CLINICAL RESEARCH

European Heart Journal (2010) 31, 10071012

doi:10.1093/eurheartj/ehp538

Prevention

The risk of tendon xanthomas in familial

hypercholesterolaemia is influenced by variation
in genes of the reverse cholesterol transport
pathway and the low-density lipoprotein
oxidation pathway
1
Department 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 with
familial hypercholesterolaemia (FH). Therefore, xanthomas and atherosclerosis may result from the same pathophysiological mechanisms. Reverse cholesterol transport (RCT) and low-density lipoprotein (LDL) oxidation are pathophysiological pathways of atherosclerosis, and it is well established that genetic variation in these pathways influences
CVD risk. We therefore determined whether genetic variation in these pathways is also associated with the occurrence of tendon xanthomas in FH patients.
.....................................................................................................................................................................................
Methods
Four genetic variants in each pathway were genotyped in 1208 FH patients. We constructed a gene-load score for
and results
both 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 were
associated with the presence of xanthomas (OR 1.24, 95% CI 1.08 1.41, Ptrend 0.0015).
.....................................................................................................................................................................................
Conclusion
The presence of tendon xanthomas in FH patients is associated with genetic variation in the RCT and LDL oxidation
pathways. These results support the hypothesis that xanthomas and atherosclerosis share pathophysiological
mechanisms.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Xanthomas Familial hypercholesterolaemia Atherosclerosis Reverse cholesterol transport LDL oxidation

Introduction
Tendon xanthomas are cholesterol deposits in tendons. Their
presence is a clinical sign of familial hypercholesterolaemia (FH,
OMIM # 143890), an inherited disorder characterized by high lowdensity lipoprotein (LDL) cholesterol levels and premature cardiovascular disease (CVD). Recently, we demonstrated that the presence of xanthomas associates with a three-fold higher risk of CVD
in patients with FH.1 This finding suggests that xanthomas and
atherosclerosis share pathophysiological mechanisms.

Reverse cholesterol transport (RCT) and LDL oxidation are

two important pathophysiological pathways of atherosclerosis.
The RCT pathway promotes cholesterol efflux from the arterial
wall macrophages to the liver.2 The ATP-binding cassette transporter A1 (ABCA1) enhances the cholesterol flux from macrophages
to apolipoprotein AI (ApoAI) on high-density lipoprotein (HDL)
particles. After transfer of cholesterol esters from these HDL particles to LDL particles by cholesteryl-ester transfer protein
(CETP), the cholesterol is cleared together with its LDL particle
by 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.nl

Published 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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Daniella M. Oosterveer 1, Jorie Versmissen 1, Mojgan Yazdanpanah 1,

Joep C. Defesche 2, John J.P. Kastelein 2, and Eric J.G. Sijbrands 1*

1008
of these proteins in this candidate pathway has been associated
with CVD risk factors and the risk of CVD.2 6
Similarly, genes in the LDL oxidation pathway also significantly
influence atherosclerosis. Apolipoprotein AIV (ApoAIV), paraoxonase 1 (PON1), nitric oxide synthase 3 (NOS3), and cystathione
b-synthase (CBS) are antioxidants, which directly or indirectly neutralize free radicals and therefore diminish and prevent LDL oxidation, an important step in the formation of atherosclerotic
plaques.7 9
Because the effect of a single genetic variant is usually small and
can be compensated by variants in other genes in the same
pathway, pathway-based analyses are more informative.10
A number of studies suggest that the RCT pathway and the LDL
oxidation pathway influence each other.11 13 We therefore
determined the combined effect of genetic variation in each of
these two pathways on the risk of tendon xanthomas and assessed
interaction between these pathways.

Study population
Heterozygous FH patients were recruited from 27 lipid clinics in the
Netherlands between 1989 and 2002. Detailed information on the
study design and population has been described previously.14,15
Briefly, when FH is suspected in a patient, a blood sample is routinely
submitted to a central laboratory for LDLR mutation analysis. Out of a
random selection of 2400 unrelated patients with clinical FH, we
included 1208 patients with genetically confirmed heterozygous FH
and with information on the presence or absence of xanthomas. The
majority was of Caucasian descent (99%). The Ethics Committee of
each participating hospital approved the protocol, and informed
consent was required to be included in the study.
The level of total cholesterol, HDL cholesterol, and triglycerides
was measured using standard methods in patients withdrawn from
lipid-lowering medication at least 6 weeks before blood sampling.
The LDL cholesterol levels were calculated using the Friedewald
formula.16

Xanthoma definition
The presence of tendon xanthomas was defined as (i) the presence of
one or more unequivocal tendon xanthomas, (ii) the presence of a
clear thickening of both Achilles tendons determined by observation
and palpation during physical examination,17 (iii) a history of surgical
removal of one or more xanthomas. As the presence of xanthomas
was doubted in 68 patients, these patients were excluded from the
study. The characteristics of these patients did not differ from those
in the population examined (data not shown).

Selection and genotyping of the single

nucleotide polymorphisms
On the basis of a panel of CVD candidate single nucleotide polymorphisms (SNPs)18 and existing data derived from literature, we
selected four genes of the RCT pathway and four genes of the LDL
oxidation pathway. The selection of the variants in these genes was
based on functionality and/or associations with CVD in earlier
studies and HapMap tagging. In the RCT pathway, we chose
rs1800977 of the ABCA1 gene,2,19 rs670 of the ApoA1 gene,2,20,21
rs708272 of the CETP gene,2,22 24 and rs5742911 of the LDLR
gene.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 rs5742905

of the CBS gene32 34 of the LDL oxidation pathway.
The risk allele for each polymorphism was chosen on the basis of
the previous studies examining associations with CVD or intermediate
traits: the C allele of rs1800977 of the ABCA1 gene,19 the A allele of
rs670 of the ApoA1 gene,21 the T allele of rs708272 the CETP
gene,35,36 the A allele of rs854560 of the PON1 gene,28 the C allele
of rs3918226 of the NOS3 gene,37 and the T allele of rs5742905 of
the CBS gene.34 Owing to discrepancies in literature and to lack of
studies with a population similar to ours, we chose the risk allele for
the following polymorphisms based on our data: the G allele of the
LDLR gene polymorphism and the T allele of rs675 of the ApoA4 gene.
DNA was extracted from peripheral blood samples using standard
methods.38 To genotype the ApoA1 SNP rs670, we used TaqMan
allelic discrimination Assays-By-Design (Applied Biosystems, Foster
City, CA, USA). The forward and reverse primer sequences were
50 -CAGGACCAGTGAGCAGCAA-30 and 50 -GGCTGGGAGGCTGA
TAAGC-30 , respectively. The probes used were VIC-CCAGCCC
TGGCCCT and FAM-CCAGCCCTGGCCCT. The polymorphisms
of the other genes were performed previously by Roche Molecular
Systems, CA, USA. The genotypes were generated using PCR and
immobilized probe assay, as described previously.18 Results were
scored blinded to the presence of xanthomas.

Statistical analysis
All statistical analyses were performed with SPSS 14.0 for Windows.
The x2 test and t-test were used to compare characteristics
between patients with xanthomas and those without. Hardy
Weinberg equilibriums (HWE) of the polymorphisms were tested
using a x2 test.
We used logistic regression models to calculate odds ratios
(ORs) and 95% confidence intervals (CIs) for each individual polymorphism, adjusted for age and sex (Model 1) and additionally
adjusted for body mass index (BMI) and untreated LDL cholesterol
levels (Model 2).
Haplotype analysis was not possible because of low linkage disequilibrium (LD) between the different polymorphisms, as one would
expect of polymorphisms located at different chromosomes or
located more than 35 000 000 bp apart from each other. Low LD is
a prerequisite for gene-load score analysis.
To calculate the gene-load score, the absence of risk alleles was
coded 0, the presence of one risk allele was coded as 1, and the presence of two risk alleles was coded as 2. To assess the combined effects
of the four polymorphisms of the RCT pathway genes, a variable was
created which included these four polymorphisms. This resulted in a
score ranging from 0 to 8, representing the total number of risk
alleles. Similarly, we assessed the combined effects of the risk alleles
of the four LDL oxidation pathway genes. Because very few individuals
carried no or little risk alleles for the RCT pathway, we combined 0
and 1 risk alleles into one group in our logistic regression analyses
(adjusted for age and sex). For LDL oxidation pathway gene-load
score, we combined 1 to 3 risk alleles into one group.
To assess if there was evidence for multiplicative interaction
between the pathways, we performed a logistic regression analysis
including the grouped RCT pathway, the grouped LDL oxidation,
and an interaction term, adjusted for age and sex.

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

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Methods

D.M. Oosterveer et al.

1009

Genetic background of tendon xanthomas in FH patients

higher BMI, higher total cholesterol and LDL cholesterol

levels than those without xanthomas (P , 0.01). The frequency
of CVD was higher among patients with xanthomas (P , 0.01).

Table 1 General characteristics of the study

population
Characteristics

Xanthomas

.................................

Present

P-value

Absent

................................................................................
Number
Age (years)

567
43.0 + 12.1

641
40.3 + 12.7

Male gender (%)

45.9

47.0

Smoking (%)
Body mass index (kg/m2)

69.9
24.9 + 3.5

67.4
24.2 + 3.2

Diabetes (%)

2.1

1.3

,0.01
0.70
0.37
,0.01
0.24

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

22.8
9.45 + 1.91

0.72
,0.01

No statin treatment (%)a

Total cholesterol (mmol/L)

24.2
10.74 + 2.10

LDL cholesterol (mmol/L)

8.65 + 1.99

7.37 + 1.88

,0.01

HDL cholesterol (mmol/L)

Triglycerides (mmol/L)

1.13 + 0.32
1.48 + 0.76

1.15 + 0.33
1.39 + 0.72

0.41
0.08

Continuous variables: mean + standard deviation.

a
No statin treatment was defined as no statin treatment before cardiovascular
event.

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

0.921.34

0.27

(ref)
1.12

0.931.36

0.24

0.45

(ref)
1.11

0.861.43

0.43

0.02

(ref)
1.23

1.021.49

0.03

1.051.62

0.02

1.031.64

0.03

0.881.29

0.53

0.921.91

0.13

0.981.89

0.07

...............................................................................................................................................................................
The reverse cholesterol transport pathway
ABCA1 (rs1800977)

T
C

0.34
0.66

(ref)
1.11

ApoA1 (rs670)

G
A

0.85
0.15

(ref)
1.10

CETP (rs708272)

C
T

0.57
0.43

(ref)
1.25

LDLR (rs5742911)

A
G

0.76
0.24

(ref)
1.28

1.041.58

0.02

(ref)
1.30

A
T

0.19
0.81

(ref)
1.31

1.051.64

0.02

(ref)
1.29

T
A
T
C
C
T

0.37
0.63
0.07
0.93
0.90
0.10

(ref)
1.10
(ref)
1.36
(ref)
1.34

0.861.40
1.041.50

...............................................................................................................................................................................
The LDL oxidation pathway
ApoA4 (rs675)
PON1 (rs854560)
NOS3 (rs3918226)
CBS (rs5742905)

Logistic Model 1: adjusted for age and sex.

Logistic Model 2: adjusted for age, sex, LDL cholesterol and BMI.

0.921.32

0.31

0.951.93

0.09

0.981.84

0.07

(ref)
1.06
(ref)
1.32
(ref)
1.36

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Systolic blood pressure

(mmHg)

HDL cholesterol level was not associated with xanthomas

(P 0.41).
All polymorphisms were in HWE, with the exception of the CBS
rs5742905 polymorphism, which was not in HWE due to absence
of the TT genotype (P 0.00053).
The odds of xanthomas per individual SNP in each pathway
is shown in Table 2. In the RCT pathway, the T allele of
the CETP rs708272 polymorphism was significantly associated
with the presence of xanthomas (OR 1.25, 95% CI 1.04 1.50,
P 0.02). More carriers of a G allele of the LDLR rs5742911
polymorphism had xanthomas than non-carriers (additive
model: 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 T
allele of the ApoA4 polymorphism had a higher odds of xanthomas
than those carrying A alleles (OR 1.31, 95% CI 1.05 1.64, P
0.02). The other polymorphisms were not significantly associated
with xanthomas.
Figure 1 shows the gene-load scores of the RCT and the
LDL oxidation pathway. 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). Each increase in the
number of risk alleles in the LDL oxidation pathway was
accompanied by an increase in the frequency of xanthomas (OR
1.24, 95% CI 1.08 1.41, Ptrend 0.0015). Although these
associations were significant, the areas under the ROC curves
(AUCs) found were relatively small: for the RCT pathway 0.56
(95% CI 0.52 0.60) and for the LDL oxidation pathway 0.55
(95% CI 0.52 0.59).
We did not find evidence for multiplicative interaction between
the RCT and the LDL oxidation pathway (OR interaction 0.98,

1010

D.M. Oosterveer et al.

Discussion

pathophysiological pathways. *P , 0.0125. The numbers

between bars below the columns denote the number of patients
with that particular number of risk genotypes. In the figure 98.8%
confidence intervals are given. (A) Gene-load score of the reverse
cholesterol transport pathway. (B) Gene-load score of the LDL
oxidation pathway. (C) Both pathways combined.

95% CI 0.87 1.11, P 0.78). In line with this, combining the geneload scores of both pathways showed a significant trend similar to
the ORs as the individual pathways (OR 1.24, 95% CI 1.12 1.37,
P , 0.01, Figure 1C).

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Figure 1 The risk of xanthomas per gene-load score of two

In this study, we have demonstrated that variants in genes of the

RCT pathway and the LDL oxidation pathway are associated
with the presence of tendon xanthomas in FH patients. The
odds of xanthomas increased with increasing numbers of risk
alleles in the genes of both pathways.
In addition to the higher risk of CVD in FH patients with xanthomas compared with that in those without xanthomas,1 there are
other findings suggesting that xanthomas and atherosclerosis are
related and share pathophysiological pathways. The composition
of xanthomas and atherosclerotic plaques show similarities as
both lesions consist of connective tissue matrix containing macrophages transformed into foam cells.39 In addition, both xanthomas
and plaques can be prevented or reduced by the use of cholesterollowering drugs.40 42 Recently, we have shown that variation in the
5-lipoxygenase activation protein gene, which is involved in inflammation, contributed to the risk of xanthomas in FH.43 The present
study is the first study to extend the analysis of xanthomas to
genetic variation in the RCT and LDL oxidation pathways.
Previously, genetic variation in these pathways has been related to
atherosclerosis or one of its risk factors.8,19 22,24 28,32,35,36,44
These findings strongly suggest that xanthomas and atherosclerosis
share pathogenic mechanisms.
We assigned the same score to each risk allele of each polymorphism. As an alternative method, we also made a weighted
gene score for each patient, in which the absence of risk alleles
was coded 0 and the presence of one or two risk alleles were
coded as 1  LN(OR) and 2  LN(OR), respectively.45 The resulting continuous gene score was divided into five equal groups of
patients. The results of this alternative approach were similar to
the unweighted approach (data not shown).
In gene-load score analyses, additive risk-allele effects are used
as markers of the susceptibility of particular pathway to deteriorate
and contribute to signs and symptoms. The dosage effect observed
in both pathways supports involvement of these pathways in the
development of xanthomas. The effect of both the RCT pathway
and the LDL oxidation pathway clearly showed that the odds of
xanthomas increased according to the number of risk alleles.
Although the previous literature has suggested an interaction
between the studied pathways, we did not find a multiplicative
interaction between the pathways.11 13 In line with this, combining
the gene scores of both pathways showed a trend similar to that of
the individual pathways.
The AUC values demonstrate that the gene-load scores of these
pathways contributed modestly to xanthomas, and other (unknown)
pathogenetic factors need to be elucidated to understand the pathogenesis of xanthomas more completely. On the other hand, we may
have underestimated the contribution of the two pathways by studying 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 the
presence of xanthomas (Table 1). Perhaps the functionality of
HDL particles is of more importance than the quantity. Previously
a probucol study indeed demonstrated that the reduced HDL
particle size was linked to xanthoma regression.46 Unfortunately,
we had no data about the subdivision of the particles.

1011

Genetic background of tendon xanthomas in FH patients

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

Conflict of interest: none declared.

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In our study, the CBS rs5742905 polymorphism was out of

HWE. Because homozygosity of the CBS rs5742905 T allele
leads to CBS deficiency that causes hyperhomocystinemia
(OMIM #236200),32,33 the absence of TT carriers in our population can be explained by the preferential referral of CBS-deficient
patients to other outpatient clinics. The results of the analyses,
including and excluding the CBS variant, were similar (data not
shown).
To maintain sufficient patients per number of risk alleles, we
could not include all genes involved both pathways. For example,
both lecithin-cholesterol acyltransferase and scavenger-receptor
B1 (SR-B1) are known to be involved in the RCT pathway too.
Lecithin-cholesterol acyltransferase converts cholesterol in HDL
particles to cholesterol esters, and SR-B1 binds HDL particles
and facilitates removal of remaining cholesterol esters from these
particles. However because of discrepancies in literature about
their importance in the RCT pathway and because of differences
in outcome per sex and age of polymorphism analysis,47 51 we
did not genotype a variant in these genes. Variation in another
important candidate gene, the E2 E3E4 variation in the apolipoprotein E (ApoE) gene, was previously found not to be associated
with xanthomas.52 In addition, we decided not to include any polymorphisms of genes involved in the more down-stream bile acid
synthesis and excretion.
It is well known that the natural history of atherosclerosis differs
between the two sexes. Similar to CVD, the male gender is also
associated with the presence of xanthomas.1 In the present
study, the percentage of males was not significantly different
between the FH patients with and without tendon xanthomas.
Stratified analyses of the individual polymorphisms demonstrated
that all variants with the exception of one increase the odds of
xanthomas more in males than in females (data not shown).
However, because all ORs were in the same direction in both
sexes, and the CIs of these ORs clearly overlapped, we decided
to perform a combined analysis corrected for sex (and age)
preserving optimal power for the current analyses.
The presence of xanthomas can be determined by physical
examination of the tendons without any discomfort for the
patients. 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 available
and we excluded all patients in whom we had doubts about the
presence of xanthomas. Therefore, it could be argued that we
did not study the presence of xanthomas, but instead analysed
the occurrence of severe xanthomas. Nonetheless, this diagnosis
is relevant for daily clinical practice.
In conclusion, we observed that the presence of tendon xanthomas in FH patients is influenced by genetic variation in the RCT and
LDL oxidation pathways. Our results support the hypothesis that
xanthomas and atherosclerosis share pathophysiological pathways.
The presence of xanthomas may therefore indicate FH patients
with a more severe CVD risk.

1012

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