Atherosclerosis 185 (2006) 97–107

The association of common SNPs and haplotypes in the CETP andMDR1 genes with lipids response to fluvastatin in

familial hypercholesterolemia

Dani Bercovicha,1, Yechiel Friedlanderb,1, Sigal Korema, Asaf Houminerb,Amnon Hoffmanc, Lilach Kleinbergc, Chen Shochata,

Eran Leitersdorfd, Vardiella Meinerd,e,∗a The Laboratory for Molecular Genetics and Pharmacogenetics, Migal-Galilee Technology Center, Kiryat-Shmona, Israel

b Department of Social Medicine, Hebrew University-Hadassah School of Public Health, Jerusalem, Israelc Department of Pharmaceutics, School of Pharmacy, The Heberew University, Jerusalem, Israel

d Center for Research, Prevention, and Treatment of Atherosclerosis, Department of Medicine B, Hadassah University Hospital, Jerusalem, Israele Department of Human Genetics, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel

Received 1 September 2004; received in revised form 5 May 2005; accepted 16 May 2005Available online 5 July 2005

A

O ransporter(M ith 40 mgd xamine theaR ; meant s in bothg and MDR1g associatedw ssion modeliC may leadt©

K plotypes;H

1

z

dary

ility-C.rideDL-eir

sider-

0d

bstract

bjective: To examine whether genetic polymorphisms in the cholesteryl-ester transfer protein (CETP) and the P-glycoprotein drug tMDR1), are associated with variable lipid response to fluvastatin.

ethods: Lipid levels were determined in a compliance-monitored clinical study at baseline and following 20 weeks of treatment wose of fluvastatin in 76 FH patients. CETP and MDR1 SNP genotyping was performed and linear regression was used to essociations between common SNPs and haplotypes and lipid response.esults: Treatment with 40 mg of fluvastatin resulted in mean low density lipoprotein cholesterol (LDL-C) reduction of 21.5%

riglyceride (TG) reduction of 8.3%; and a mean high-density lipoprotein cholesterol (HDL-C) increase of 13.4%. Five tagging SNPenes were used to reconstruct five and six haplotypes accounting for 71.4% and 90.2% of the observed haplotypes in the CETPenes, respectively. CETP-H13 and MDR1-h4 were associated with an increase in LDL-C response. CETP-H5 was significantlyith decreased TG and HDL-C response, whereas MDR1-h10 was associated with decreased TG response. A multivariate regre

ndicated an independent additive effect of CETP-H5 and MDR1-h10 on the level of TG response.onclusions: CETP and MDR1 have independent effects on lipid changes following fluvastatin treatment. The results of this study

o an improved understanding of the genetic determinants of lipids response to treatment.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Hypercholesterolemia familial; Pharmacogenetics; Cholestrol ester transfer protein; MDR; Single nucleotide polymorphism; Haydroxymethylglutaryl-CoA reductase inhibitors

. Introduction

The development of 3-hydroxy-3-methylglutaryl coen-yme A (HMG CoA) reductase inhibitors or “statins”, led

∗ Corresponding author. Tel.: +972 2 6777618; fax: +972 2 6777499.E-mail address: vmeiner@hadassah.org.il (V. Meiner).

1 Contributed equally to this work.

to important improvements in the primary and seconprevention of coronary artery disease[1]. Statins differ inabsorption, plasma protein binding, excretion, and soluband exhibit variable dose-related efficacy in reducing LDLFurthermore, statins cause a small reduction in triglycelevels and a minor increase in high-density cholesterol (HC). Monitoring different subgroups of patients and thresponse to statins demonstrated that in addition to con

021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2005.05.025

98 D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107

able variability most patients do not reach established goals[2]. Inter-individual variability in drug response is a majorclinical problem and is of concern during drug development.It has been shown that in addition to known environmen-tal factors, sequence polymorphic variants in genes encod-ing drug-metabolizing enzymes, drug transporters, or drugtargets, may have important pharmacokinetic and pharma-codynamic consequences and thus affect drug response[3].One such group of studies focused on the variations in the P-glycoprotein drug transporter (MDR1) gene[4]. MDR1 playsan important role in the bioavailability of common drugs inmedical treatment, such as digoxin, doxorubicin, and vinblas-tine which serve as its substrates and thus MDR1 genetic vari-ants are potential underlying molecular parameters respon-sible for inter-individual differences in drug metabolism andresponse[5]. The modulation of MDR1 activity by vari-ous statins was reported by Bogman et al.[6] and recentlyfollowed by a study that analyzed the association betweenpolymorphisms inMDR1 and the variability in response toatorvastatin[7]. In this study, two prevalent MDR1 polymor-phisms were examined in 344 hypercholesterolemic patientsthat were treated with 10 mg atorvastatin. One of the polymor-phisms was significantly associated with a smaller reductionin LDL-C and with a larger increase in HDL-C followingtherapy. Moreover, haplotype analysis using these polymor-phisms identified a subgroup of treated patients with eveng statinw andi tatinie eenC ffecto

asso-c stert int tingt ndV ighHd itsi eptoru Ps thesep tedp P,T od-u ADp nalp witht dom-i eticp tedt ionsw them tin

treatment in 98 patients with untreated dyslipidemia[21].These studies examined the effect of statins on heterogeneouspatient groups that required the treatment for various formsof dyslipidemias. In contrast to multifactorial dyslipidemias,familial hypercholesterolemia (FH) offers a suitable mono-genic model for the assessment of lipid lowering efficacy[22]. We sought to examine specific genetic determinantsthat modulate the response to lipid lowering agents in agroup of molecularly defined, compliance-monitored, FHindividuals by analyzing the association between responseto fluvastatin and SNP haplotypes in the CETP and theMDR1.

2. Methods

2.1. Phenotypic characterization of FH patients

In this study, we analyzed patients that were enrolledin single-blind study with fluvastatin. All participants gavetheir informed consent, and the study was approved bothby the Institutional Review Board and the Israeli Ministryof Health Ethics Committee. Study participants were het-erozygous FH patients (men and women >18 years old) whowere ascertained through screening of all available familymembers for plasma lipid and lipoprotein levels. Their LDL-c dL)d ridel i-c ptorm uslyd 59o eno-t cuso ages,g or-m ucedi ryc en-d ta on-i inedb eksa ns,p 0 mg.P ing2 stiga-t velsw least1 kits( rageo elsw ula[ tiona % and9

reater response to atorvastatin. In contrast to atorvahich is metabolized by members of the CYP3A family

s thus subjected to MDR1 mediated transport, fluvass removed mainly via metabolism by CYP2C9[8]. How-ver, in healthy individuals there is no association betwYP2C9 genetic variants and the cholesterol lowering ef fluvastatin[9].

Among the various genes for which SNPs have beeniated with differential statin response is cholesteryl-eransfer protein (CETP)[10]. CETP plays a central rolehe in the remodeling of plasma lipoproteins by promohe transfer of cholesteryl esters from HDL-C to LDL-C aLDL-C [11]. CETP deficient patients demonstrate hDL-C levels and reduced levels of LDL-C[12,13]. In CETPeficiency the metabolic basis for low LDL-C levels is

ncreased rate of clearance which may suggest LDL recp-regulation[14]. The discovery of multitude of CETequence variations led to association studies linkingolymorphisms with coronary artery disease (CAD) relahenotypes[15–19]. An extensively investigated CETP SNaqI site in intron 1, was shown to be associated with a mlation of the antiatherogenic effect of pravastatin in Catients[10]. The latter variant together with an additioromoter CETP polymorphism was shown to correlate

he degree of response to atorvastatin in a 30-week ranzed double-blind placebo-controlled trial in 217 diabatients [20]. Recently, Winkelmann et al. demonstra

hat haplotypes derived from CETP SNP combinatere more informative than single variants in predictingagnitude of change in HDL-C and TG following sta

holesterol levels had to remain >4.9 mmol/L (190 mg/espite a prior intensive dietary treatment and triglyce

evels <3.4 mmol/L (300 mg/dL). Genomic DNA from clinally diagnosed FH patients was extracted and LDL receutation was identified using SSCP analysis as previoescribed[23]. Specific mutation was characterized inf 76 patients (78%). In 17 of the patients (22%) the ph

ype co-segregated with haplotypes linked to the LDLR lon chromosome 19p. Detailed data regarding specificender BMI and baseline lipid levels in addition to infation on each of the characterized mutations is introd

n Supplementary Table 1. The patients underwent dietaonsultation and were instructed to follow the recommations of the NCEP step II diet[24] with calorie contendjusted to maintain a stable body weight. In order to m

tor dietary compliance 3-day food records were obtaefore and during the study. Following a period of 8 wefter discontinuation of all cholesterol lowering medicatioatients received fluvastatin capsules at daily doses of 4lasma lipid and lipoprotein levels at baseline and follow0 weeks were measured and used for the current inve

ion. Plasma total cholesterol, triglyceride, and HDL-C leere determined from blood samples collected after at2 h of fasting using commercially available diagnosticBoehringer Mannheim). Lipid levels represented an avef two blood collections over 48 hours. Plasma LDL-C levere calculated according to the Friedewald–Levy form

25]. Mean weekly compliance to the prescribed medicas assessed by capsule counting ranged between 929%.

D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107 99

2.2. SNP and haplotype characterization

For the CETP gene all exons and flanking intronic bound-aries in addition to the 5′UT region (−3000 nucleotides)were screened using a WAVE apparatus (Transgenomic Inc.,Omaha, NE) dHPLC mutation detection system as described[26]. A targeted MDR1 screen, based on previously publishedSNPs, comprised 16 fragments of which 10 were exons andflanking regions (2, 3, 5, 6, 12, 13, 17, 22, 25, and 27), and 6were introns (2, 3, 5, 12, 13, and 22).Supplementary Tables2 and 3present detailed description of the identified CETPand MDR1 SNPs, respectively. PCR primers for the screenedfragments and conditions for the dHPLC mutation detectionare available upon request. dHPLC detected SNPs were con-firmed by sequencing twice at each DNA strand.

The method for selecting the analyzed SNPs was differ-ent for the CETP and MDR1 genes and is outlined below.Following sequencing of the CETP promoter region, the cod-ing sequence, and the 3′ UTR we identified 26 SNPs in ourstudied population. Data regarding CETP human variations(http://droog.gs.washington.edu/) provided us with means toevaluate all the pairwise linkage disequilibrium (LD) betweenloci and to generate bins from which we selected promoterSNPs that are shown to tag and capture the common varia-tion in the 5′ gene region. Data regarding calculated LD ispresented inSupplementary Tables 4 and 5. A cut-point ofr , outo beens asso-ct kedt elya lipidp DR1g studi AF(

ought do ars av ion-m tod

2

unt-i forH oodl tudyp ct forv nullh mongt anal-y ala ls for

the continuous outcome variables, which were standardizedto have a mean zero and standard deviation, one. Given twoalleles at the putative candidate gene loci, we have param-eterized the different SNPs’ effect through a single ordinalvariable 0, 1, or 2, representing a possible additive effect ofthe variant allele. We performed a hypothesis test that collec-tively assessed the association of the patterns of variation inthe entire candidate gene with standardized percent responseusing the pre-selected SNPs to characterize common haplo-types. Once a genetic association with the percent responsehas been identified in this manner, the corresponding 95%confidence intervals (CI) for each SNP were calculated. Thisjoint testing of all major alleles at a locus has several impor-tant advantages[30]. Assuming the SNPs tested capture mostof the common allelic variation within a gene, there is greaterpower to detect the complex pattern of genetic variation (hap-lotypes) associated with the response even in the presence ofsome degree of allele misclassification, compared to separatehypothesis testing for each SNP. Testing each SNP individu-ally might miss the greater part of the response attributable toa candidate genetic locus, particularly among the evolution-ary more complex genes, where combinations of SNPs defineeach haplotype. Furthermore, plausible contrasts or group-ings of alleles defined by experimental and computationalbiology may reduce exposure misclassification compared totesting of individual alleles. Thus, linear regression modelsw n thes cate-g whow e inp ed asa

es inh

a

w ev ali eens ragee nt thatd d then hap-l ic:a ono luesr plo-t ypicv uta-t e

mα

O aplo-

2 > 0.64 was used to determine bins of SNPs in high LDf which only single tagSNP needs to be typed. It hashown that this threshold maximizes the power to detectiations with SNPs that are in LD with the tagSNPs[27]. Tohese tagging SNPs we added Taq1B (although tightly lino −525 variation) and Ex14/I405V that were extensivnalyzed in the past for their association with plasmahenotypes. Since no such data is available for the Mene, as outlined above we sequenced the gene in our

ed population and selected all common SNPs [>10% Mmean allele frequency) on an average].

We then used a Bayesian approach, implemented thrhe software package PHASE[28] to infer haplotypes basen the chosen tagSNPs. The Bayesian approach appealid or more so compared to the well-known expectataximum (EM) algorithm, and tends to be well suitedatasets with large numbers of polymorphisms.

.3. Statistical analysis

Allele frequencies were estimated by the “gene cong” method and then the genotypes were testedardy–Weinberg equilibrium by chi-square tests. Bl

ipids and lipoproteins at baseline and at the end of the seriod were each adjusted by linear regression to correariation due to the differences in gender and age. Theypothesis of equality of mean percent response levels a

he different genotype groups was tested by the one-waysis of variance[29]. Then, the great majority of statisticnalysis was done within the context of regression mode

-

s

ere used similarly to examine the associations betweetandardized response variables and the three orderedories of haplotypes. In multivariate analyses subjectsere found to be homozygous for the common allelrevalent SNPs or for common haplotypes were selectreference group.We used average excess in order to test for differenc

aplotype effects.For haplotypej, the average excess

j =∑

i=lnTijyi∑

i=lnTij

− y

hereTij is the number of copies (0, 1, or 2) of haplotypjaried by individuali, yi the phenotypic value of individu, and y is the sample mean of the phenotype. It has bhown that under Hardy–Weinberg equilibrium the avexcess is equal to the least square regression coefficieefines the linear relationship between the phenotype anumber of copies of each haplotype. For contrasting two

otype average excesses,αj andαk, we used the test statist2d = (αj − αk)2. We determined the empirical distributif this statistic by permuting the vector of phenotype vaandomly among the individuals bearing the tested haypes, such that any pairing of haplotypes with phenotalues was equally probable. For each of 10,000 permions of the data, a new value,α2

p, was calculated in the sam

anner asα2d ; thep-value for testing the null hypothesis,H0:

j =αk, was the proportion of 10,000 trials withα2p ≥ α2

d .nly subjects carrying at least one of the contrasted h

100 D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107

types are included in the permutations. This is required forthe test to be statistically valid. The two contrasted haplo-types completely determine membership in the reference setfor permutations[31].

3. Results

We observed variability in plasma lipids and lipopro-teins following 20 weeks of fluvastatin treatment witha sex- and age-adjusted mean reduction [range] of21.5% [(−56.6%)–22.4%] and 8.3% [(−52.2%)−75.6%],in LDL-C and TG, respectively, and 13.8% increase[(−18.1%)–46.1%], in HDL-C. In order to analyze this vari-ability we genotyped DNA samples obtained from 76 FHindividuals (47% males) enrolled in this single-blind clinicalstudy. The mean age in the study group was 43 years (range18–73) and mean BMI 25.6 kg/m2. Supplementary Table 1summarizes the clinical and genetic information of the ana-lyzed group.

Five SNPs in the CETP and MDR1 genes were selectedfor the study as outlined inTable 1. None of the genotypedCETP single variations demonstrated an association with thedegree of LDL-C response, as shown inTable 2, yet a singleMDR1 variation, designated Int 17, was shown to be associ-ated with decrease LDL-C response to fluvastatin (p = 0.049).S o-t TwoC tedw uenta orr nse( mon‘

ithls thata ngleC e of as -c L-C

response (b = 0.439; 95% CI = 0.02–0.86), when comparedto the response of homozygotes for the−2600 common‘G’ allele or to the response of homozygotes for the com-mon Taq1B ‘G’ allele (b = 0.452; 95% CI = 0.02–0.88).On the other hand, the less frequent Ex14/I405V allelewas significantly associated with increased HDL-C response(b =−0.46, 95% CI =−0.91–0.00) when compared to theresponse of homozygotes for the−2600 ‘G’ allele. In addi-tion, the presence of a single copy of the less frequentEx14/I405V allele had a significant independent effect onTG response (b = 0.49, 95% CI = 0.02–0.96) when comparedto the response of homozygotes for the common alleles ofCETP−2600 and Taq1B (b = 0.499, 95% CI = 0.04–0.96).

In the MDR1 gene, under a univariate model, both Int13and Int17 SNPs were associated with a decrease in LDL-Cresponse. Using an additive model, when other SNPs wereincluded, and when compared to the response of homozy-gotes for the MDR1 Int13 common allele, the presence ofa single copy of the less frequent allele of MDR1 Int17SNP was significantly associated with less LDL-C reduc-tion following pharmacological treatment (b =−0.467; 95%CI =−0.93–0.00). On the other hand, the presence of themore frequent allele in the MDR1 Int13 SNP was signifi-cantly associated with favorable LDL-C response (b = 0.770;95% CI = 0.05–1.49), when the selected reference group inthe multivariate model was subjects who were found to beh L-C R1g

ivid-u o-t linedb esw withl lenth andC le7 plo-t H5.Ua o the

TA ted for

G le frequ

C .300.430.330.390.31 ine

M .17/0.390.09.280.33

upplementary Table 6summarizes the lipid and lipoprein response to treatment by solitary SNP genotypes.ETP SNPs, namely−867 and Ex14/I405V, were associaith the magnitude of HDL-C response. The less freqllele of −867, the ‘A’ allele, was shown to confer poesponse (p = 0.03), whereas a marginally favorable respop = 0.053) was obtained when associating the less comA’ allele in Ex14/I405V with HDL-C and TG changes.

The associations of the collectively five CETP SNPs wipids and lipoprotein response are shown inTable 3. Ashown for the separate effect, there was no evidencedjusted LDL-C response differed considerably by siETP genotypes. Under an additive model, the presencingle copy of the less common−867 ‘A’ allele was assoiated with a significant independent decrease in HD

able 1llele frequencies, nucleotide and amino acid changes in SNPs selec

ene Polymorphism Position Alle

ETP −2600 5′UTR 0.70/0−867 Promoter 0.57/−525 Promoter 0.67/Taq1B Intron 1 0.61/Ex14/I405V Int13/+16 0.69/

DR1 Int 5 Exon 6/−25 0.83/0Ex 13/G412G Int 12/+12 0.61Int 13 Exon 13/+44 0.91/Int 17 Exon18/−76 0.72/0Ex22/A893S Int22/-9 0.67/

omozygous for the common Int17 allele. Adjusted HDand TG response did not differ considerably by MD

enotypes.Haplotypes were constructed and assigned to all ind

als as described in Section2. Of the 32 possible haplypes corresponding to designated CETP variations outy “−2600/−867/–525/Taq1B/EX14/I405V”, 16 haplotypere estimated to be present. The univariate association

ipids and lipoproteins changes of the five most prevaaplotypes (CETP-H2, CETP-H5, CETP-H7, CETP-H8,ETP-H13) was examined (Table 4). Supplementary Tabintroduces the numbers of individuals for each ha

ype. Only two individuals were homozygotes for CETP-nder an additive model, CETP-H13 (∧GAAGA∧), showedsignificant increase in LDL-C response, as compared t

the genotype analysis

ency (+/−) Nucleotide Amino acid change

+ −G A –G A –A C –G A –G A Valine for Isoleuc

G T –T C –C T –

T A –C A Serine for Alanine

D.B

ercovichetal./A

therosclerosis185

(2006)97–107

101Table 2Age- and gender-adjusted lipid and lipoprotein concentrations after fluvastatin treatment according to TaqI and−525 SNPs

Taq1B −525

All genotypes(n = 75)

GG (n = 22) AG (n = 46) AA (n = 7) p-Value All genotypes(n = 76)

AA (n = 26) AC (n = 49) CC (n = 1) p-Value

Baseline (mg/dL)Total cholesterol 367.08± 57.86 362.59± 65.79 365.90± 55.21 388.98± 51.31 0.568 367.47± 57.57 359.29± 54.19 371.26± 59.86 394.46 .626HDL-cholesterol 32.66± 8.42 29.92± 6.80 33.63± 8.76 34.96± 9.83 0.178 32.66± 8.37 31.24± 8.52 33.65± 8.17 21.07 .188LDL-cholesterol 301.86± 55.03 300.37± 63.73 299.95± 52.39 319.04± 46.5 0.692 302.29± 54.79 294.48± 51.00 305.70± 57.13 338.11 .570Triglycerides 163.38± 31.67 159.55± 43.23 163.32± 69.14 175.89± 61.76 0.832 163.12± 61.00 166.74± 58.55 160.91± 63.34 177.39 .902

Treatment (mg/dL)Total cholesterol 297.79± 56.31 293.98± 71.20 298.54± 48.89 305.71± 57.43 0.879 297.92± 55.95 296.04± 61.39 298.43± 53.98 322.00 .899HDL-cholesterol 35.26± 9.86 32.55± 9.49 36.68± 9.73 34.41± 11.48 0.267 35.25± 9.80 34.05± 11.15 36.16± 8.95 22.02 .270LDL-cholesterol 229.13± 54.11 229.93± 72.31 227.89± 45.06 234.71± 50.26 0.951 229.41± 53.81 226.94± 62.06 230.55± 50.12 238.00 .951Triglycerides 146.66± 62.02 135.59± 47.34 149.08± 67.00 165.52± 76.80 0.498 146.31± 61.68 151.34± 67.12 141.30± 57.32 260.66 .140

Change (%)Total cholesterol 15.57± 9.82 15.83± 11.51 14.99± 9.42 18.57± 6.87 0.671 15.61± 9.76 14.84± 9.07 15.97± 10.27 17.74 .873HDL-cholesterol 8.43± 13.67 7.31± 14.55 7.58± 12.86 17.53± 14.67 0.181 8.45± 13.58 8.10± 15.91 8.59± 12.49 10.84 .974LDL-cholesterol 16.24± 11.58 16.32± 13.81 15.81± 11.02 18.85± 8.22 0.815 16.26± 11.51 15.72± 10.95 16.42± 11.97 22.67 .832Triglycerides 15.69± 24.81 23.15± 20.46 12.62± 26.49 12.48± 23.90 0.248 15.87± 24.69 17.03± 27.52 16.59± 21.62 −49.50 .026

Age (years): 42.42± 14.01; BMI (kg/m2); 26.02± 3.53.

Table 3Multivariate regression coefficients (95% confidence intervals) from models of plasma lipid responsea on CETP and MDR1 SNPs

Gene SNP Reference =−2600 Reference = Taq1B

LDL-C HDL-C TG LDL-C HDL-C TG

CETP −2600 – – – 0.349 (−0.049–0.747) 0.059 (−0.329–0.447) −0.299 (−0.693–0.095)−867 0.220 (−0.223–0.663) 0.439 (0.020–0.858) 0.086 (−0.345–0.517) 0.310 (−0.129–0.749) 0.452 (0.023–0.881) 0.136 (−0.299–0.571)−525 0.133 (−0.335–0.601) −0.006 (−0.449–0.437) 0.193 (−0.264–0.650) 0.197 (−0.260–0.654) 0.007 (−0.440−0.454) 0.228 (−0.225–0.681)Taq1B 0.061 (−0.343–0.465) 0.249−0.133–0.631) −0.226 (−0.620–0.168) – – –Ex14/I405V −0.062 (−0.544–420) −0.457 (−0.914–0.000) 0.490 (0.020–0.960) −0.023 (−0.489–0.443) −0.434 (−0.891–0.023) 0.499 (0.036–0.962)

Gene SNP Reference = Int 13 Reference = Int 17

LDL HDL TG LDL HDL TG

MDR1 Int 5 0.260 (−0.261–0.781) 0.221 (−0.324–0.766) −0.229 (−0.792–0.334) 0.594 (−0.025–0.213) 0.560 (−0.091–0.211) −0.537 (−1.211–137)Ex13/G412G −0.413 (−0.979–0.153) 0.093 (−0.499–0.682) −0.236 (−0.848–0.376) −0.333 (−0.899–233) 0.171 (−0.425–0.767) −0.293 (−0.908–0.322)Int 13 – – – 0.770 (0.053–1.487) 0.616 (−0.139–0.371) −0.509 (−1.289–0.271)Int 17 −0.467 [(−0.930)–(−0.004)] 0.404 (−0.080–0.888) 0.181 (−0.319–0.681) – – –Ex22/A893S −0.166 (−0.750–0.418) 0.198 (−0.414–0.810) −0.088 (−0.719–0.543) −0.027 (−0.615–561) 0.285 (−0.334–0.904) −0.210 (−0.849–0.570)

Values in bold fonts are statistically significant.a Standardized age- and sex-adjusted percent change.

102 D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107

Table 4Effects of CETP haplotypes on lipids responsea, difference in average excess, andp-value for the differences in effects

Haplotype −2600 −867 −525 Taq1B Ex14/I405 V

Hapfreq

Lipidchange (%)

p-Value r2 Difference in average excessb andp-valuec

CETP-H7 CETP-H5 CETP-H8 CETP-H13

LDL-CCETP-H2 G G C G G 0.260 0.182 0.428 0.008 0.038 0.002 0.023 0.224

0.49 0.84 0.50 0.09

CETP-H7 G G A G G 0.136 −0.157 0.495 0.006 0.028 0.000 0.4960.59 0.98 0.03

CETP-H5 G A A A G 0.130 0.182 0.488 0.006 0.081 0.1810.39 0.20

CETP-H8 A A A A A 0.104 −0.190 0.503 0.006 0.5040.06

CETP-H13 G A A G A 0.084 0.673 0.026 0.064

HDL-CCETP-H2 G G C G G 0.260 0.067 0.771 0.001 0.136 0.251 0.173 0.023

0.19 0.02 0.07 0.58

CETP-H7 G G A G G 0.136 −0.367 0.108 0.034 0.757 0.002 0.0480.005 0.89 0.49

CETP-H5 G A A A G 0.130 0.720 0.005 0.101 0.841 0.4240.006 0.05

CETP-H8 A A A A A 0.104 −0.485 0.084 0.039 0.0710.47

CETP-H13 G A A G A 0.084 −0.142 0.644 0.003

TriglycerideCETP-H2 G G C G G 0.260 −0.247

CETP-H7 G G A G G 0.136 0.105

CETP-H5 G A A A G 0.130 −0.409

CETP-H8 A A A A A 0.104 0.184

CETP-H13 G A A G A 0.084 0.171

Values in bold fonts are statistically significant.a Standardized age- and sex-adjusted percent change.b The upper number designates difference in average excess (deviation froc The lower number designatesp-value as determined from a permutation tes

overall sample mean response. This haplotype was least fre-quent (8.4%) and explained 6.4% of the variation in LDL-Cresponse. Altogether BMI, age and gender explained 5.4%of the variability in LDL-C. The LDL-C response of sub-jects harboring CETP-H13 was significantly greater than theLDL-C response of subjects with the CETP-H7 (∧GGAGG∧)and marginally greater than the LDL-C response of CETP-H8 subjects (∧AAAAA ∧). Univariate analysis indicated thatCETP-H5 (∧GAAAG∧), was significantly associated withHDL-C and TG response (p = 0.005 and 0.002, respectively.This haplotype (prevalence—13%) explained 10% and 12%of the variations in HDL-C and TG, respectively. In compar-ison, BMI, age and gender explained 11.7% and 2.2% of thevariability in HDL-C and TG, respectively. Subjects harbor-ing the CETP-H5 haplotype showed significantly less favor-able response in HDL-C, as compared to the overall sample

m s har-bT sig-n mplem l hap-l

pes( lo-t iationw ento 1-h ase zy-g ana R1-h L-

0.285 0.015 0.097 0.239 0.162 0.1580.29 0.03 0.08 0.16

0.372 0.011 0.640 0.008 0.0070.015 0.78 0.8

0.002 0.124 0.796 0.7850.01 0.01

0.199 0.022 0.0000.98

0.270 0.016

m the population mean).t.

ean response, and to the HDL-C response of subjectoring each of the other haplotypes (Table 4). Similarly, theG response of subjects harboring CETP-H5 showed aificantly lower response as compared to the overall saean TG response and as compared to other individua

otypes.Of the 32 possible reconstructed MDR1 haploty

Int5/EX13 G412G/ Int13/Int17/EX22 A893S), 12 hapypes were estimated to be present. Univariate associth lipids and lipoproteins response to fluvastatin treatmf the six most prevalent haplotypes (MDR1-h1, MDR2, MDR1-h3, MDR1-h4, MDR1-h9, and MDR1-h10) wxamined (Table 5). There were nine and eight homootes for MDR1-h2 and MDR1-h4, respectively. Underdditive model, the frequent (19.5%) haplotype—MD4 (∧GCCTA∧), was associated with an increase in LD

D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107 103

Table 5Effects of MDR1 haplotypes on lipids responsea, difference in average excess, andp-value for the differences in effects

Haplotype Int 5 Ex13/G412G

Int 13 Int 17 Ex22/A893S

Hapfreq

Lipidchange(%)

p-Value r2 Difference in average excessb andp-valuec

MDR1-h1 MDR1-h3 MDR1-h2 MDR1-h10 MDR1-h4

LDL-CMDR1-h9 G T C A C 0.052 −0.352 0.349 0.012 0.005 0.005 0.058 0.084 0.560

0.86 0.85 0.49 0.54 0.06MDR1-h1 G C C A A 0.130 −0.332 0.204 0.021 0.000 0.030 0.048 0.460

0.99 0.42 0.58 0.03MDR1-h3 T T T T C 0.084 −0.291 0.342 0.012 0.028 0.047 0.460

0.61 0.04MDR1-h2 G T C T C 0.383 −0.130 0.454 0.008 0.002 0.257

0.89 0.05MDR1-h10 T T C A C 0.058 −0.030 0.933 0.000 0.210

0.24MDR1-h4 G C C T A 0.195 0.379 0.025 0.065

HDL-CMDR1-h9 G T C A C 0.052 −0.234 0.535 0.005 0.010 0.002 0.030 0.570 0.020

0.81 0.90 0.63 0.12 0.72MDR1-h1 G C C A A 0.130 −0.147 0.574 0.004 0.002 0.005 0.430 0.002

0.88 0.75 0.10 0.88MDR1-h3 T T T T C 0.084 −0.191 0.534 0.005 0.015 0.498 0.008

0.68 0.10 0.79MDR1-h2 G T C T C 0.383 −0.062 0.721 0.002 0.340 0.001

0.07 0.90MDR1-h10 T T C A C 0.058 0.619 0.081 0.040 0.380

0.12MDR1-h4 G C C T A 0.195 −0.059 0.732 0.002

TriglycerideMDR1-h9 G T C A C 0.052 0.178 0.503 0.010 0.001 0.028 0.024 0.172 0.017

0.90 0.44 0.02 0.12 0.55MDR1-h1 G C C A A 0.132 0.304 0.052 0.080 0.038 0.034 0.197 0.026

0.29 0.12 0.04 0.33MDR1-h3 T T T T C 0.086 −0.027 0.919 0.000 0.000 0.061 0.001

0.95 0.30 0.86MDR1-h2 G T C T C 0.375 −0.027 0.887 0.000 0.067 0.000

0.14 0.86MDR1-h10 T T C A C 0.059−0.461 0.016 0.119 0.081

0.18MDR1-h4 G C C T A 0.197 0.347 0.252 0.028

Values in bold fonts are statistically significant.a Standardized age- and sex-adjusted percent change.b The upper number designates difference in Average Excess (Deviation from the Population Mean).c The lower number designatesp-value as determined from a permutation test.

C response when compared to the overall sample meanresponse. This haplotype explained 6.5% of the variationin LDL-C response as compared to 5.4% of the variationthat was explained by BMI age and gender. The LDL-C response of subjects harboring the MDR1-h4 haplotypewas significantly greater than the response of subjects har-boring MDR1-h1 (∧GCCAA∧); of subjects with MDR1-h2(∧GTCTC∧); and of subjects with MDR1-h3 (∧TTTTC∧).There were no significant associations between the adjustedHDL-C response and MDR1 haplotypes (Table 5). Univariateassociation has shown that under an additive model, the lessfrequent (6%) haplotype—MDR1-h10 (∧TTCAC∧), showeda significant decrease in TG response, as compared to theTG response of the overall sample. This haplotype explainedapproximately 12% of the variation in TG response as com-pared to 2.2% of the TG variation that was explained by BMI

age and gender. The permutation tests indicated that the TGresponse of subjects harboring the MDR1-h10 haplotype, wassignificantly lower than the TG response of subjects harbor-ing MDR1-h1 haplotype (∧GCCAA∧) (Table 5).

Under a multivariate additive model, the presence of asingle copy of the CETP-H13 haplotype showed a signifi-cantly independent increase in LDL-C response (b = 0.713;95% CI = 0.10–1.33), when compared to the response of areference group including CETP-H2 and a combined groupof uncommon haplotypes (Table 6). Similar findings wereobserved when the reference group was selected to beCETP-H7 and a combined group of uncommon haplotypes.CETP-H5 haplotype was associated with a decreased HDL-C response (b = 0.62, 95% CI = 0.09–1.14) when comparedwith the CETP-H7 and a combined group of uncommon hap-lotypes reference group. In accordance with the univariate

104D

.Bercovich

etal./Atherosclerosis

185(2006)

97–107Table 6Multivariate regression coefficients (95% confidence intervals) from models of plasma lipids responsea on CETP and MDR1 haplotypes

Haplotype Reference = CETP-H2;otherb Reference = CETP-H7;otherb

LDL-C HDL-C TG LDL-C HDL-C TG

CETPCETP-H2 – – – 0.215 (−0.230–660) 0.121 (−0.316–0.558) −0.269 (−0.700–0.162)CETP-H7 −0.132 (−0.587–0.323) −0.295 (−0.736–0.146) 0.062 (−0.389–0.513) – – –CETP-H5 0.223 (−0.330–0.779) 0.531 (−0.006–1.068) −0.704 [(−1.255)–(−0.153)] 0.255 (−0.280–0.790) 0.615 (0.090–1.140) −0.709 [(−1.232)–(−0.186)]CETP-H8 0.021 (−0.579–0.621) −0.375 (−0.957–0.207) 0.212 (−0.370–0.794) 0.002 (−0.596–0.600) −0.339 (−0.927–0.249) 0.258 (−0.318–0.834)CETP-H13 0.713 (0.098–1.328) −0.156 (−0.754–0.442) 0.307 (−0.289–0.903) 0.721 (0.108–1.334) −0.140 (−0.742–0.462) 0.301 (−0.289–0.891)

Haplotype Reference = MDR1-h2;otherb Reference = MDR1-h4;otherb

LDL-C HDL-C TG LDL-C HDL-C TG

MDR1MDR1-h2 – – – −0.249 (−0.604–0.106) −0.127 (−0.488–0.234) −0.075 (−0.259–0.109)MDR1-h4 0.286 (−0.075–0.647) −0.058 (−0.428–0.312) −0.011 (−0.197–0.175) – – –MDR1-h1 −0.321 (−0.893–0.251) −0.130 (−0.716–0.456) 0.154 (−0.142–0.450) −0.513 (−1.050–0.024) −0.112 (−0.661–0.437) 0.154 (−0.120–0.428)MDR1-h3 −0.272 (−0.885–0.341) −0.134 (−0.763–0.495) −0.096 (−0.412–0.220) −0.503 (−1.138–0.132) −0.186 (−0.835–0.463) −0.132 (−0.453–0.189)MDR1-h10 −0.133 (−0.888–0.622) 0.513 (−0.261–1.287) −0.298 (−0.686–0.090) −0.309 (−1.036–0.418) 0.543 (−0.198–1.284) −0.292 (−0.662–0.078)MDR1-h9 −0.393 (−1.152–0.366) −0.201 (−0.979–0.577) 0.141 (−0.249–0.531) −0.569 (−1.326–0.188) −0.216 (−0.988–0.556) 0.126 (−0.258–0.510)

Values in bold fonts are statistically significant.a Standardized age- and sex-adjusted percent change.b ‘Other’ refers to a combined group of uncommon haplotypes.

Table 7Multivariate regression coefficients (95% confidence intervals) from models of plasma lipids responsea on CETP and MDR1 haplotypes

Haplotype Reference = CETP-H2 Reference = CETP-H7

LDL-C HDL-C TG LDL-C HDL-C TG

CETP-H 2 – – – −0.275 (−0.192–0.742) 0.138 (−0.327–0.602) −0.272 (−0.733–0.189)CETP-H 7 −0.190 (−0.653–0.274) −0.303 (−0.757–0.150) 6.8× 10−2 (−0.397–0.533) – – –CETP-H 5 0.194 (−0.366–0.754) 0.607 (0.060–1.155) −0.725 [(−1.288)–(−0.162)] −0.252 (−0.290–0.793) 0.696 (0.158–1.234) −0.744 [(−1.281)–(−0.207)CETP-H 8 0.186 (−0.456–0.827) −0.409 (−1.036–0.218) 0.153 (−0.477–0.784) 0.159 (−0.482–0.799) −0.381 (−1.018–0.256) 0.204 (−0.423–0.381)CETP-H 13 0.827 (0.185–1.468) −0.195 (−0.823–0.432) 0.181 (−0.450–0.811) 0.834 (0.196–1.472) −0.185 (−0.819–0.449) 0.176 (−0.448–0.801)

MDR1-h 2 −4.5× 10−2 (−0.594–0.503) −0.425 (−0.961–0.112) −0.365 (−0.910–0.180) −5.8× 10−2 (−0.604–0.487) −0.439 (−0.982–0.103) −0.353 (−0.893–0.187)MDR1-h 4 0.229 (−0.331–0.790) −0.385 (−0.933–0.164) −0.298 (−0.850–0.253) 0.241 (−0.317–0.798) −0.378 (−0.932–0.177) −0.308 (−0.854–0.238)MDR1-h 1 −0.466 (−1.152–0.219) −0.101 (−0.771–0.570) −9.9× 10−2 (−0.773–0.575) −0.412 (−1.099–0.274) −6.6× 10−2 (−0.749–0.616) −0.149 (−0.821–0.523)MDR1-h 3 −0.216 (−0.980–0.549) −0.568 (−1.315–0.180) −0.417 (−1.168–0.335) −0.190 (−0.951–0.570) −0.543 (−1.299–0.212) −0.435 (−1.179–0.309)MDR1-h 10 −0.156 (−0.964–0.652) 0.364 (−0.426–1.155) −0.817 [(−1.612)–(−0.022)] −0.167 (−0.971–0.637) 0.385 (−0.414–1.184) −0.792 [(−1.579)–(−0.005)MDR1-h 9 −0.632 (−1.479–0.215) −0.483 (−1.312–0.345) 0.102 (−0.731–0.935) −0.680 (−1.531–0.170) −0.542 (−1.298–0.393) 0.181 (−0.652–1.013)

Values in bold fonts are statistically significant.a Standardized age- and sex-adjusted percent change.

D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107 105

analysis, the presence of a single copy of CETP-H5 haplo-type was associated with a significant independent effect onTG response.

The results from multivariate association of the collec-tively six MDR1 haplotypes with lipids and lipoproteinsresponse are also shown inTable 6. This analysis providedno suggestion that adjusted LDL-C, HDL-C, and TG percentchanges upon fluvastatin treatment differed considerably byMDR1 haplotypes.

As shown for the separate multivariate models, the pres-ence of a single copy of CETP-H13 haplotype demon-strated a significant independent association with LDL-Cresponse (b = 0.827; 95% CI = 0.19–1.47;b = 0.834; 95%CI = 0.20–1.47), when either CETP-H2 or CETP-H7 and acombined group of uncommon haplotypes served as ref-erence groups (Table 7). When analyzing HDL-C and TGchanges following pharmacological treatment, CETP-H5was significantly associated with poor HDL-C and TGresponse. When the multivariate model included all com-mon haplotypes in both genes, the presence of MDR1-h10haplotype was found to be independently associated with TGresponse suggesting an additive effect of the two genes onthe level of response to fluvastatin.

4. Discussion

ffecto onset t ofC TGr esultsf Ani fromt y oft FHp hichp redd con-t ande iza-t

oversa thed therh ns inT onc asn teinr ped oorH om-p ec lyd L-C

response to fluvastatin (p = 0.03) (shown inSupplementaryTable 6). However, upon analyzing haplotypes, both CETP-H5 and CETP-H8 are comprised of the−867 ‘A’ allele, yetone is highly associated with poor response (p = 0.005) andthe other with a modest improved HDL-C response to fluvas-tatin (p = 0.08).

The association of CETP haplotypes with response tostatins was first demonstrated by Winkelmann et al.[21].These authors showed in 98 patients with untreated dys-lipidemia that individual SNPs and CETP haplotypes weresignificantly associated with CETP enzyme mass and activ-ity. In addition, they demonstrated a significant associationbetween certain CETP haplotypes and the effect of statinson HDL-C and triglycerides. Our study substantiates theirfindings and in addition demonstrates an effect on LDL-Clowering properties of fluvastatin. In the two studies the over-all changes in lipid levels following treatment were differentprobably due to the mixed nature of the studied population,and the types of statins used. In our study we analyzed arelatively homogenous group of FH patients and they wereall treated with fluvastatin. It may be argued that generallyFH would require more intensive lipid reduction, yet fluvas-tatin that was administered in the current study may be abetter agent to reveal subtle differences compared to a “super-statin”. Moreover, by initial focusing on individuals with anextreme phenotype, such as FH on one hand, and by reducingt andd sis isi

od-e ectH thea ls isn asedc r-v rce-t as-t

ertainM nder’M pesi ge inL thatc . Wec iffer-e 3A 4a socia-t tic( ated[ d ont atinc ringe s int treat-m is 2S was

In the current study, we demonstrate an independent ef CETP haplotypes on the degree of plasma lipid resp

o fluvastatin. In addition, we observed an additive effecETP and MDR1 polymorphisms on the magnitude of

esponse. The capacity to demonstrate such effects rrom the nature of the described intervention study.mportant advantage of our single center study stemshe clinical settings that it offers. First, the homogeneithe studied population, being molecularly characterizedatients, suggests a common pathological process for wharmacological intervention is introduced. The monitoiet and the constant dose of medication, together with

rolled and optimized compliance merge the phenotypenvironmental effects to a recognized level of standard

ion.Our study reemphasizes the preference of haplotypes

ingle polymorphisms. Previous studies[10,20,32]revealedn association between the CETP TaqIB variant andegree of response to lipid modifying agents. On the oand, de Grooth et al. did not demonstrate that variatioaqIB could predict the beneficial effect of pravastatinoronary events[33]. In our study, TaqIB polymorphism wot associated with the degree of plasma lipid and lipoproesponse (Tables 2 and 3). When using our CETP haplotyata, both CETP-H5 and CETP-H8 which represent ‘pDL response’ and ‘better HDL response’ haplotypes crise the ‘A’ TaqIB variant. Similarly, if we examine thontribution of the−867 CETP ‘A’ allele, we may incorrectraw a conclusion that it is associated with a higher HD

he effect of ‘non-genetic factors’, such as complianceiet on the other hand, the accuracy of the genetic analy

ncreased.CETP is a key protein involved in the intravascular rem

ling of HDL thus it is understandable that it may affDL-C and TG levels in response to statins. However,ssociation between genetic variants and LDL-C leveot straightforward. CETP deficient subjects show increatabolic rates of LDL[14] and in accord with this obseation a recent paper showed that CETP inhibition by torapib leads to a greater LDL-C reduction following atorvatin administration[34].

In the present study, we present data that associates cDR1 haplotypes and the degree of response. A ‘respoDR1-h4 that accounts for 20% of the analyzed haploty

s shown to be responsible for 6.5% of the observed chanDL-C response which is comparable to the variabilityan be accounted for by BMI, age, and gender (5.4%)hose to analyze genetic variants in MDR1 despite its dnt metabolism path that involves CYP2C9 and not CYPs other statins, such as atorvastatin. Previously, the as

ion betweenCYP2C9 polymorphisms and pharmacokinePK) properties and efficacy of fluvastatin was evalu9]. The PK of both enantiomers of fluvastatin dependehe CYP2C9 genotype yet differences in plasma fluvastoncentrations were not reflected by cholesterol loweffect. Kajinami et al. demonstrated that polymorphism

he MDR1 gene influence the response to atorvastatinent[7]. These authors used for their haplotype analysNPs, namely G2677T and C3435T. The former SNP

106 D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107

also analyzed in our study (Ex22/A893S) yet for haplotypeconstruction we used additional SNPs distributed through-out the gene. Several statins, yet not fluvastatin, modulateMDR1 properties and may introduce an uncontrolled variablewhen assessing the genetic effect of MDR1[6]. Although inour studies MDR1 was considered mainly as modulating PKproperties, evidence suggests that MDR1 may participate inlipid translocation across membranes and as such may alsomodulate pharmacodynamic (PD) properties[35,36]. On theother hand, CETP which was initially considered a PD mod-ulator was shown to participate in lipophilic drug distributionand metabolism and thus may also serve as a PK modulator[37,38].

In conclusion, the independent additive effect of SNP hap-lotypes in CETP and MDR1 bring up the cumulative effectof genetic PD and PK factors, such as CETP and MDR1.We suggest that the integration of data obtained from geneticpolymorphisms in CETP and MDR1 may serve as a paradigmfor quantitative prediction of individual response to statins,incorporating both PK and PD variance.

Some study limitations need to be considered when dis-cussing the results of the current investigation. These relatemainly to the modest number of the patients enrolled in thestudy and to the fact that fluvastatin was the only statin used.Thus, it is impossible to generalize the data to other statins andto polygenic dyslipidemias. Also, our study requires replica-t eticP statinr

A

n bef is.2

R

easeeath

broad–57.ent

e theher-rch

is formics

ncer

udyalshar-

[6] Bogman K, PeyerAK, Torok M, Kusters E, Drewe J. HMG-CoAreductase inhibitors and P-glycoprotein modulation. Br J Pharmacol2001;132:1183–92.

[7] Kajinami K, Brousseau ME, Ordovas JM, Schaefer EJ. Polymor-phisms in the multidrug resistance-1 (MDR1) gene influence theresponse to Atorvastatin treatment in a gender-specific manner. AmJ Cardiol 2004;93:1046–50.

[8] Williams D, Feely J. Pharmacokinetic–pharmacodynamic drug inter-actions with HMG-CoA reductase inhibitors. Clin Pharmacokinet2002;41:343–70.

[9] Kirchheiner J, Kudlicz D, Meisel C, et al. Influence of CYP2C9 poly-morphisms on the pharmacokinetics and cholesterol-lowering activityof (−)-3S,5R-fluvastatin and (+)-3R,5S-fluvastatin in healthy volun-teers. Clin Pharmacol Ther 2003;74:186–94.

[10] Kuivenhoven JA, Jukema JW, Zwinderman AH, et al. The role ofa common variant of the cholesteryl ester transfer protein genein the progression of coronary atherosclerosis. N Engl J Med1998;338:86–93.

[11] Bruce C, Tall AR. Cholesteryl ester transfer protein, reverse choles-terol transport and atherosclerosis. Curr Opin Lipidol 1995;6:306–11.

[12] Brown ML, Inazu A, Hesler CB, et al. Molecular basis of lipidtransfer protein deficiency in a family with increased high-densitylipoproteins. Nature 1989;342:448–51.

[13] Koizumi J, Mabuchi H, Yoshimura A, et al. Deficiency of serumcholesteryl-ester transfer activity in patients with familial hyperal-phalipoproteinaemia. Atherosclerosis 1985;58:175–86.

[14] Ikewaki K, Nishiwaki M, Sakamoto T, et al. Increased catabolic rateof low density lipoproteins in humans with cholesteryl ester transferprotein deficiency. J Clin Invest 1995;96:1573–81.

[15] Kuivenhoven JA, de Knijff P, Boer JMA, et al. Heterogeneityntra-

Biol

[ ro-n theper-

[ smaor-

Res

[ unc-ans-nsityreg-

[ hiphism

ype 2

[ terylrvas-

[ ofying96.

[ mia.lers of

[ ilial

[ panelrol in

ent

ion in additional patient groups and obviously more genK and PD genes should be studied as modulators of

esponse.

ppendix A. Supplementary data

Supplementary data associated with this article caound, in the online version, atdoi:10.1016/j.atheroscleros005.05.025.

eferences

[1] The Long-Term Intervention with Pravastatin in Ischaemic Dis(LIPID) Study Group. Prevention of cardiovascular events and dwith pravastatin in patients with coronary heart disease and arange of initial cholesterol levels. N Engl J Med 1998;339:1349

[2] Pearson TA, Laurora I, Chu H, Kafonek S. The lipid treatmassessment project (L-TAP): a multicenter survey to evaluatpercentages of dyslipidemic patients receiving lipid-lowering tapy and achieving low-density lipoprotein cholesterol goals. AIntern Med 2000;160:459–67.

[3] Evans WE, Johnson JA. Pharmacogenomics: the inherited basinterindividual differences in drug response. Annu Rev GenoHum Genet 2001;2:9–39.

[4] Leveque D, Jehl F. P-glycoprotein and pharmacokinetics. AnticaRes 1995;15:331–6.

[5] Fellay J, Marzolini C, Meaden ER, et al. Swiss HIV Cohort Stresponse to antiretroviral treatment in HIV-1-infected individuwith allelic variants of the multidrug resistance transporter 1: a pmacogenetics study. Lancet 2002;359(9300):30–6.

at the CETP gene locus: influence on plasma CETP concetions and HDL cholesterol levels. Arterioscler Thromb Vasc1997;17:560–8.

16] Bruce C, Sharp DS, Tall AR. Relationship of HDL and conary heart disease to a common amino acid polymorphism icholesteryl ester transfer protein in men with and without hytriglyceridemia. J Lipid Res 1998;39:1071–8.

17] Bernard S, Moulin P, Lagrost L, et al. Association between plaHDL-cholesterol concentration and TaqIB CETP gene polymphism in non-insulin-dependent diabetes mellitus. J Lipid1998;39:59–65.

18] Dachet C, Poirier O, Cambien F, Chapman J, Rouis M. New ftional promoter polymorphism, CETP/-629, in cholesteryl ester trfer protein (CETP) gene related to CETP mass and high-delipoprotein cholesterol levels: role of Sp1/Sp3 in transcriptionalulation. Arterioscler Thromb Vasc Biol 2000;20:507–15.

19] Kawasaki I, Tahara H, Emoto M, Shoji T, Nishizawa Y. Relationsbetween TaqIB cholesteryl ester transfer protein gene polymorpand macrovascular complications in Japanese patients with tdiabetes. Diabetes 2002;51:871–4.

20] van Venrooij FV, Stolk RP, Banga JD, et al. Common cholesester transfer protein gene polymorphisms and the effect of atotatin therapy in type 2 diabetes. Diab Care 2003;26:1216–23.

21] Winkelmann BR, Hoffmann MM, Nauck M, et al. Haplotypesthe cholesteryl ester transfer protein gene predict lipid-modifresponse to statin therapy. Pharmacogenomics J 2003;3:284–

22] Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesteroleIn: Scriver SR, Beaudet AL, Valle D, Sly WS, Childs B, KinzKW, Vogelstein B, editors. The metabolic and molecular baseinherited disease. New York: McGraw-Hill; 2001. p. 2863–913.

23] Reshef A, Nissen H, Triger L, et al. Molecular genetics of famhypercholesterolemia in Israel. Hum Genet 1996;98:581–6.

24] National Cholesterol Education Program. Report of the experton detection, evaluation, and treatment of high blood cholesteadults (Adult Treatment Panel II). Washington, DC: US Departmof Health and Human Services; 1993.

D. Bercovich et al. / Atherosclerosis 185 (2006) 97–107 107

[25] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the con-centration of low density lipoprotein cholesterol in plasma withoutuse of the preparative ultracentrifuge. Clin Chem 1972;18:499–502.

[26] Bodamer OA, Bercovich D, Schlabach M, et al. Use of denaturingHPLC to provide efficient detection of mutations causing familialhypercholesterolemia. Clin Chem 2002;48:1913–8.

[27] Carlson CS, Eberle MA, Rieder MJ, et al. Selecting a maxi-mally informative set of single-nucleotide polymorphisms for asso-ciation analyses using linkage disequilibrium. Am J Hum Genet2004;74:106–20.

[28] Stephens M, Smith NJ, Donnelly P. A new statistical method forhaplotype reconstruction from population data. Am J Hum Genet2001;68:978–89.

[29] Snedecor GW, Cochran WG. Statistical methods. Ames: Iowa StateUniversity Press; 1980.

[30] Burt RD, Vaughan TL, McKnight B, et al. Associations betweenhuman leukocyte antigen type and nasopharyngeal carcinoma in Cau-casians in the United States. Cancer Epidemiol Biomarkers Prev1996;5:879–87.

[31] Edgington ES. Randomization tests. New York: Marcel Deckker Inc.;1995.

[32] Carlquist JF, Muhlestein JB, Horne BD, et al. The cholesteryl estertransfer protein Taq1B gene polymorphism predicts clinical benefitof statin therapy in patients with significant coronary artery disease.Am Heart J 2003;146:1007–14.

[33] de Grooth GJ, Zerba KE, Huang SP, et al. The cholesteryl estertransfer protein (CETP) TaqIB polymorphism in the cholesterol andrecurrent events study: no interaction with the response to pravas-tatin therapy and no effects on cardiovascular outcome: a prospectiveanalysis of the CETP TaqIB polymorphism on cardiovascular out-come and interaction with cholesterol-lowering therapy. J Am CollCardiol 2004;43:854–7.

[34] Brousseau ME, Schaefer EJ, Wolfe ML, et al. Effects of an inhibitorof cholesteryl ester transfer protein on HDL cholesterol. N Engl JMed 2004;350:1505–15.

[35] Kirchheiner J, Kudlicz D, Meisel C, et al. Influence of CYP2C9 poly-morphisms on the pharmacokinetics and cholesterol-lowering activityof (−)-3S,5R-fluvastatin and (+)-3R,5S-fluvastatin in healthy volun-teers. Clin Pharmacol Ther 2003;74:186–94.

[36] van Helvoort A, Smith AJ, Sprong H, et al. MDR1 P-glycoprotein isa lipid translocase of broad specificity, while MDR3 P-glycoproteinspecifically translocates phosphatidylcholine. Cell 1996;87:507–17.

[37] Raggers RJ, van Helvoort A, Evers R, van Meer G. The humanmultidrug resistance protein MRP1 translocates sphingolipid analogsacross the plasma membrane. J Cell Sci 1999;112:415–22.

[38] Kwong M, Wasan KM. Cholesteryl ester transfer protein facilitatesthe movement of water-insoluble drugs between lipoproteins: a novelbiological function for a well-characterized lipid transfer protein.Biochem Pharmacol 2002;64:1669–75.