy controls. Premature CAD was defined as history of myocardialinfarction, angioplasty, revascularization surgery or coronary stenosisN50% on angiography, diagnosed before age 55 in men and beforeage 65 in women. Controls were apparently healthy asymptomaticindividuals without family history of premature CAD, recruitedfrom blood bank donors and through brochures posted in Social Ser-vices centers. Exclusion criteria for controls included congestiveheart failure, liver, renal, thyroid or oncological disease. The selec-tion of the patients and controls of the GEA study was described ina previous study (Villarreal-Molina et al., 2012). Demographic, clin-ical, anthropometric, biochemical parameter and cardiovascularrisk factors were evaluated in both patients and controls. Anthropomet-ric parameters were measured by trained personnel and included waistcircumference and body mass index (BMI) calculated as weight inkilograms divided by height in square meters. Blood pressure wasmeasured three different times by sphygmomannometry and the aver-age of the last twomeasurements was obtained. Obesity was defined as

BMI ≥30 kg/m2. Central obesity, hypoalphalipoproteinemia, hypertri-glyceridemia, andmetabolic syndromewere defined using Adult Treat-ment Panel III (ATP-III) criteria (2002). Hypercholesterolemia wasdefined as total cholesterol (TC) levels ≥200 mg/dL. Hypertensionwas defined as systolic blood pressure ≥140 mm Hg and/or diastolicblood pressure≥90mmHg or the use of oral antihypertensive therapy.Type 2 diabetesmellitus (T2DM)was diagnosed according to theWorldHealth Organization criteria.

Computed tomography of the chest and abdomen

Computed tomography of the chest and abdomen was performedusing a 64-channel multi-detector helical computed tomographysystem (Somatom Sensation, Siemens) and interpreted by experi-enced radiologists. Scans were read to assess and quantify thefollowing: 1) coronary artery calcification (CAC) score using theAgatston method (Mautner et al., 1994); 2) total abdominal, subcu-taneous and visceral adipose tissue areas as described by Kvistet al. (1988) in order to calculate visceral to subcutaneous adiposetissue ratio (VAT/SAT); and 3) hepatic to splenic attenuation ratio(LSAR) as described by Longo et al. (1993). The tomography was per-formed in all the patients and in a group of 949 healthy controls.However, 273 of apparently healthy individuals presented CAC pos-itive (CAC score N0) and were considered as individuals withsubclinical atherosclerosis. These individuals were not consideredfor the analysis. The control group only included individuals withnegative CAC (n = 676).

Genotyping of HIF1A

Genotyping was performed on DNA obtained from peripheralblood mononuclear cells (Lahiry and Nurnberger, 1991). TheHIF1A (rs11549465, rs11549467, and rs2057482) polymorphismswere genotyped using 5′ exonuclease TaqMan genotyping assayson an ABI Prism 7900HT Fast Real-Time PCR system, according tomanufacturer's instructions (Applied Biosystems, Foster City, USA)(Table 1 displays single nucleotide polymorphism—SNPs—studied).

Statistical analysis

All calculations were performed using SPSS version 18.0 (SPSS,Chicago, IL) statistical package. Means ± SD and frequencies of base-line characteristics were calculated. Chi-square tests were used tocompare frequencies and ANOVA and Students t-test were used tocompare means. ANOVA was used to determine associations be-tween the polymorphisms and metabolic variables, adjusting forage and gender. Logistic regression analysis was used to test for asso-ciations of polymorphisms with CAD under inheritance models. Themost appropriate inheritance model was selected based on Akaikeinformation criteria (AIC). Multiple logistic models were constructedin order to identify the variables that explain better risk of develop-ing premature CAD between the studied groups. Models were con-structed including one variable at time, and final models includedvariables with biological relevance or with statistical significance orboth. Confounding bias was accepted when changes in estimatedodds ratios (ORs) were equal or larger than 10%. When a principal ef-fect model was reached, effect modification was also tested and in-teractions terms were constructed between the polymorphismsand different variables; the terms were included in the modelwhen the significance of the p-value was larger or equal to 0.20.Hosmer–Lemeshow Goodness of Fit test was performed for eachmultiple logistic model. Genotype frequencies did not show deviationfrom the Hardy–Weinberg equilibrium (HWE) (P N 0.05). Pairwiselinkage disequilibrium (LD, D′) estimations between polymorphismsand haplotype reconstruction were performed with Haploview version

Table 1HIF1A polymorphisms studied.

No.a dbSNPb Chr. Allele change(Minor allele)

Residue change Chr. position (bp) Location in gene

1 rs11549465 14 CCA-(T)CA P [Pro]-S [Ser] 62207557 Exon-92 rs11549467 14 GCA-(A)CA A [Ala]-T [Thr] 62207575 Exon-93 rs2057482 14 (T)-C —— 62213848 UTR-3

a Order of the polymorphisms is according to the chromosomal position.b SNP ID in database dbSNP.

407A. López-Reyes et al. / Experimental and Molecular Pathology 96 (2014) 405–410

4.1 (Broad Institute of the Massachusetts Institute of Technology andHarvard University, Cambridge, MA, USA).

Functional prediction analysis

We predicted the potential effect of the HIF1A polymorphism as-sociated with premature CAD in our population using bioinformaticstools, including FastSNP (Yuan et al., 2006), SplicePort: An Interac-tive Splice Site Analysis Tool (http://www.spliceport.cs.umd.edu/SplicingAnalyser2.html), SNPs3D (http://www.snps3d.org/), PESX:Putative Exonic Splicing enhancers/Silencers (http://cubweb.biology.columbia.edu/pesx/), and ESEfinder release 3.0 (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home).

Results

General characteristics of the population are shown in Tables 2and 3.

Association of polymorphisms with premature CAD

Observed and expected frequencies in the polymorphic sites were inHWE. The distribution of HIF1A (rs11549465, rs11549467) polymor-phismwas similar in patientswith premature CAD, and healthy controlsin all the models analyzed (data not shown). However, the distributionof HIF1A rs2057482 was different in the studied groups. Under a domi-nant model adjusted for age, VAT/SAT ratio, hypertension, T2DM, HDL-

Table 2Clinical characteristics in premature CAD patients and healthy controls.

Controls(n = 676)

Premature CAD(n = 949)

P value⁎

Age (years) 52.1 ± 9.2 53.6 ± 7.3 b0.001Gender (male%/female%) 38.8/61.2 80.0/20.0 b0.001BMI (kg/m2) 28.3 ± 4.5 36.9 ± 5.0 b0.001Obesity (%) 29.9 36.0 0.002Waist circumference (cm) 93.2 ± 11.7 98.1 ± 11.0 b0.001Central obesity (%) 79.1 83.1 0.041Total abdominal fat (cm2) 443.8 ± 147.1 440.2 ± 142.8 0.370Subcutaneous abdominal fat (cm2) 295.6 ± 114.0 261.8 ± 100.3 b0.001Visceral abdominal fat (cm2) 148.2 ± 64.3 178.4 ± 72.6 b0.001VAT/SAT ratio 0.609 ± 0.319 0.702 ± 0.350 b0.001Hypertension (%) 20.1 68.0 b0.001Diastolic blood pressure (mm Hg) 72.5 ± 9.2 73.1 ± 10.3 0.111Systolic blood pressure (mm Hg) 117.1 ± 17.0 119.1 ± 18.9 0.364Heart rate (bpm) 65.3 ± 9.2 65.4 ± 11.3 0.760Antihypertensive treatment (%) 11.2 75.0 b0.001Smoking habitsCurrent smokers (%) 23.0 12.4 b0.001Former smokers (%) 32.1 62.4 b0.001

Consumption of alcohol≥6 g/day (%) 73.0 55.0 b0.001Former alcohol (%) 7.3 22.0 b0.001

The variables are expressed as the mean ± standard deviation (SD).CAD; coronary artery disease, BMI = body mass index. VAT/SAT ratio: visceral tosubcutaneous adipose tissue ratio.⁎ P values were estimated using the MannWhitneyU-test for continuous variables and

Pearson's Chi-square test for categorical values.

C, hypercholesterolemia, and hypertriglyceridemia, the HIF1Ars2057482 T allele was associated with decreased risk of prematureCAD when compared to healthy controls (OR = 0.616, 95% CI =0.409–0.928, Pdom = 0.020) (Table 4). The statistical power estimatedwith QUANTO software (http://hydra.usc.edu/GxE/) to detect asso-ciation between premature CAD and controls was 0.9347 for HIF1Ars2057482.

Association of the polymorphisms with cardiovascular risk factors

The association of the studied polymorphisms with cardiovascu-lar risk factors was evaluated comparing CAD patients and healthycontrols. In this analysis adjusted by age, the HIF1A rs2057482 T al-lele was associated with decreased risk of obesity (OR = 0.562,95% CI: 0.424–0.745, P b 0.001), central obesity (OR = 0.752, 95%CI: 0.583–0.970, P = 0.028), hypertension (OR = 0.115, 95% CI:0.091–0.146, P b 0.001), hypercholesterolemia (OR = 0.497, 95%CI: 0.400–0.617, P b 0.001), and hypertriglyceridemia (OR = 0.566,95% CI: 0.463–0.692, P b 0.001) and increased risk of T2DM (OR =4.766, 95% CI: 3.557–6.386, P b 0.001) (Table 5).

Association of the polymorphisms with metabolic parameters

The effect of the studied polymorphisms on various metabolicparameters was explored separately in controls (CAC score = 0),and premature CAD. Under a dominant model adjusted by age, theHIF1A rs2057482 T polymorphism was associated with high VAT/SATratio (OR = 0.474, 95% CI: 0.270–0.832, P = 0.009) and high HDL-C

Table 3Biochemical parameters in premature CAD patients and healthy controls.

Controls(n = 676)

Premature CAD(n = 949)

P value ⁎

TC ≥200 mg/dL (n%) 39.1 24.4 b0.001Total cholesterol (mg/dL) 190.6 ± 35.9 168.7 ± 48.1 b0.001HDL-C (mg/dL) 48.1 ± 14.2 40.1 ± 10.6 b0.001LDL-C (md/dL) 116.1 ± 31.5 97.7 ± 39.5 b0.001ApoA1 (md/dL) 139.1 ± 41.2 120.5 ± 26.9 b0.001ApoB (mg/dL) 89.2 ± 27.6 83.8 ± 31.5 b0.001Hypertriglyceridemia (%) 45.0 58.5 b0.001Triglycerides (mg/dL) 165.6 ± 106.0 192.5 ± 122.5 b0.001Statin and/or fibrate treatment (%) 9.0 67.3 b0.001T2DM (%) 9.6 34.1 b0.001Glucose (mg/dL)⁎ 96.1 ± 30.2 112.0 ± 44.4 b0.001HOMA-IR⁎ 5.1 ± 8.9 6.6 ± 5.7 b0.001Antidiabetic treatment 5.8 27.1 b0.001Hepatic steatosis (%) 32.0 30.0 0.403Alanine transaminase (IU/L) 27.6 ± 11.2 28.0 ± 10.9 0.011Aspartate transaminase (IU/L) 27.6 ± 17.1 28.9 ± 17.5 0.053Alkaline phosphatase (IU/L) 84.1 ± 23.9 80.0 ± 25.6 b0.001Gamma-glutamyl transpeptidase (IU/L) 34.7 ± 31.6 44.7 ± 43.1 b0.001Serum uric acid (mg/dL) 5.5 ± 1.4 6.4 ± 1.6 b0.001Blood creatinine (mg/dL) 0.839 ± 0.198 0.974 ± 0.244 b0.001

The variables are expressed as the mean ± standard deviation (SD).CAD: coronary artery disease; T2DM: type 2 diabetes mellitus; HOMA-RI: homeostaticmodel of insulin resistance.⁎ P values were estimated using theMannWhitney U-test for continuous variables and

Pearson's Chi-square test for categorical values.

Table 4Association of the HIF1A rs2057482 polymorphism with premature CAD.

HIFA1rs2057482

Genotype frequency (%) MAF Model OR (95%CI) *P value

C/C T/C T/T

Controls(n = 676)

547 (0.810) 120 (0.177) 9 (0.013) 0.102 Codominant 1Codominant 2DominantRecessiveHeterozygousAdditive

0.605 (0.398–0.920)0.428 (0.072–2.541)0.616 (0.409–0.928)0.472 (0.091–3.034)0.610 (0.401–0.926)0.620 (0.422–0.911)

0.0190.3510.0200.5260.0200.015

Premature CAD(n = 949)

809 (0.853) 134 (0.141) 6 (0.006) 0.077

Association was tested using logistic regression adjusting by age, VAT/SAT ratio, hypertension, T2DM, HDL-C levels, hypercholesterolemia and hypertriglyceridemia. R2 = 0.5171.CAD: coronary artery disease; MAF: minor allele frequency.OR = odds ratio, CI = confidence interval. In bold is showed the dominant model.

408 A. López-Reyes et al. / Experimental and Molecular Pathology 96 (2014) 405–410

levels in healthy controls (OR = 0.986, 95% CI: 0.973–0.999, P = 0.04)(Table 6).

Haplotype analysis and SNP functional prediction

The HIF1A polymorphisms (rs11549465 and rs2057482) were inhigh linkage disequilibrium (D′ N 0.8 and r2 N 0.9); however, the dis-tribution of the haplotypes was similar in patients with prematureCAD and healthy controls (data not shown).

Based on SNP functional prediction software, the HIF1A rs2057482polymorphism seems to be functional. This SNP is located in theUTR-3′ region in a potential splicing regulatory sequence with highaffinity for the SRSC35 protein.

Discussion

Cardiovascular diseases, notably, coronary artery disease, haveemerged as one of the major causes of death worldwide (Murrayand Lopez, 1997). A number of risk factors have been identified.Recently, attention has been given to genes implicated in oxidativestress and hypoxic states that generate an imbalance between oxygensupply and demand occurring as a result of atherosclerotic plaqueformation and the inflammatory processes taking place within thevascular endothelium (Sluimer and Daemen, 2009).

In the present study, three polymorphisms of HIF1A (rs11549465,rs11549467, and rs2057482) gene were analyzed in order to establishits role as susceptibility markers to develop premature CAD, highlight-ing that our control group only included individuals without coronaryartery calcification. This represents an important difference with previ-ously reported studies. In our study, the HIF1A rs2057482 polymor-phism was associated with decreased risk of developing prematureCAD. This polymorphism has been studied in several diseases oftenwith conflicting results. In a previous study, Hlatky et al. (2007) deter-mined seven polymorphisms (rs11549465, rs4902080, rs2057482,rs41362550, rs10873142, rs41508050, and rs11549467) of the HIF1Agene in patients with myocardial infarction and stable angina. They

Table 5Association of the rs2057482 HIF1A polymorphism with cardiovascular risk factors.

MAFPremature CAD

Obesity (n = 541) 0.008Central obesity (n = 1323) 0.009Hypertension (n = 780) 0.062Hypercholesterolemia (n = 494) 0.000Hypertriglyceridemia (n = 856) 0.054T2DM (n = 390) 0.092Hepatic steatosis (n = 475) 0.148

All associations were tested using logistic regression adjusting by age.CAD: coronary artery disease; OR: odds ratio; CI: confidence interval; T2DM: type 2 diabetes m

reported that the rs11549465 TT, rs1087314 CC and rs41508050 CTgenotypes were more common in patients who presented with sta-ble angina rather than myocardial infarction. For the rs11549465polymorphism, the minor allele Twas more prevalent in stable angi-na, and the C allele was more prevalent in myocardial infarction. Inour study, we analyzed three of the polymorphisms studied byHlatky et al. (2007) (rs11549465, rs2057482, and rs11549467) ob-serving that the HIF1A rs2057482 T allele was associated with a de-creased risk of premature CAD when compared to healthy controls.The functional prediction software used here predicted that HIF1Ars2057482 polymorphism is functional. This SNP is located in theUTR-3′ region in a potential splicing regulatory sequence with highaffinity for the SRSC35 protein. The results obtained using the infor-matics software agree with the genetic results and confirm the func-tional effect of the HIF1A rs2057482 polymorphisms. In a previousstudy, a significantly increased risk of cervical cancer was associatedwith the CC genotype of rs2057482 polymorphism. Moreover, thecarriers of CT + TT genotypes had significantly decreased HIF-1mRNA expression levels compared to those with CC genotype (Fuet al., 2014). As was commented, the HIF-1α triggers VEGF expression,which increases plaque capillary density and extends the depth of neo-vascularization to the intima (Celletti et al., 2001), resulting in muralhemorrhage and plaque rupture, and inducing acute coronary syn-drome (Shah, 2003). Considering this effect, we hypothesized that theHIF1A rs2057482 T allele may weaken the ability of HIF-1α transcrip-tional activation ability to affect the expression of VEGF, resulting in re-ducing plaque capillary density and plaque rupture and decreasing theincidence of CAD. The different results observed in other polymor-phisms could be due to ethnic differences and the diseases that werecompared. While Hlatky et al. (2007) compared myocardial infarctionand stable angina patients; we compared patients with prematureCAD, and healthy controls. Nevertheless, it is important to considerthat the role of HIF1A gene polymorphisms as a marker for several dis-eases remains controversial. Recently, Bahadori et al. (2010) investigat-ed the relationship between genotypes of two HIF1A polymorphisms(rs11549465, and rs11549467) and peripheral artery disease (PAD) in

MAFControls

OR (95% CI) p

0.190 0.562 (0.424–0.745) b0.0010.150 0.752 (0.583–0.970) 0.0280.074 0.115 (0.091–0.146) b0.0010.172 0.497 (0.400–0.617) b0.0010.066 0.566 (0.463–0.692) b0.0010.000 4.766 (3.557–6.386) b0.0010.196 0.915 (0.735–1.140) 0.430

ellitus; MAF: minor allele frequency.

Table 6Association of the rs2057482 HIF1A polymorphism with quantitative metabolic parameters in healthy controls.

C/CGenotype

T/C + T/TGenotypes

OR (95% CI) P

BMI (kg/m2) 28.3 ± 4.6 28.3 ± 4.3 1.001 (0.960–1.044) 0.963Waist circumference (cm) 93.1 ± 11.4 93.5 ± 13.2 0.997 (0.981–1.014) 0.756Total abdominal fat (cm2) 439.7 ± 143.5 461.5 ± 161.3 0.999 (0.998–1.000) 0.144Subcutaneous abdominal fat (cm2) 293.2 ± 114.1 305.9 ± 113.4 0.999 (0.997–1.001) 0.268Visceral abdominal fat (cm2) 146.6 ± 60.5 155.5 ± 78.7 0.998 (0.995–1.001) 0.171VAT/SAT ratio 0.593 ± 0.315 0.677 ± 0.330 0.474 (0.270–0.832) 0.009Total cholesterol (mg/dL) 190.5 ± 36.0 191.3 ± 35.7 0.999 (0.994–1.005) 0.809HDL-C (mg/dL) 47.5 ± 13.9 50.4 ± 14.8 0.986 (0.973–0.999) 0.040LDL-C (md/dL) 115.9 ± 31.4 116.8 ± 31.8 0.999 (0.993–1.005) 0.774ApoA1 (md/dL) 138.6 ± 42.2 141.1 ± 37.1 0.998 (0.994–1.003) 0.555ApoB (mg/dL) 89.4 ± 27.8 88.4 ± 27.2 1.001 (0.994–1.008) 0.701Triglycerides (mg/dL) 169.1 ± 112.7 150.4 ± 69.7 1.002 (0.999–1.005) 0.059Glucose (mg/dL)* 96.2 ± 29.1 95.7 ± 34.7 0.999 (0.993–1.006) 0.870HOMA-IR* 5.1 ± 9.7 4.8 ± 3.5 1.004 (0.975–1.033) 0.784Serum uric acid (mg/dL) 5.5 ± 1.4 5.4 ± 1.4 1.029 (0.894–1.184) 0.690Blood creatinine (mg/dL) 0.835 ± 0.198 0.856 ± 0.197 0.599 (0.224–1.601) 0.307

All associations were tested using logistic regression adjusted by age.OR = odds ratio, CI = confidence interval.VAT/SAT ratio: visceral to subcutaneous adipose tissue ratio. In bold are showed the significant associations.

409A. López-Reyes et al. / Experimental and Molecular Pathology 96 (2014) 405–410

an Austrian population. In their study, no significant associations werefound between the two polymorphic sites and PAD.

Novel in the present study is the association between HIF1Ars2057482 polymorphism and different cardiovascular risk factors.In our study, the T allele was associated with decreased risk of obesity,central obesity, hypertension, hypercholesterolemia, and hypertriglyc-eridemia. On the other hand, individuals with TC + TT genotypespresented high VAS/SAT ratio and increased levels of HDL-C than indi-viduals with CC genotype. This is the first report on the associations ofthis polymorphismwith risk factors; hence, confirmation of these find-ings by other groups is desirable.

Study limitations need to be addressed. This study only included theanalysis of three polymorphisms of the HIF1A gene. Considering thatthis is the first work to report an association of HIF1A polymorphismswithmetabolic parameters and cardiovascular risk factors in prematureCAD, replication in another group of patients is necessary. The predictedfunctional consequences of the rs2057482 polymorphism, using infor-matics tools, need to be tested experimentally.

In summary, our data suggest that the contribution genetic of HIF1Ars2057482 polymorphism plays an important role in the risk of devel-oping premature CAD. TheHIF1A rs2057482 polymorphismalsowas as-sociated with metabolic parameters and cardiovascular risk factors.Additional studies in populations with different ethnic backgroundcould help to further define the precise role of these genetic markersas risk factors for the development of CAD.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported in part by grants from the ConsejoNacional de Ciencia y Tecnología (Project number 156911), MexicoCity, Mexico. We thank Vicente Morales Oyaverde for their supportin proofreading the manuscript.

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