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CLINICAL RESEARCHPrevention

The risk of tendon xanthomas in familialhypercholesterolaemia is influenced by variationin genes of the reverse cholesterol transportpathway and the low-density lipoproteinoxidation pathwayDaniella M. Oosterveer1, Jorie Versmissen1, Mojgan Yazdanpanah1,Joep C. Defesche2, John J.P. Kastelein2, and Eric J.G. Sijbrands1*1Department of Internal Medicine D435, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands; and 2Department of Vascular Medicine,Amsterdam Medical Center, Amsterdam, The Netherlands

Received 9 July 2009; revised 27 October 2009; accepted 17 November 2009; online publish-ahead-of-print 16 December 2009

Aims The presence of tendon xanthomas is a marker of high risk of cardiovascular disease (CVD) among patients withfamilial hypercholesterolaemia (FH). Therefore, xanthomas and atherosclerosis may result from the same pathophy-siological mechanisms. Reverse cholesterol transport (RCT) and low-density lipoprotein (LDL) oxidation are patho-physiological pathways of atherosclerosis, and it is well established that genetic variation in these pathways influencesCVD risk. We therefore determined whether genetic variation in these pathways is also associated with the occur-rence of tendon xanthomas in FH patients.

Methodsand results

Four genetic variants in each pathway were genotyped in 1208 FH patients. We constructed a gene-load score forboth pathways. The odds of xanthomas increased with the number of the risk alleles in the RCT pathway (OR 1.21,95% CI 1.081.36, Ptrend 0.0014). Similarly, higher numbers of risk alleles in the LDL oxidation pathway wereassociated with the presence of xanthomas (OR 1.24, 95% CI 1.081.41, Ptrend 0.0015).

Conclusion The presence of tendon xanthomas in FH patients is associated with genetic variation in the RCT and LDL oxidationpathways. These results support the hypothesis that xanthomas and atherosclerosis share pathophysiologicalmechanisms.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -Keywords Xanthomas Familial hypercholesterolaemia Atherosclerosis Reverse cholesterol transport LDL oxidation

IntroductionTendon xanthomas are cholesterol deposits in tendons. Theirpresence is a clinical sign of familial hypercholesterolaemia (FH,OMIM # 143890), an inherited disorder characterized by high low-density lipoprotein (LDL) cholesterol levels and premature cardio-vascular disease (CVD). Recently, we demonstrated that the pres-ence of xanthomas associates with a three-fold higher risk of CVDin patients with FH.1 This finding suggests that xanthomas andatherosclerosis share pathophysiological mechanisms.

Reverse cholesterol transport (RCT) and LDL oxidation aretwo important pathophysiological pathways of atherosclerosis.The RCT pathway promotes cholesterol efflux from the arterialwall macrophages to the liver.2 The ATP-binding cassette transpor-ter A1 (ABCA1) enhances the cholesterol flux from macrophagesto apolipoprotein AI (ApoAI) on high-density lipoprotein (HDL)particles. After transfer of cholesterol esters from these HDL par-ticles to LDL particles by cholesteryl-ester transfer protein(CETP), the cholesterol is cleared together with its LDL particleby the LDL receptor (LDLR) on the liver. Variation in the genes

* Corresponding author. Tel: 31 10 70 33 283, Fax: 31 10 70 33 269, Email: e.sijbrands@erasmusmc.nlPublished on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

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of these proteins in this candidate pathway has been associatedwith CVD risk factors and the risk of CVD.2 6

Similarly, genes in the LDL oxidation pathway also significantlyinfluence atherosclerosis. Apolipoprotein AIV (ApoAIV), paraoxo-nase 1 (PON1), nitric oxide synthase 3 (NOS3), and cystathioneb-synthase (CBS) are antioxidants, which directly or indirectly neu-tralize free radicals and therefore diminish and prevent LDL oxi-dation, an important step in the formation of atheroscleroticplaques.7 9

Because the effect of a single genetic variant is usually small andcan be compensated by variants in other genes in the samepathway, pathway-based analyses are more informative.10

A number of studies suggest that the RCT pathway and the LDLoxidation pathway influence each other.11 13 We thereforedetermined the combined effect of genetic variation in each ofthese two pathways on the risk of tendon xanthomas and assessedinteraction between these pathways.

Methods

Study populationHeterozygous FH patients were recruited from 27 lipid clinics in theNetherlands between 1989 and 2002. Detailed information on thestudy design and population has been described previously.14,15

Briefly, when FH is suspected in a patient, a blood sample is routinelysubmitted to a central laboratory for LDLR mutation analysis. Out of arandom selection of 2400 unrelated patients with clinical FH, weincluded 1208 patients with genetically confirmed heterozygous FHand with information on the presence or absence of xanthomas. Themajority was of Caucasian descent (99%). The Ethics Committee ofeach participating hospital approved the protocol, and informedconsent was required to be included in the study.

The level of total cholesterol, HDL cholesterol, and triglycerideswas measured using standard methods in patients withdrawn fromlipid-lowering medication at least 6 weeks before blood sampling.The LDL cholesterol levels were calculated using the Friedewaldformula.16

Xanthoma definitionThe presence of tendon xanthomas was defined as (i) the presence ofone or more unequivocal tendon xanthomas, (ii) the presence of aclear thickening of both Achilles tendons determined by observationand palpation during physical examination,17 (iii) a history of surgicalremoval of one or more xanthomas. As the presence of xanthomaswas doubted in 68 patients, these patients were excluded from thestudy. The characteristics of these patients did not differ from thosein the population examined (data not shown).

Selection and genotyping of the singlenucleotide polymorphismsOn the basis of a panel of CVD candidate single nucleotide poly-morphisms (SNPs)18 and existing data derived from literature, weselected four genes of the RCT pathway and four genes of the LDLoxidation pathway. The selection of the variants in these genes wasbased on functionality and/or associations with CVD in earlierstudies and HapMap tagging. In the RCT pathway, we chosers1800977 of the ABCA1 gene,2,19 rs670 of the ApoA1 gene,2,20,21

rs708272 of the CETP gene,2,2224 and rs5742911 of the LDLRgene.2,25,26 We selected rs675 of the ApoA4 gene,9,27 rs854560 of

the PON1 gene,28 31 rs3918226 of the NOS3 gene,8 and rs5742905of the CBS gene32 34 of the LDL oxidation pathway.

The risk allele for each polymorphism was chosen on the basis ofthe previous studies examining associations with CVD or intermediatetraits: the C allele of rs1800977 of the ABCA1 gene,19 the A allele ofrs670 of the ApoA1 gene,21 the T allele of rs708272 the CETPgene,35,36 the A allele of rs854560 of the PON1 gene,28 the C alleleof rs3918226 of the NOS3 gene,37 and the T allele of rs5742905 ofthe CBS gene.34 Owing to discrepancies in literature and to lack ofstudies with a population similar to ours, we chose the risk allele forthe following polymorphisms based on our data: the G allele of theLDLR gene polymorphism and the T allele of rs675 of the ApoA4 gene.

DNA was extracted from peripheral blood samples using standardmethods.38 To genotype the ApoA1 SNP rs670, we used TaqManallelic discrimination Assays-By-Design (Applied Biosystems, FosterCity, CA, USA). The forward and reverse primer sequences were50-CAGGACCAGTGAGCAGCAA-30 and 50-GGCTGGGAGGCTGATAAGC-30, respectively. The probes used were VIC-CCAGCCCTGGCCCT and FAM-CCAGCCCTGGCCCT. The polymorphismsof the other genes were performed previously by Roche MolecularSystems, CA, USA. The genotypes were generated using PCR andimmobilized probe assay, as described previously.18 Results werescored blinded to the presence of xanthomas.

Statistical analysisAll statistical analyses were performed with SPSS 14.0 for Windows.The x2 test and t-test were used to compare characteristicsbetween patients with xanthomas and those without. HardyWeinberg equilibriums (HWE) of the polymorphisms were testedusing a x2 test.

We used logistic regression models to calculate odds ratios(ORs) and 95% confidence intervals (CIs) for each individual poly-morphism, adjusted for age and sex (Model 1) and additionallyadjusted for body mass index (BMI) and untreated LDL cholesterollevels (Model 2).

Haplotype analysis was not possible because of low linkage disequi-librium (LD) between the different polymorphisms, as one wouldexpect of polymorphisms located at different chromosomes orlocated more than 35 000 000 bp apart from each other. Low LD isa prerequisite for gene-load score analysis.

To calculate the gene-load score, the absence of risk alleles wascoded 0, the presence of one risk allele was coded as 1, and the pres-ence of two risk alleles was coded as 2. To assess the combined effectsof the four polymorphisms of the RCT pathway genes, a variable wascreated which included these four polymorphisms. This resulted in ascore ranging from 0 to 8, representing the total number of riskalleles. Similarly, we assessed the combined effects of the risk allelesof the four LDL oxidation pathway genes. Because very few individualscarried no or little risk alleles for the RCT pathway, we combined 0and 1 risk alleles into one group in our logistic regression analyses(adjusted for age and sex). For LDL oxidation pathway gene-loadscore, we combined 1 to 3 risk alleles into one group.

To assess if there was evidence for multiplicative interactionbetween the pathways, we performed a logistic regression analysisincluding the grouped RCT pathway, the grouped LDL oxidation,and an interaction term, adjusted for age and sex.

ResultsTable 1 shows the general characteristics of our study population.The FH patients with tendon xanthomas were older, had

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higher BMI, higher total cholesterol and LDL cholesterollevels than those without xanthomas (P, 0.01). The frequencyof CVD was higher among patients with xanthomas (P, 0.01).

HDL cholesterol level was not associated with xanthomas(P 0.41).

All polymorphisms were in HWE, with the exception of the CBSrs5742905 polymorphism, which was not in HWE due to absenceof the TT genotype (P 0.00053).

The odds of xanthomas per individual SNP in each pathwayis shown in Table 2. In the RCT pathway, the T allele ofthe CETP rs708272 polymorphism was significantly associatedwith the presence of xanthomas (OR 1.25, 95% CI 1.041.50,P 0.02). More carriers of a G allele of the LDLR rs5742911polymorphism had xanthomas than non-carriers (additivemodel: OR 1.28, 95% CI 1.041.58, P 0.02, Table 2).

Table 2 shows that, in the LDL oxidation pathway, carriers of a Tallele of the ApoA4 polymorphism had a higher odds of xanthomasthan those carrying A alleles (OR 1.31, 95% CI 1.051.64, P 0.02). The other polymorphisms were not significantly associatedwith xanthomas.

Figure 1 shows the gene-load scores of the RCT and theLDL oxidation pathway. The odds of xanthomas increased withthe number of the risk alleles in the RCT pathway (OR 1.21,95% CI 1.081.36, Ptrend 0.0014). Each increase in thenumber of risk alleles in the LDL oxidation pathway wasaccompanied by an increase in the frequency of xanthomas (OR1.24, 95% CI 1.081.41, Ptrend 0.0015). Although theseassociations were significant, the areas under the ROC curves(AUCs) found were relatively small: for the RCT pathway 0.56(95% CI 0.520.60) and for the LDL oxidation pathway 0.55(95% CI 0.520.59).

We did not find evidence for multiplicative interaction betweenthe RCT and the LDL oxidation pathway (OR interaction 0.98,

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Table 1 General characteristics of the studypopulation

Characteristics Xanthomas P-value

Present Absent

Number 567 641

Age (years) 43.0+12.1 40.3+12.7 ,0.01

Male gender (%) 45.9 47.0 0.70

Smoking (%) 69.9 67.4 0.37

Body mass index (kg/m2) 24.9+3.5 24.2+3.2 ,0.01

Diabetes (%) 2.1 1.3 0.24

Systolic blood pressure(mmHg)

131.9+17.7 131.5+18.0 0.69

Diastolic blood pressure(mmHg)

80.3+10.8 79.5+10.3 0.21

CVD (%) 27.0 19.0 ,0.01

No statin treatment (%)a 24.2 22.8 0.72

Total cholesterol (mmol/L) 10.74+2.10 9.45+1.91 ,0.01

LDL cholesterol (mmol/L) 8.65+1.99 7.37+1.88 ,0.01

HDL cholesterol (mmol/L) 1.13+0.32 1.15+0.33 0.41

Triglycerides (mmol/L) 1.48+0.76 1.39+0.72 0.08

Continuous variables: mean+ standard deviation.aNo statin treatment was defined as no statin treatment before cardiovascularevent.

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Table 2 The odds of xanthomas and variants in genes of two pathophysiological pathways

Gene (rs number) Allele Frequency Model 1a Model 2b

OR 95% CI P-value OR 95% CI P-value

The reverse cholesterol transport pathway

ABCA1 (rs1800977) T 0.34 (ref) (ref)C 0.66 1.11 0.921.34 0.27 1.12 0.931.36 0.24

ApoA1 (rs670) G 0.85 (ref) (ref)A 0.15 1.10 0.861.40 0.45 1.11 0.861.43 0.43

CETP (rs708272) C 0.57 (ref) (ref)T 0.43 1.25 1.041.50 0.02 1.23 1.021.49 0.03

LDLR (rs5742911) A 0.76 (ref) (ref)G 0.24 1.28 1.041.58 0.02 1.30 1.051.62 0.02

The LDL oxidation pathway

ApoA4 (rs675) A 0.19 (ref) (ref)T 0.81 1.31 1.051.64 0.02 1.29 1.031.64 0.03

PON1 (rs854560) T 0.37 (ref) (ref)A 0.63 1.10 0.921.32 0.31 1.06 0.881.29 0.53

NOS3 (rs3918226) T 0.07 (ref) (ref)C 0.93 1.36 0.951.93 0.09 1.32 0.921.91 0.13

CBS (rs5742905) C 0.90 (ref) (ref)T 0.10 1.34 0.981.84 0.07 1.36 0.981.89 0.07

aLogistic Model 1: adjusted for age and sex.bLogistic Model 2: adjusted for age, sex, LDL cholesterol and BMI.

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95% CI 0.871.11, P 0.78). In line with this, combining the gene-load scores of both pathways showed a significant trend similar tothe ORs as the individual pathways (OR 1.24, 95% CI 1.121.37,P, 0.01, Figure 1C).

DiscussionIn this study, we have demonstrated that variants in genes of theRCT pathway and the LDL oxidation pathway are associatedwith the presence of tendon xanthomas in FH patients. Theodds of xanthomas increased with increasing numbers of riskalleles in the genes of both pathways.

In addition to the higher risk of CVD in FH patients with xantho-mas compared with that in those without xanthomas,1 there areother findings suggesting that xanthomas and atherosclerosis arerelated and share pathophysiological pathways. The compositionof xanthomas and atherosclerotic plaques show similarities asboth lesions consist of connective tissue matrix containing macro-phages transformed into foam cells.39 In addition, both xanthomasand plaques can be prevented or reduced by the use of cholesterol-lowering drugs.40 42 Recently, we have shown that variation in the5-lipoxygenase activation protein gene, which is involved in inflam-mation, contributed to the risk of xanthomas in FH.43 The presentstudy is the first study to extend the analysis of xanthomas togenetic variation in the RCT and LDL oxidation pathways.Previously, genetic variation in these pathways has been related toatherosclerosis or one of its risk factors.8,19 22,24 28,32,35,36,44

These findings strongly suggest that xanthomas and atherosclerosisshare pathogenic mechanisms.

We assigned the same score to each risk allele of each poly-morphism. As an alternative method, we also made a weightedgene score for each patient, in which the absence of risk alleleswas coded 0 and the presence of one or two risk alleles werecoded as 1 LN(OR) and 2 LN(OR), respectively.45 The result-ing continuous gene score was divided into five equal groups ofpatients. The results of this alternative approach were similar tothe unweighted approach (data not shown).

In gene-load score analyses, additive risk-allele effects are usedas markers of the susceptibility of particular pathway to deteriorateand contribute to signs and symptoms. The dosage effect observedin both pathways supports involvement of these pathways in thedevelopment of xanthomas. The effect of both the RCT pathwayand the LDL oxidation pathway clearly showed that the odds ofxanthomas increased according to the number of risk alleles.Although the previous literature has suggested an interactionbetween the studied pathways, we did not find a multiplicativeinteraction between the pathways.11 13 In line with this, combiningthe gene scores of both pathways showed a trend similar to that ofthe individual pathways.

The AUC values demonstrate that the gene-load scores of thesepathways contributed modestly to xanthomas, and other (unknown)pathogenetic factors need to be elucidated to understand the patho-genesis of xanthomas more completely. On the other hand, we mayhave underestimated the contribution of the two pathways by study-ing only a few genetic variants of each pathway.

HDL plays an important role in the RCT pathway; however,HDL cholesterol levels itself were not associated with thepresence of xanthomas (Table 1). Perhaps the functionality ofHDL particles is of more importance than the quantity. Previouslya probucol study indeed demonstrated that the reduced HDLparticle size was linked to xanthoma regression.46 Unfortunately,we had no data about the subdivision of the particles.

Figure 1 The risk of xanthomas per gene-load score of twopathophysiological pathways. *P, 0.0125. The numbersbetween bars below the columns denote the number of patientswith that particular number of risk genotypes. In the figure 98.8%confidence intervals are given. (A) Gene-load score of the reversecholesterol transport pathway. (B) Gene-load score of the LDLoxidation pathway. (C) Both pathways combined.

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In our study, the CBS rs5742905 polymorphism was out ofHWE. Because homozygosity of the CBS rs5742905 T alleleleads to CBS deficiency that causes hyperhomocystinemia(OMIM #236200),32,33 the absence of TT carriers in our popu-lation can be explained by the preferential referral of CBS-deficientpatients to other outpatient clinics. The results of the analyses,including and excluding the CBS variant, were similar (data notshown).

To maintain sufficient patients per number of risk alleles, wecould not include all genes involved both pathways. For example,both lecithin-cholesterol acyltransferase and scavenger-receptorB1 (SR-B1) are known to be involved in the RCT pathway too.Lecithin-cholesterol acyltransferase converts cholesterol in HDLparticles to cholesterol esters, and SR-B1 binds HDL particlesand facilitates removal of remaining cholesterol esters from theseparticles. However because of discrepancies in literature abouttheir importance in the RCT pathway and because of differencesin outcome per sex and age of polymorphism analysis,47 51 wedid not genotype a variant in these genes. Variation in anotherimportant candidate gene, the E2E3E4 variation in the apolipo-protein E (ApoE) gene, was previously found not to be associatedwith xanthomas.52 In addition, we decided not to include any poly-morphisms of genes involved in the more down-stream bile acidsynthesis and excretion.

It is well known that the natural history of atherosclerosis differsbetween the two sexes. Similar to CVD, the male gender is alsoassociated with the presence of xanthomas.1 In the presentstudy, the percentage of males was not significantly differentbetween the FH patients with and without tendon xanthomas.Stratified analyses of the individual polymorphisms demonstratedthat all variants with the exception of one increase the odds ofxanthomas more in males than in females (data not shown).However, because all ORs were in the same direction in bothsexes, and the CIs of these ORs clearly overlapped, we decidedto perform a combined analysis corrected for sex (and age)preserving optimal power for the current analyses.

The presence of xanthomas can be determined by physicalexamination of the tendons without any discomfort for thepatients. However, there are no data on inter-observer variability,which could be a problem in the detection of small xanthomas.Xanthomas can be identified in patients with ultrasonography.Unfortunately, we had no ultrasonography of the tendons availableand we excluded all patients in whom we had doubts about thepresence of xanthomas. Therefore, it could be argued that wedid not study the presence of xanthomas, but instead analysedthe occurrence of severe xanthomas. Nonetheless, this diagnosisis relevant for daily clinical practice.

In conclusion, we observed that the presence of tendon xantho-mas in FH patients is influenced by genetic variation in the RCT andLDL oxidation pathways. Our results support the hypothesis thatxanthomas and atherosclerosis share pathophysiological pathways.The presence of xanthomas may therefore indicate FH patientswith a more severe CVD risk.

FundingThis work was supported by the Dutch Heart Foundation (2006B190).

Conflict of interest: none declared.

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