genetic variation in liver x receptor alpha and risk of...

Genetic Variation in Liver X Receptor Alpha and Risk ofIschemic Vascular Disease in the General Population

Stefan Stender, Ruth Frikke-Schmidt, Aristomenis Anestis, Dimitris Kardassis, Amar A. Sethi,Børge G. Nordestgaard, Anne Tybjærg-Hansen

Objective—Although animal studies indicate that liver X receptor alpha (LXR�) might influence risk of atherosclerosis,data in humans remain scarce. We tested the hypothesis that genetic variation in LXR� associates with risk of ischemicvascular disease and/or plasma lipid and lipoprotein levels in the general population.

Methods and Results—We studied 10,281 white persons of Danish ancestry from a general population cohort, including1,986 in whom ischemic heart disease (IHD) developed, and 989 in whom ischemic cerebrovascular disease developed.We examined another 51,429 white persons of Danish ancestry from a general population study, including 3,789 withIHD. We genotyped 10 genetic variants identified by resequencing LXR�. Homozygosity for �840AA/�115AA(�2.7%) predicted hazard ratios of 1.3 (95% confidence interval, 1.0–1.7) for IHD, 1.6 (1.2–2.2) formyocardial infarction, and 1.7 (1.3–2.4) for ischemic cerebrovascular disease. The corresponding odds ratios in thesecond cohort were 1.1 (0.9–1.4) for IHD and 1.5 (1.1–2.0) for myocardial infarction. In the combined studies, oddsratios were 1.2 (1.0–1.4) for IHD and 1.5 (1.2–1.9) for myocardial infarction. Homozygosity for �840AA/�115AA didnot associate with lipid or lipoprotein levels. LXR� �1830T�C (tagging the haplotype �1830C/�840A/�115A, allr2�0.97) associated with 91% increased transcriptional activity.

Conclusion—This study suggests that functional genetic variation in LXR� predicts risk of ischemic vascular disease inthe general population. (Arterioscler Thromb Vasc Biol. 2011;31:00-00.)

Key Words: coronary heart disease � epidemiology � gene mutations � ischemic heart disease � stroke

Liver X receptor alpha (LXR�) is a nuclear receptor thatplays a central role in both lipid metabolism and inflam-

mation.1 LXR� is activated by increased intracellular levelsof oxysterols, a breakdown product of cholesterol, andorchestrates the removal of cholesterol from peripheral tis-sues by regulating genes involved in reverse cholesteroltransport.2 Other downstream effects of LXR� activationinclude increases in catabolism, biliary excretion, and con-version to fatty acids of cholesterol.3 Furthermore, LXR�modulates the immune response via an antiinflammatoryeffect on macrophages.4 Because plasma cholesterol levelsas well as inflammatory processes are key players in thedevelopment of the atherosclerotic plaque, a possible roleof LXR� in ischemic cardiovascular and ischemic cere-brovascular disease (ICVD) has been the focus of intenseresearch.1 Several studies have demonstrated that LXR�agonists attenuate the development of atherosclerosis inmice and protect against neuronal damage in animalmodels of ischemic stroke (IS).5,6 Further underpinning apossible role of LXR� in ischemic vascular disease is the

observation that LXR� knockout mice display an in-creased susceptibility to atherosclerosis and IS.6,7 How-ever, data pertaining to LXR� biology in humans remainscarce.

We hypothesized that genetic variation in LXR� mightinfluence risk of ischemic vascular disease and/or plasmalipid and lipoprotein levels in the general population. To testthis hypothesis, we resequenced the LXR� gene in 190individuals and genotyped the identified genetic variants inthe Copenhagen City Heart Study (CCHS), a study of 10,281white individuals of Danish ancestry from the general popu-lation followed for up to 33 years. We subsequently exam-ined risk of ischemic heart disease (IHD), myocardialinfarction (MI), ICVD, and IS, plasma lipid and lipopro-tein levels, and biochemical markers of inflammation as afunction of LXR� genotype. Any genetic associationidentified with risk of ischemic vascular disease wasvalidated in the Copenhagen General Population Study(CGPS), a study comprising 51,429 white individuals ofDanish ancestry from the general population.

Received on: January 20, 2011; final version accepted on: August 25, 2011.From the Department of Clinical Biochemistry (S.S., R.F.-S., A.A.S., A.T.-H.), Rigshospitalet, Copenhagen University Hospitals and Faculty of Health

Sciences, University of Copenhagen, Denmark; Department of Biochemistry (A.A., D.K.), University of Crete Medical School, Heraklion, Greece; PacificBiometrics, Inc (A.A.S.), Seattle, WA; Department of Clinical Biochemistry (A.A.S., B.G.N.) and The Copenhagen General Population Study (R.F.-S.,B.G.N., A.T.-H.), Herlev Hospital, and The Copenhagen City Heart Study (B.G.N., A.T.H.), Bispebjerg Hospital, Copenhagen University Hospitals andFaculty of Health Sciences, University of Copenhagen, Denmark.

Correspondence to Anne Tybjærg-Hansen, Department of Clinical Biochemistry KB3011, Section for Molecular Genetics, Rigshospitalet, CopenhagenUniversity Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail [email protected]

© 2011 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.111.223867

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MethodsStudies were approved by institutional review boards and Danishethical committees and conducted according to the Declaration ofHelsinki. Written informed consent was obtained from all partici-pants. All participants were white and of Danish descent: Thenational Danish Central Person Register registers date and place ofbirth and death, gender and descent, and all immigration/emigrationto/from Denmark of all inhabitants in Denmark. Danish descent isdefined as an individual born in Denmark with Danish citizenshipwith both parents also born in Denmark with Danish citizenship.Because there was virtually no immigration before the 1990s withthe exception from nearby Scandinavian countries, this is an ethni-cally extremely homogeneous population. Furthermore, at the timeof examination, the examiner registered the ethnicity of the partici-pants in both studies.

Study Cohorts

The CCHSThe CCHS8–10 is a study of the Danish general population initiatedfrom 1976 to 1978 with follow-up examinations in 1981 to 1984,1991 to 1994, and 2001 to 2003. Individuals were randomly selectedto represent the Danish general population aged 20 to 80� years. Weincluded 10,281 participants who gave blood for DNA analysis at the1991 to 1994 and/or 2001 to 2003 examinations in the presentanalysis. Of these, 1986 developed IHD, 950 suffered an MI, 989developed ICVD, and 764 had IS during follow-up. All endpointsand data collection were recorded in the follow-up period fromJanuary 1976 through May 2009. Follow-up was up to 33 years andwas 100% complete;ie, no participants were lost to follow-up.

The CGPSThe CGPS8,11 is a mainly cross-sectional study of the Danish generalpopulation initiated in 2003 and is still recruiting. However, asendpoints were collected until May 2009, this study is also partlyprospective. The aim is to recruit a total of 100,000 participantsascertained exactly as in the CCHS but with a focus on allmultifactorial diseases including IHD. At the time of genotyping forthe present study, 51,429 participants had been included. Of these,3789 had IHD and 1554 had suffered an MI. Information ondiagnoses of IHD and MI was ascertained as in the CCHS. However,ICVD and IS diagnoses were not validated in the CGPS, and theseendpoints were therefore not included. For a detailed description ofthe diagnostic criteria, please see the Supplemental Methods, avail-able online at http://atvb.ahajournals.org.

Laboratory AnalysesPlasma levels of total cholesterol, LDL cholesterol, apolipoproteinB, HDL cholesterol, apolipoprotein A1, triglycerides, and fibrinogenwere measured using standard hospital assays (Konelab, Helsinki,Finland, and Boehringer Mannheim, Mannheim, Germany). LDLcholesterol was calculated using the Friedewald equation if thetriglyceride level was less than 4 mmol per liter (354 mg perdeciliter) and was measured directly for higher triglyceride levels.High-sensitivity C-reactive protein was measured by nephelometry(Dako, Glostrup, Denmark).

Other CovariatesBody mass index was weight in kilograms (kg) divided by height inmeters squared (m2). Hypertension, diabetes mellitus, smokingstatus, and use of lipid-lowering drugs were dichotomized anddefined as hypertension (systolic blood pressure �140 mm Hg ordiastolic blood pressure �90 mm Hg or use of antihypertensivemedication) versus no hypertension, diabetes (self-reported disease,use of insulin, use of oral hypoglycemic drugs, and/or nonfastingplasma glucose �11.0 mmol/L) versus no diabetes, current smokersversus nonsmokers, and use of lipid-lowering drugs (yes/no).

Gene SequencingWe sequenced LXR� in individuals from the CCHS with the lowest1% (n�95) and highest 1% (n�95) levels of plasma HDL choles-terol for age (in 10-year age groups) and sex.8,12 Ten PCR fragmentscovering 1,000 basepairs of the region upstream of the ATGtranslation start site (first A�nucleotide 36 in exon 2, position47280768 on chromosome 11, NCBI genome build 37.1), and all 9protein-coding exons and exon-intron boundaries were amplified andsequenced on an ABI PRISM 3100 Genetic Analyzer (AppliedBiosystems Inc.).

GenotypingAn ABI PRISM 7900HT Sequence Detection System (AppliedBiosystems) was used for genotyping by TaqMan-based assays. Allpromoter and nonsynonymous variants (n�10) identified by rese-quencing were genotyped in the CCHS, and one variant with effecton all ischemic endpoints in the CCHS (�115G�A, taggingthe haplotype �1830C[rs3758674]/�840A[rs61856015]/�115A[rs12221497], Supplemental Figure I, available online at http://atvb.ahajournals.org), was further genotyped in the CGPS. LXR��1830T�C, which was not incorporated in the original rese-quencing, was genotyped in 683 individuals from the CCHS toconfirm near complete linkage with �840C�A and �115G�Aas previously reported13 (Supplemental Figure I, available onlineat http://atvb.ahajournals.org).

LXR� �1830T>C Expression AnalysisThe region of the human LXR� promoter between nucleotides�2009 and �1168 (relative to the ATG start codon) was amplifiedby PCR using human genomic DNA as a template and the followingprimers: Forward (�2009): 5� CGGGGTACCCTATAGTCTCAG-TAGCTG 3�; Reverse (�1168): 5� CCAAGCTTTGTCCA-GAAGTCTCGGT 3� and was cloned at the Kpn I/Hind III sites ofthe plasmid pGL4.10 (Promega). The LXR� (�1830T�C)-Lucplasmid was generated by overlap extension PCR using the follow-ing internal mutagenesis primers: Forward: 5� GCAGATTTCCTAC-CAAAGGCTCTCC 3�; Reverse: 5� GGAGAGCCTTTGGTAG-GAAATCTGC 3� along with the external primers described above.The amplified PCR fragment was cloned at the Kpn I/HinD III siteof the pGL4.10 vector. All plasmids were verified by sequencing.Transient transfections of HepG2 cells with the LXR�-Luc reportervectors, along with an expression vector for beta galactosidase (CMV-�-gal), were performed using the calcium phosphate precipitationmethod. Normalization for transfection efficiency was performed by�-galactosidase assays, and results were expressed as the normalizedrelative percent promoter activity from 5 independent transfections.Luciferase assays were performed using the luciferase assay kit fromPromega Corp. according to the manufacturer’s instructions.

Statistical AnalysisStata software, version 10 (Stata Corp, College Station, TX) wasused for all analyses. Two-sided probability values less than 0.05were considered significant. The Mann-Whitney U test or Kruskal-Wallis analysis of variance was used for continuous variables, andPearson �2 test was used for categorical values. Trend tests were byCuzick nonparametric test for trend. Risk of IHD, MI, ICVD, or ISas a function of LXR� genotype was examined prospectively in theCCHS using left truncation (or delayed entry). Cox regressionmodels with age as the time scale, adjusted for age, sex, body massindex, hypertension, diabetes, smoking, use of lipid lowering drugs,plasma levels of total cholesterol, HDL cholesterol, and triglycerideswere used to estimate hazard ratios. Proportionality of hazards overtime was assessed by plotting �ln[�ln(survival)] versus ln(analysistime). There was no suspicion of nonparallel lines. Competing risk ofany death was accounted for by censoring at the date of death. In theCGPS, odds ratios for IHD and MI (adjusted for all covariates listedabove) were calculated by conditional logistic regression analysis.Interaction of genotype with all covariates listed above was evalu-ated by including 2-factor interaction terms between genotype andcovariates, one at a time, in the Cox regression model.

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ResultsBaseline characteristics of participants in the CCHS and theCGPS by disease status are presented in Table 1.

Resequencing and GenotypingLXR� is known to regulate several genes involved in reversecholesterol transport, and genetic variation in this gene mighttherefore affect HDL cholesterol levels. Therefore, to increasethe likelihood of identifying genetic variants with effect on HDLcholesterol levels, we resequenced 1000 basepairs upstream ofthe ATG start site and all 9 protein-coding exons in individuals

with the lowest 1% (n�95; 190 alleles) and highest 1% (n�95;190 alleles) HDL cholesterol levels in the CCHS.8,12 Of the 13variants identified using this approach, 7 had minor allelefrequencies �1%, and of these 4 were new (�524G�A,�288A�G, �44C�A, 385G�A/G129R; Table 2).

We genotyped the 9 variants located upstream from theATG translation start site and G129R, the rare nonsynony-mous variant, in the entire CCHS (n�10,281). The genotypedvariants tag 43% (3/7) of common variants across the LXR�region according to HapMap. The genotyping succes rate was�99.9%. All variants were in Hardy-Weinberg equilibrium

Table 1. Characteristics of the Participants in the 2 General Population Studies by Disease Status

Copenhagen City Heart Study Copenhagen General Population Study

No Event IHD ICVD No Event IHD

No. 7,673 1,986 989 47,640 3,789

Women (%) 4,466 (58) 975 (49)‡ 529 (53)‡ 26,986 (57) 1,480 (39)‡

Age (years) 54 (39–66) 68 (60–74)‡ 68 (62–74)‡ 56 (46–65) 68 (60–76)‡

Total cholesterol (mmol/L) 5.8 (5.0–6.7) 6.4 (5.6–7.3)‡ 6.4 (5.7–7.2)‡ 5.6 (4.9–6.3) 5.2 (4.4–6.0)‡

LDL cholesterol (mmol/L) 3.5 (2.8–4.3) 4.0 (3.2–4.8)‡ 4.0 (3.2–4.7)‡ 3.2 (2.6–3.9) 2.8 (2.1–3.6)‡

Apolipoprotein B (mg/dL) 83 (68–100) 93 (79–110)‡ 93 (79–109)‡ 107 (88–131) 106 (85–129)‡

HDL cholesterol (mmol/L) 1.5 (1.2–1.9) 1.4 (1.1–1.7)‡ 1.5 (1.2–1.9)† 1.6 (1.3–2.0) 1.4 (1.2–1.8)‡

Apolipoprotein A1 (mg/dL) 142 (129–162) 134 (117–155)‡ 139 (121–161)* 157 (139–177) 156 (139–177)‡

Triglycerides (mmol/L) 1.4 (1.0–2.1) 1.8 (1.3–2.6)‡ 1.8 (1.3–2.5)‡ 1.4 (1.0–2.1) 1.6 (1.2–2.4)‡

Body mass index (kg/m2) 24 (22–27) 26 (24–29)‡ 26 (23–29)‡ 26 (23–28) 27 (25–30)‡

Hypertension (%) 4,276 (56) 1,492 (75)‡ 778 (79)‡ 26,089 (55) 2,898 (77)‡

Diabetes mellitus (%) 205 (3) 176 (9)‡ 91 (9)‡ 1,540 (3) 445 (12)‡

Current smokers (%) 3,549 (46) 1,032 (52)† 491 (50) 10,343 (22) 832 (22)

Lipid lowering therapy (%) 32 (0.4) 75 (4)‡ 23 (2)‡ 3,274 (7) 1,650 (45)‡

Values are median and (interquartile range) or No. and (percentage). IHD indicates ischemic heart disease; ICVD, ischemic cerebrovascular disease. *P�0.05,†P�0.01, ‡P�0.001 for participants with IHD or ICVD vs no event.

Table 2. Genetic Variation in the Promoter and Protein-Coding Exons of LXR� in Individuals From the General Population WithExtreme High Density Lipoprotein Cholesterol Levels

Gene RegionNucleotide

Substitution

No. of Alleles(Allele Frequency in %)

P ValueAllele Frequency in

General Population* in %Amino Acid

Residue rs No./ReferenceLow HDL-C

(n�190 Alleles)High HDL-C

(n�190 Alleles)

Promoter �840C�A 32 (17) 39 (21) 0.29 16.3 rs61896015

Promoter �524G�A 0 1 (0.5) 0.32 0.01

Promoter �379T�C 0 2 (1) 0.16 0.4 (13)

Promoter �288A�G 0 2 (1) 0.16 0.1

Promoter �238C�T 0 1 (0.5) 0.32 0.1 rs77536828

Promoter �171A�G 2 (1) 5 (3) 0.25 2.0 rs112624241

Promoter �115G�A 32 (17) 39 (21) 0.29 16.3 rs12221497

Promoter �44C�A 1 (0.5) 1 (0.5) 1.00 0.1

Promoter (exon 2)† �6G�A 17 (9) 32 (17) 0.01 17.2 rs11039155

Exon 3 297C�T 18 (9) 32 (17) 0.01 S99S rs2279238

Exon 3 385G�A 0 1 (0.5) 0.32 0.04 G129R

Intron 4 IVS4�26G�A 1 (0.5) 0 0.32 rs55993545

Intron 5 IVS5 �55A�G 6 (3) 8 (4) 0.58 rs41275186

*The Copenhagen City Heart Study. †Untranslated region of exon 2. The first A of the ATG translation start site in liver X receptor alpha (LXR�) is located at nucleotide36 in exon 2 (at position 47280768 on chromosome 11, NCBI genome build 37.1). HDL-C indicates HDL cholesterol; IVS, intervening sequence.

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(probability values from 0.71–0.99). Three common variants,�1830T�C (rs3758674), �840C�A (rs61896015), and�115G�A (rs12221497) were in strong pairwise linkagedisequilibrium (all pairwise r2�0.97, Supplemental Figure I,available online at http://atvb.ahajournals.org). The minorallele frequency (16%) and the genotype distribution of�115G�A in both our studies (Table 3) were very similar tofrequencies in a Swedish population,14 and to frequenciesin other European populations.13,15,16 Furthermore, 4 com-mon ancestry informative17 SNPs in, respectively, APOE(�22/�32/�42/�33/�43/�44; rs429358 and rs7412), CETP(Taq1b, rs708272), and APOB (XbaI, rs693) were equallydistributed between the 3 LXR� �115G�A genotypes inour studies (Supplemental Table I, available online athttp://atvb.ahajournals.org).

Risk of Ischemic Vascular DiseaseThe cumulative incidences of IHD, MI, ICVD, and IS wereincreased in �840AA/�115AA homozygotes versus�840CC/�115GG homozygotes in the CCHS (Log-rankprobability values from 0.02 to �0.001; Figure 1). Homozy-gosity for �840AA/�115AA predicted risk of IHD (hazardratio [HR] 1.3; 95% confidence interval [CI]: 1.0–1.7;P�0.02), MI (HR 1.6; 95 CI: 1.2–2.2; P�0.005), ICVD (HR1.7; 95% CI: 1.3–2.4; P�0.001), and IS (HR 1.9; 95% CI:1.4–2.7; P�0.001) versus �840CC/�115GG homozygotes(Figure 2, left column). The corresponding HRs using arecessive model (�840AA/�115AA versus �840CC/�115GG and �840CA/�115GA combined) were 1.4 (95%CI: 1.1–1.7) for IHD, 1.6 (95% CI: 1.1–2.2) for MI, 1.7 (95%CI: 1.3–2.4) for ICVD, and 1.9 (95% CI: 1.4–2.7) for IS. Ofthe 989 participants in the CCHS who developed ICVD, 367(37%) also developed IHD during follow-up. In order to testwhether the observed association of �840AA/�115AAwith ICVD and IS in the CCHS could be explained by anoverlap of diagnoses, we excluded all participants withIHD (n�1,986). In this setting, �840AA/�115AA stillpredicted risk of ICVD (HR 1.5; 95% CI: 1.0 –2.4;P�0.05) and IS (HR 1.9; 95% CI: 1.2–3.0; P�0.01). Noneof the remaining variants associated consistently with

disease endpoints (Supplemental Figure II, available on-line at http://atvb.ahajournals.org).

In the CGPS, homozygosity for �840AA/�115AAversus �840CC/�115GG associated with odds ratios(ORs) for IHD and MI, of 1.1 (95% CI: 0.9 –1.4; P�0.29)and 1.5 (95% CI: 1.1–2.0; P�0.008; Figure 2, middlecolumn). The corresponding ORs using a recessive modelwere 1.1 (95% CI: 0.9 –1.4) for IHD, and 1.5 (95% CI:1.1–2.0) for MI. In the combined studies, homozygosityfor �840AA/�115AA versus �840CC/�115GG associ-ated with risk of IHD (OR 1.2; 95% CI: 1.0 –1.4; P�0.02)and MI (OR 1.5; 95% CI: 1.2–1.9; P�0.001; Figure 2,right column).

Table 3. Plasma Lipid, Lipoprotein, and Apolipoprotein Levels, and Markers of Inflammation as a Function of LXR��840C>A/�115G>A Genotype in the General Population*

The Copenhagen City Heart Study�840C�A/�115G�A Genotype

P

The Copenhagen General Population Study�840C�A/�115G�A Genotype

PCC/GG CA/GA AA/AA CC/GG CA/GA AA/AA

Total no. (%) 7,198 (70) 2,818 (27) 265 (3) 35,999 (70) 14,033 (27) 1,397 (3)

Total cholesterol, mmol/L 6.05 (0.02) 6.05 (0.03) 5.92 (0.08) 0.32 5.63 (0.003) 5.63 (0.009) 5.60 (0.030) 0.61

LDL cholesterol, mmol/L 3.68 (0.01) 3.70 (0.02) 3.55 (0.07) 0.23 3.24 (0.005) 3.23 (0.008) 3.20 (0.030) 0.28

Apolipoprotein B, mg/dL 88 (0.3) 88 (0.5) 85 (1.5) 0.20 112 (0.2) 112 (0.3) 112 (0.9) 0.09

HDL cholesterol, mmol/L 1.56 (0.006) 1.57 (0.008) 1.58 (0.030) 0.62 1.63 (0.003) 1.64 (0.004) 1.64 (0.010) 0.10

Apolipoprotein A1, mg/dL 143 (0.3) 143 (0.6) 144 (1.8) 0.98 159 (0.2) 160 (0.2) 159 (0.8) 0.50

Triglycerides, mmol/L 1.85 (0.02) 1.81 (0.03) 1.75 (0.07) 0.27 1.73 (0.006) 1.71 (0.010) 1.75 (0.030) 0.15

hsCRP, mg/L 3.2 (0.08) 3.4 (0.12) 3.7 (0.46) 0.91 2.8 (0.03) 2.8 (0.04) 2.8 (0.25) 0.35

Fibrinogen, �mol/L 9.3 (0.03) 9.4 (0.05) 9.3 (0.16) 0.50 11.6 (0.02) 11.6 (0.02) 11.7 (0.08) 0.60

*The Copenhagen City Heart Study and the Copenhagen General Population Study. Values are mean and (standard error of the mean). P values by Kruskal Wallisanalysis of variance. LXR� indicates liver X receptor alpha; hsCRP, high-sensitivity C-reactive protein.

Figure 1. Cumulative incidence of ischemic vascular disease inthe Copenhagen City Heart Study as a function of age andLXR� �840C�A/�115G�A genotype. IHD indicates ischemicheart disease; MI, myocardial infarction; ICVD, ischemic cere-brovascular disease; IS, ischemic stroke.




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There were no interactions between �840C�A/�115G�A genotype and covariates from Table 1 or totalcholesterol, HDL cholesterol, triglycerides, high-sensitivityC-reactive protein, and fibrinogen on risk of ischemic cardio-vascular disease.

Lipids, Lipoproteins, and Inflammatory MarkersLXR� �840C�A/�115G�A genotype did not associatewith plasma levels of total cholesterol, LDL cholesterol,apolipoprotein B, HDL cholesterol, apolipoprotein A1, tri-glycerides, high-sensitivity C-reactive protein, or fibrinogenin the CCHS or in the CGPS (Table 3). None of the remaining9 variants genotyped in the CCHS were associated with lipidor lipoprotein levels (Supplemental Figure III, availableonline at http://atvb.ahajournals.org), or with inflammatorymarkers (data not shown).

LXR� �1830T>C Promoter FunctionThe relative luciferase activity driven by the LXR� promoterwith the mutant C at position �1830 relative to the ATG startsite was increased by 91% compared with the wild-typepromoter with T at position �1830 (P�0.005; Figure 3,mean of 5 experiments).

DiscussionThe principal finding of this study is that homozygosity foran in vitro functional genetic variant in the promoterregion of LXR� predicted increased risk of ischemic

Figure 2. Hazard/odds ratios and 95% confidence intervals for ischemic vascular disease in the Copenhagen City Heart Study (CCHS),Copenhagen General Population Study (CGPS), and in the combined studies (CCHS�CGPS) as a function of LXR� �840C�A/�115G�A genotype. IHD indicates ischemic heart disease; MI, myocardial infarction; ICVD, ischemic cerebrovascular disease; IS,ischemic stroke.

Figure 3. LXR� promoter expression as a function of�1830T�C genotype. Transfection efficiencies were normalizedusing beta galactosidase assays. The normalized relative % pro-moter activity (�standard deviation) from 5 independent experi-ments is shown. Probability value by Mann-Whitney U test.




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vascular disease without affecting plasma lipid or lipopro-tein levels.

Our study is the first to examine the association of geneticvariation in LXR� with risk of ischemic vascular disease inthe general population. Other studies have reported associa-tions with obesity,14 life span,18 and plasma lipid lev-els13,15,19,20 for common genetic variants in LXR�. LXR� andLXR� have similar functions in lipid metabolism and inflam-mation, but whereas LXR� is expressed in the liver, adiposetissue, kidney, adrenals, and macrophages, LXR� is ex-pressed ubiquitously.1 Genetic variation in LXR� has beenassociated mainly with obesity14 and type 2 diabetes,21–23

both well-known risk factors for ischemic vascular disease.Constructs based on the region harboring �840C�A and

�115G�A (immediately upstream of the ATG start site) didnot show any luciferase activity, likely reflecting a lack ofpromoter function for this region. A functional promoterregion of LXR� has previously been identified approximately�3000 to �1300 basepairs upstream from the ATG startcodon.24 This region harbors the SNP �1830T�C shown tobe in strong linkage disequilibrium with �840C�A and�115G�A.13 We genotyped �1830T�C in 683 individualsfrom the CCHS and found a similar high degree of linkagebetween this variant and �840C�A and �115G�A in ourstudy (all pairwise r2�0.97), and hypothesized that�1830T�C could be the functional variant underlying theincreased risk observed in �840AA/�115AA homozygotes.Indeed, luciferase assays showed a strong increase in LXR�promoter activity associated with the rare C-allele of�1830T�C. The implication of these data are that an in vitrofunctional promoter variant in LXR� associates with elevatedrisk of ischemic vascular disease.

Animal studies suggest that LXR� activation exerts anatheroprotective effect.5–7 Intriguingly, genetic variation thatincreased LXR� transcription in vitro associated with in-creased risk of ischemic vascular disease in our study inhumans. Mechanistically, this might be explained by theincrease in production of atherogenic triglyceride-rich verylow density lipoproteins associated with LXR� activation inmice.25 In support of this, Robitaille et al13 (n�784, mean age49 years) found that �840C�A/�115G�A associated withslightly elevated plasma levels of total cholesterol and tri-glycerides in heterozygotes and homozygotes combined ver-sus noncarriers. Legry et al20 recently reported that LXR��115G�A associated with 10% lower HDL cholesterollevels (a marker of increased triglyceride levels in epidemi-ological studies) in a study of European adolescents(n�1144, mean age 15 years). In contrast, we genotyped�115G�A (tagging the haplotype �1830C/�840A/�115A)in more than 60,000 individuals (mean age 56 years) and didnot detect any association with plasma lipid or lipoproteinlevels. Possible explanations for this include differences instudy designs, in characteristics of participants (fasting versusnonfasting triglyceride, body mass index, diabetes, and age),or possible interactions with environmental factors, especiallydiet.13,20 However, it is worth noting that genetic variants witheffect on risk of ischemic cardiovascular disease withoutinfluencing plasma lipid and lipoprotein levels are not un-known.26–28 Because a 2-fold difference in transcription

levels does not appear to affect plasma lipid levels in thepresent study, these results seem to suggest that, at least inman, non-lipid–related effects of LXR� are more importantthan lipid-related effects in modulating risk of ischemicvascular disease.

The association between �840C�A/�115G�A and is-chemic cardiovascular disease exhibits recessive inheritance.One might perhaps have expected additive inheritance from apromoter variant. However, we have previously shown thatan in vitro functional promoter variant in ZNF202 (a regula-tory gene implicated in lipid metabolism, not unlike LXR�),associates with risk of IHD in a recessive manner.27

LXR� has not been implicated in genomewide associationstudies of ischemic cardiovascular disease29 or stroke.30 Thevariants showing association with ischemic endpoints in ourstudy are well covered both in HapMap (�115G�A) and oncommercially available genotyping chips. However, the re-cessive nature of the association, the low frequency of thehomozygote at-risk genotype (2.7%), and the relatively mod-erate risk estimates (�1.9 for all endpoints) observed in thepresent study together make it unlikely that this associationwill be picked up in the discovery phase of most genomewideassociation studies, because of lack of power and/or thestringent probability values in these studies.31,32

Of potential clinical relevance, homozygosity for�840AA/�115AA might eventually be used as part of agenetic risk prediction algorithm.32 We suggest that addi-tional validation of the association should be carried out byother groups before including the variant in such algorithms.

Associations between a rare homozygote genotype and riskare extremely susceptible to population stratification. How-ever, (1) all participants were white and of Danish descent;(2) minor allele and genotype frequencies of the �115G�Agenotype were similar to frequencies in other Scandinavianand European countries; (3) all SNPs studied were in Hardy-Weinberg equilibrium in both our studies (deviation fromHardy-Weinberg equilibrium may indicate population strati-fication33); and (4) the genotype distribution of ancestryinformative SNPs in APOE, CETP, and APOB did not differby �115G�A genotype, suggesting that �115AA homozy-gotes were of the same ancestry as other members of thecohort. In summary, population stratification is highly un-likely in our studies.

In conclusion, this study suggests that functional geneticvariation in LXR� predicts risk of ischemic vascular diseasein the general population. Although additional replication inyet other cohorts is warranted, the data presented here bridgethe gap between animal and human studies, and add to thegrowing body of evidence in favor of LXR� as an importantplayer in ischemic vascular disease.

AcknowledgmentsWe thank technicians Mette Refstrup and Karin Møller Hansen forexpert technical assistance. We are indebted to the staff andparticipants of the Copenhagen City Heart Study and the Copenha-gen General Population Study for their important contributions.

Sources of FundingThis work was supported by a Specific Targeted Research Projectgrant from the European Union, Sixth Framework Programme




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Priority (FP-2005-LIFEHEALTH-6) contract 037631, the DanishMedical Research Council (Copenhagen), the Research Fund atRigshospitalet, Copenhagen University Hospital (Copenhagen),Chief Physician Johan Boserup and Lise Boserup’s Fund (Copenha-gen), Ingeborg and Leo Dannin’s Grant (Copenhagen), and HenryHansen’s and Wife’s Grant (Copenhagen).

DisclosuresNone.

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Børge G. Nordestgaard and Anne Tybjærg-HansenStefan Stender, Ruth Frikke-Schmidt, Aristomenis Anestis, Dimitris Kardassis, Amar A. Sethi,

General PopulationGenetic Variation in Liver X Receptor Alpha and Risk of Ischemic Vascular Disease in the

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1

Supplemental Material

Supplemental Methods

Information on diagnoses of IHD (World Health Organization, International Classification of

Diseases, 8th edition codes 410-414 and 10th edition codes I20-I25) was collected and verified by

reviewing all hospital admissions and diagnoses entered in the national Danish Patient Registry, all

causes of death entered in the national Danish Causes of Death Registry, and medical records from

hospitals and general practitioners. IHD was fatal or non-fatal MI or characteristic symptoms of

angina pectoris, including revascularization procedures; death from other causes lead to censoring.

A diagnosis of MI required the presence of elevated cardiac enzymes together with at least one of

the following: symptoms of ischemia or electrocardiographic changes indicative of MI1,2. Potential

cases with ICVD including IS (World Health Organization International Classification of Diseases,

8th and 10th revisions: codes 431-438 and I60-I69, G45) were gathered from the national Danish

Patient Registry and the national Danish Causes of Death Registry. For each person registered with

ischemic cerebrovascular disease, hospital records were requested. To also include nonfatal

nonhospitalized ICVD, participants were asked at study examinations whether they previously had

a stroke. If affirmative, further information was obtained from that person’s general practitioner.

Experienced medical doctors with special neurological interest reviewed all potential cases.

Possible stroke events (hospitalized as well as non-hospitalized) were validated using the World

Health Organization definition of stroke: an acute disturbance of focal or global cerebral function

with symptoms lasting longer than 24 hours or leading to death with presumably no other reasons

than of vascular origin. To distinguish among infarction (=IS), intracerebral hemorrhages, and

subarachnoid hemorrhages, either CT or MRI scan, autopsy, spinal fluid examination, or surgical

description was necessary. The event was diagnosed as IS if the scan did not visualize an infarction

2

or hemorrhage, but the person had symptoms that met the criteria of the stroke definition. The

diagnosis of stroke was not applied in cases where a scan revealed signs of prior cerebrovascular

disease, but without history of any symptoms. The diagnostic criteria for ICVD were IS, transient

ischemic attack (focal neurological symptoms lasting less than 24 hours), or amaurosis fugax

(transient blindness on one eye only).

3

Supplemental References

(1) Thygesen K, Alpert JS, White HD. Universal definition of myocardial infarction. J Am Coll

Cardiol 2007;50:2173-95.

(2) Fox K, Garcia MA, Ardissino D, Buszman P, Camici PG, Crea F, Daly C, De Backer G,

Hjemdahl P, Lopez-Sendon J, Marco J, Morais J, Pepper J, Sechtem U, Simoons M, Thygesen

K, Priori SG, Blanc JJ, Budaj A, Camm J, Dean V, Deckers J, Dickstein K, Lekakis J,

McGregor K, Metra M, Morais J, Osterspey A, Tamargo J, Zamorano JL. Guidelines on the

management of stable angina pectoris: executive summary: The Task Force on the

Management of Stable Angina Pectoris of the European Society of Cardiology. Eur Heart J

2006;27:1341-81.

4

Supplemental Figure Legends

Supplemental Figure I.

Pairwise linkage disequilibrium between all 5 LXRα SNPs (minor allele frequency >1%) identified

by resequencing and genotyped in the Copenhagen City Heart Study. Disequilibrium statistics are

reported as D´ above the diagonal, and as r2 below the diagonal. The remaining 6 LXRα variants

were rare and thus not included. * 1830T>C only genotyped in 683 individuals.

Supplemental Figure II.

Hazard ratios and 95% confidence intervals for ischemic vascular disease in the Copenhagen City

Heart Study as a function of LXRα genotype. IHD=ischemic heart disease, MI=myocardial

infarction, ICVD=ischemic cerebrovascular disease, IS=ischemic stroke.

Supplemental Figure III.

Plasma lipid, lipoprotein and apolipoprotein levels as a function of LXRα genotype in the

Copenhagen City Heart Study. Numbers above bars indicate P values by Mann Whitney U test or

Kruskal-Wallis analysis of variance. ApoA1=apolipoprotein A1, ApoB=apolipoprotein B,

HDL=high density lipoprotein, LDL=low density lipoprotein.

5

Supplemental Table I. Frequencies of four ancestry informative SNPs by LXRα genotype in the CCHS and CGPS combined. Overall LXRα -840C>A/-115G>A genotype

Ancestry informative SNP Genotype No. (%) GG/CC GA/CA AA/AA P-value

APOB XbaI CC 13,662 (22.9) 23.0 22.6 21.3

rs693 CT 30,335 (50.6) 50.5 51.3 50.4

TT 15,793 (26.5) 26.5 26.1 28.2 0.16

APOE ε22/ε32/ε42/ε33/ε43/ε44 ε22 415 (0.7) 0.7 0.6 0.8

rs429358 and rs7412 ε32 7,668 (12.5) 12.5 12.6 12.7

ε42 1,806 (3.0) 3.0 2.9 2.9

ε33 33,937 (55.4) 55.5 55.3 55.6

ε43 15,617 (25.5) 25.5 25.7 25.4

ε44 1,740 (2.8) 2.9 2.8 2.6 0.96

CETP Taq1B GG 12,756 (31.5) 31.3 32.0 30.2

rs708272 GA 20,002 (49.4) 49.7 48.6 48.9

AA 7,761 (19.2) 19.0 19.3 21.0 0.18

Frequencies in %. P-values by Chi-square test. CCHS=Copenhagen City Heart Study; CGPS=Copenhagen General Population Study.

APOB rs693 was genotyped in 59,790; APOE rs429358 and rs7412 were genotyped in 61,183; CETP rs708272 was genotyped in 40,519

individuals.

6

Supplemental Figure I.

-183

0T>

C*

-840

C>

A

-171

A>

G

-115

G>

A

-6G

>A

D’ R2

-1830T>C* 1.0 1.0 1.0 1.0

= 1.0

>0.90

-840C>A 0.97 1.0 1.0 1.0

0.90

>0.80

-171A>G 0 0 1.0 1.0

<0.90

<0.80

-115G>A 0.97 0.99 0 1.0

<0.70

<0.20

-6G>A 0.04 0.04 0.09 0.04

<0.1

<0.01

7

Supplemental Figure II.

IHDn=1,986

Hazard ratio

0.5 1 2 3

MIn=950

0.5 1 2 3

ICVDn=989

0.5 1 2 3

ISn=764

0.5 1 2 3

LXR genotype n

7,2082,806

266

10,2783

10,20477

10,26120

10,25229

9,877398

4

7,1982,818

265

10,25724

7,0462,936

298

10,2729

-840

-524

-379

-288

-238

-171

-115

-44

-6

385

CCCAAA

GGGAAA

GGGA

CCCT

AAAG

TTTC

GGGA

CCCA

GGGAAA

AAAGGG

1,38453567

n events n events n events n events

64826438

67826843

52020836

1,38753168

65026238

67726943

52020836

1,35257459

63828427

67729022

52222814

1,9806

9411

9854

7604

1,895910

903470

950390

735290

1,9824

9500

9881

7640

1,9833

9500

9881

7631

1,96521

94010

9827

7586

1,9851

9500

9890

7640

1,9842

9500

9881

7631

8

Supplemental Figure III.

To

tal c

hole

ste

rol

(mm

ol/L

)

0

2

4

6

8

LDL

cho

lest

ero

l

(m

mol

/L)

0

1

2

3

4

5

Ap

oB(m

g/dL

)

0

20

40

60

80

100

120

HD

L ch

oles

tero

l

(m

mo

l/L)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Trig

lyce

rides

(mm

ol/L

)

0

1

2

3

4

7,20

82,

806

266

10,2

78 3

10,2

04 77

10,2

61 2010

,252 29

9,87

739

8 47,

198

2,81

826

510

,257 24

7,04

62,

936

298

10,2

72 9

CC CA AA AA AG GGGG GA TT TC AA AG CC CT GG GA AA CC CA GG GA AA GG GA

-840 -524 -171-379 -238-288 -115 -44 -6 385/G129R

0.31 0.75 0.83 0.55 0.51 0.18 0.32 0.70 0.26 0.66

0.23 0.35 0.57 0.35 0.43 0.07 0.23 0.43 0.65 0.59

0.18 0.45 0.96 0.66 0.31 0.25 0.20 0.18 0.71 0.85

0.63 0.57 0.49 0.54 0.89 0.60 0.62 0.20 0.27 0.03

0.29 0.62 0.98 0.27 0.81 0.40 0.27 0.73 0.92 0.96

Ap

oA1

(mg

/dL

)

0

50

100

150

2000.97 0.87 0.61 0.56 0.73 0.57 0.98 0.27 0.28 0.07

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