Haplotype analyses of the APOA5 gene in patients with
familial combined hyperlipidemia
Gerly M. van der Vleutena, Aaron Isaacsb, Wu-Wei Zengc, Ewoud ter Avesta,
Philippa J. Talmudd, Geesje M. Dallinga-Thiee, Cornelia M. van Duijnb,
Anton F.H. Stalenhoefa, Jacqueline de Graafa,⁎
aDepartment of Medicine, Division of General Internal Medicine, 463, Radboud University Nijmegen Medical Centre,
P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands
bDepartment of Epidemiology and Biostatistics, Genetic Epidemiology Unit, Erasmus University Medical Center, Rotterdam, The Netherlands
cDepartment of Biochemistry and Molecular Biology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
dDepartment of Medicine, Division of Cardiovascular Genetics, Royal Free and University College Medical School, London, UK
eLaboratory of Vascular Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
Received 10 July 2006; received in revised form 15 October 2006; accepted 20 October 2006
Available online 26 October 2006
Background: Familial combined hyperlipidemia (FCH) is the most common genetic lipid disorder with an undefined genetic etiology.
Apolipoprotein A5 gene (APOA5) variants were previously shown to contribute to FCH. The aim of the present study was to evaluate the
association of APOA5 variants with FCH and its related phenotypes in Dutch FCH patients. Furthermore, the effects of variants in the APOA5
gene on carotid intima-media thickness (IMT) and cardiovascular disease (CVD) were examined. Materials and methods: The study population
consisted of 36 Dutch families, including 157 FCH patients. Two polymorphisms in the APOA5 gene (−1131T>C and S19W) were genotyped.
Results: Haplotype analysis of APOA5 showed an association with FCH (p=0.029), total cholesterol (p=0.031), triglycerides (p<0.001),
apolipoprotein B (p=0.011), HDL-cholesterol (p=0.013), small dense LDL (p=0.010) and remnant-like particle cholesterol (p=0.001).
Compared to S19 homozygotes, 19W carriers had an increased risk of FCH (OR = 1.6 [1.0–2.6]; p=0.026) and a more atherogenic lipid profile,
reflected by higher triglyceride (+22%) and apolipoprotein B levels (+5%), decreased HDL-cholesterol levels (−7%) and an increased prevalence
of small dense LDL (16% vs. 26%). In carriers of the −1131C allele, small dense LDL was more prevalent than in −1131T homozygotes (29% vs.
16%). No association of the APOA5 gene with IMT and CVD was evident. Conclusion: In Dutch FCH families, variants in the APOA5 gene are
associated with FCH and an atherogenic lipid profile.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Familial combined hyperlipidemia; APOA5 gene; Haplotype; Triglyceride level; Intima-media thickness; Remnant-like particle
Familial combined hyperlipidemia (FCH) is the most
common lipid disorder of unknown genetic etiology, affecting
2–5% of the general population [1,2]. Major characteristics of
FCH include elevated plasma levels of apolipoprotein B (apoB),
triglycerides (TG) and/or total cholesterol (TC). Other pheno-
types include decreased levels of high-density lipoprotein
cholesterol (HDLc) and the presence of small, dense low-
density lipoproteins (sdLDL). In addition, FCH patients have an
increased risk of cardiovascular disease (CVD) and are often
obese and insulin resistant .
Despite decades of research, the complex genetics of FCH
are still not fully understood. Several linkage analyses were
performed, leading to the identification of multiple loci for
FCH [4–9]. One repeatedly identified locus is located on
chromosome 11q, a site involved in modulating the expression
of FCH [10–12]. This region includes the apolipoprotein A1–
C3–A4–A5 gene cluster . The apolipoprotein A5 (APOA5)
gene encodes the apolipoprotein AV protein (APOAV), which
Biochimica et Biophysica Acta 1772 (2007) 81–88
⁎Corresponding author. Tel : +31 24 3618819; fax: +31 24 35 41734.
E-mail address: J.firstname.lastname@example.org (J. de Graaf).
0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
is exclusively expressed in the liver. APOAV is found on very
low-density lipoprotein (VLDL), HDL and chylomicrons and
is, compared to other apolipoproteins, present in very low
plasma concentrations [14,15]. Variations in the APOA5 gene
are related to TG levels, however, the underlying mechanism
is not yet fully understood . One hypothesis, based on
mouse studies, suggests that APOAV modulates TG levels by
guiding VLDL and chylomicrons to proteoglycan-bound
lipoprotein lipase for lipolysis and by increasing VLDL
In the human APOA5 gene, several single-nucleotide
polymorphisms (SNPs) have been identified (−1131T>C,
−3A>G, S19W (56C>G), IVS3+476G>A and 1259T>C)
[19,20]. The three major haplotypes, representing approxi-
mately 98% of all haplotypes in these populations, were
defined by the −1131T>C and the S19W SNPs [19,20]. In
Caucasians, the rare alleles of these two SNPs were associated
with elevated plasma levels of TC, TG, remnant-like particle
cholesterol (RLPc), LDL cholesterol (LDLc) and apoB,
decreased HDLc levels and the presence of sdLDL
[13,20,21]. In patients with FCH, APOA5 polymorphisms
were associated with an increased risk of FCH and related
lipid phenotypes, including TG, apoB and HDLc levels and
LDL particle size [11,22,23]. The relationship between
variants in the APOA5 gene and RLPc levels was not
previously studied in FCH patients.
Hypertriglyceridemia is an independent risk factor for the
development of CVD . Despite its effect on plasma
triglycerides, the role of APOA5 in the development of CVD
remains controversial [20,25–29]. In 372 Finnish men with
CVD, who participated in the LOCATstudy, it was demonstrated
that only the rare allele of the S19W SNP (19W) was associated
with increased progression of atherogenesis . In contrast, in
2578 subjects from the general population in the Framingham
Heart Study, an increased risk of CVD was found only for the
rare allele of the −1131T>C SNP (−1131C) [20,29]. The results
for CVD in the Framingham Heart Study, however, conflicted
with the results obtained for intima-media thickness (IMT), a
surrogate marker of CVD. Differences in IMT were associated
with the 19Wallele, but an association of the −1131C allele was
onlypresent inobese subjects. InFCHpatients,who havean
increased risk of CVD, the relationship between variants in the
APOA5 gene and CVD was not previously examined.
The aim of the present study was to investigate the
association of APOA5 gene variants (−1131T>C and S19W)
with FCH and its associated phenotypes, including RLPc levels,
using a family-based SNP and haplotype approach in well-
characterized FCH families. Furthermore, the suggested
increased risk of CVD associated with variants in the APOA5
gene was investigated in our FCH families by taking both
intima-media thickness and CVD prevalence into account.
2. Materials and methods
2.1. Study population
Back in 1994, we have recruited FCH families from the outpatient lipid
clinic of the Radboud University Nijmegen Medical Centre, ascertained
through probands, exhibiting a combined hyperlipidemia with both plasma TC
and TG levels above the age- and gender-related 90th percentile , during
several periods in which they were not treated with lipid-lowering drugs, and
despite dietary advice. Additionally, a first-degree relative possessed elevated
levels of TC and/or TG above the 90th percentile and the proband, or a first-
degree relative, suffered from premature (before the age of 60 years)
cardiovascular disease. Families were excluded when probands were
diagnosed with underlying diseases causing hyperlipidemia (i.e., diabetes
mellitus type 1 and 2, hypothyroidism, and hepatic or renal impairment), a
first-degree relative had tendon xanthomata or probands were homozygous for
the APOE2 allele. All included subjects were Caucasian and above the age of
10 years. The ascertained families had a mean size of 24 members from
multiple (between 2 and 4) generations. The present FCH study population
include the 5-year follow-up data of our original FCH families, consisting of
36 Dutch families, comprised of 611 subjects with known genealogic,
phenotypic, and genotypic data, of whom 157 individuals were diagnosed as
FCH patients . In 1999, the diagnosis of FCH was based on the nomogram
. Plasma TG and TC levels, adjusted for age and gender, and absolute
apoB levels, were applied to the nomogram to calculate a probability of being
affected with FCH. When this probability of being affected with FCH is
greater than 60%, the diagnostic phenotype is present in at least one first
degree relative, and premature CVD (before the age of 60 years) is present in
at least one individual in the family, the individual is defined as affected by
FCH. Also included in the family-based analyses were the normolipidemic
relatives (n=390), the unaffected spouses of both FCH patients and
normolipidemic relatives (n=64), and subjects without known phenotypic
and/or genotypic data (n=230) to complete the pedigree structure. After the
withdrawal of lipid-lowering medication for 4 weeks and an overnight fast,
blood was drawn by venipuncture. Body mass index (BMI) was calculated as
body weight (kilograms) divided by the square of height (meters).
Information concerning CVD was gathered through personal interviews
and physical examinations performed by the clinical investigator. When the
clinical investigator suspected the presence of CVD, further details and
confirmation of the diagnosis were sought from the participant's general
practitioner and hospital records. CVD was defined by angina pectoris (AP),
myocardial infarction (MI), stroke, peripheral vascular disease or vascular
surgery. In our study population (n=611), 56 subjects were identified with
CVD, including 26 subjects with AP, 25 with previous MI, 10 with peripheral
vascular disease, seven with stroke and 23 who underwent vascular surgery.
In total, 45% (n=25) of these individuals were diagnosed with CVD based on
the presence of two or more manifestations of CVD. The ethical committee of
the Radboud University Nijmegen Medical Centre approved the study
protocol and all procedures were in accordance with institutional guidelines.
All subjects provided written informed consent.
2.2. Biochemical analyses
Biochemical analyses were performed as previously described for this
population . In short, plasma TC and TG were determined by enzymatic,
commercially available reagents (Boehringer-Mannheim, Germany, catalog
No. 237574 and Sera Pak, Miles, Belgium, catalog No. 6639, respectively).
Total plasma apoB concentrations were measured by immunonephelometry.
HDLc was quantified by the polyethylene glycol 6000 method. LDL
subfractions were separated by single spin density gradient ultracentrifuga-
tion. A continuous variable, K, represented the LDL subfraction profile of
each individual. A negative K-value (K≤−0.1) reflected the presence of
small dense LDL . RLPc levels were measured using an immuno-
separation technique as described elsewhere . Glucose concentrations
were analyzed using the oxidation technique (Beckman®, Glucose
Analyser2, Beckman Instruments Inc., Fullerton, CA 92634, USA). Plasma
insulin concentrations were ascertained by a double antibody method. Insulin
resistance was assessed by the homeostasis model assessment (HOMA)
DNA was extracted from peripheral blood lymphocytes using a standard
technique . Genotyping for the S19Wand the −1131T>C SNPs was carried
out by PCR and restriction enzyme digestion, as previously described .
Genotyping of the S19W and −1131T>C SNPs failed in 5% and 3% of the
82G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
2.3. Carotid IMT measurements
All our FCH families were invited to participate in an on-going follow-up
program. As part of the follow-up screening, non-invasive measurements of
atherosclerosis were performed, including carotid intima-media thickness
(IMT), in all family members, including both patients and normolipidemic
relatives . For the study population described in the present study, IMT data
were availablein 23 families, including 61 FCH patient and 155 normolipidemic
relatives. An AU5 Ultrasound machine (Esaote Biomedica) with a 7.5 MHz
linear-array transducer was used to measure the IMT of both common carotid
arteries. Longitudinal images of the most distal 10 mm of both the far and the
near wall of the common carotid artery were obtained with the optimal
projection (anterolateral, lateral or posterolateral). The actual measurement of
IMT was performed offline by the sonographer using semi-automatic edge-
detection software (M'Ath®Std version 2.0, Metris, Argenteuil, France) .
All measurements were carried out in end-diastole using the R-wave of a
simultaneously recorded ECG as a reference frame. From each frame the mean
IMTwas calculated over at least 7.5 mm of the aforementioned 10 mm segment
(yielding a minimum quality index of 75%). The outcome variable was defined
as the mean IMT of the near and far wall of both common carotid arteries.
2.4. Statistical analyses
Descriptive statistics were compared using ANOVA for continuous traits
and Pearson's chi-square test for discrete traits. Prior to further statistical
analyses, extended Mendelian error-checking was performed with Pedcheck
. For families with Mendelian inconsistencies, problematic genotypes were
set to missing for the complete nuclear family or the isolated problematic
individual (1%). The parental data for both SNPs were tested for Hardy–
Weinberg equilibrium by use of Fisher's exact-test. Variables with a skewed
distribution, including TG levels, RLPc levels and the HOMA-index, were
logarithmically transformed. The HAPLOVIEW program  was used to
estimate allele and haplotype frequencies in founders, representing the unrelated
individuals of the study population, including probands and spouses. Linkage
disequilibrium (LD) and allelic association (R2) between the two SNPs were
calculated with HAPLOVIEW using the confidence interval method of Gabriel
et al. .
To study the relation of APOA5 in FCH, we used both linkage and
association analysis. The SOLAR 2.1.4 software was used to implement two-
point linkage analysis (39). The presence of linkage was tested for FCH and its
related phenotypes. As linkage analysis may have limited power to identify
common variants with moderate effects for more complex diseases, such as
FCH, we also performed association analysis .
Associations between the individual polymorphisms (1131T>C and
S19W), or haplotypes and FCH, or the related phenotypes, were analyzed
using a family based approach, with the family based association test (FBAT)
software . FBAT is attractive because it can test for linkage and/or
association while avoiding biases due to population admixture or stratification,
misspecification of the trait distribution, and/or selection based on the trait. The
FBAT test statistics utilizes a general approach to family-based association
tests, as proposed by Rabinowitz and Laird (2000), and is based on the
distribution of the offspring genotypes conditional on any trait information and
on the parental genotypes . An additive model of inheritance was used, as
the mode of inheritance of FCH is unknown and this model is particularly
robust. FBAT broke down the extended pedigrees into nuclear families
(n=219) and evaluated their contribution to the test statistics. The −e option of
FBAT, which computes the test statistic using an empirical variance estimator,
was implemented to correct for the non-independence of the nuclear families
and for the presence of linkage. The −p option, which performs a Monte-Carlo
permutation procedure, was used to estimate empirical p-values. Adjustments
for age, gender and BMI were done by calculation of the residuals. For the
haplotype analysis, the haplotype-based association test (HBAT) command of
FBAT was used, utilizing the −e and −p options. Differences were considered
statistically significant at p-value<0.05.
The explained variance in FCH by the APOA5 variants was explored using
the measured genotype method in the SOLAR Program . The measured
genotype analysis uses variance component methodology to estimate the
proportion of variance due to environmental and additive genetic effects. By
comparing models with and without the APOA5 genotypes, estimations are
calculated of what proportion of the FCH trait variance is attributable to the
genotype by examining the changes to the estimates of environmental and
additive genetic variance. p-values and the explicable proportion of variance are
To test differences between the different genotypes, generalized estimating
equation (GEE) analyses were performed in the STATA 8.0 software. The GEE
analyze is especially designed to take into account possible correlated values
within families. In the models used for the GEE analyses the link function
‘canonical’ was used and an equal-correlation population-averaged model was
used as the working correlation matrix.
The analyses were conducted using SPSS 12.0.1, PEDCHECK 1.00,
HAPLOVIEW 3.2, SOLAR 2.1.4, FBAT 3.2 and STATA 8.0 software.
3.1. Characteristics of study population
Anthropometric and metabolic characteristics of FCH
patients, normolipidemic relatives and spouses are presented
in Table 1. FCH patients were older than normolipidemic
relatives, but younger than spouses. Compared to normolipi-
demic relatives and spouses, FCH patients had a higher
prevalence of CVD and higher IMT values. When correcting
the IMT values for the age difference between FCH patients
and normolipidemic relatives, the difference in IMT remained
significant (0.78 mm (SD 0.08) in FCH versus 0.73 mm (SD
0.08) in normolipidemic relatives, p=0.000). FCH patients
were characterized by increased lipid levels, including TC, TG,
apoB, LDLc and RLPc concentrations, decreased HDLc and
an increased prevalence of sdLDL, as reflected by a K-value
below −0.1. Finally, FCH patients were more also obese, as
reflected by a higher BMI, and insulin resistant, as reflected by
relatives and spouses
Male Gender (n (%))
CVD (n (%))
Apo B (mg/L)
Continuous variables are presented as mean (SD); FCH, familial combined
hyperlipidemia; NL relatives, normolipidemic relatives; CVD, cardiovascular
disease; IMT, mean common carotid artery intima-media thickness was
measured in 61 FCH patients and 155 normilipidemic relatives; BMI, body
mass index; TC, total cholesterol; TG, triglycerides; Apo B, apolipoprotein B;
LDL-c, LDL cholesterol; HDL-c, HDL cholesterol; K-value, a value <−0.1
represents the presence of small dense LDL; RLP-c, remnant-like particle
cholesterol; HOMA, homeostasis model assessment-index; *p<0.05, compared
to NL relatives;†p<0.05, compared to spouses;‡p<0.05, compared to spouses.
83 G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
a higher HOMA-index (Table 1). The anthropometric and
metabolic characteristics of the subgroup of subjects with
IMT data are presented in Supplementary Table 1. Char-
acteristics of the subjects with IMT data did not differ
significantly from the total group as presented in Table 1 (all
p<0.05). Anthropometric and metabolic characteristics of the
subgroup of subjects with CVD are presented in Supplemen-
tary Table 2. As expected, patients with CVD were relatively
old and predominantly men. Furthermore, patients with CVD
had a relatively high IMT value and a more atherogenic lipid
and lipoprotein profile reflected by high TC and TG levels,
low HDL cholesterol levels and the presence of sdLDL.
Forty-three percent of the subjects with CVD were diagnosed
3.2. Analyses of the APOA5 gene with FCH and related lipid
In founders, rare allele frequencies for the −1131T>C and
S19W SNPs were 0.08 and 0.11, respectively. In the probands,
rare allele frequencies for the −1131T>C and S19W SNPs
were 0.10 and 0.16, respectively. The genotypic distributions
of −1131T>C and S19W were in Hardy–Weinberg propor-
tions. The alleles of the −1131T>C and the S19W SNPs were
in complete linkage disequilibrium (D'=1.00), but with very
little correlation (R2=0.01). A consequence of this linkage
disequilibrium is that, only three of the possible four different
haplotypes were observed. Haplotype analyses can, therefore,
provide additional information compared to the single SNP
analyses, since it accounts for both sources of variation. The
wildtype haplotype (APOA5*1) had a frequency of 0.83. The
frequencies of the haplotype defined by a rare allele for the
−1131T>C (APOA5*2) or by a rare allele for the S19W SNP
(APOA5*3) were 0.11 and 0.06, respectively. In total 394
individuals (92 FCH patients, 261 normolipidemic relatives
and 41 spouses) carried only common alleles for both SNPs,
who are referred to as “wildtypes”. 64 subjects (8 FCH
patients, 50 normolipidemic relatives and 6 spouses) carried
one or two rare alleles for the −1131T>C SNP and wildtype
alleles for the S19W SNP and 115 subjects (38 FCH patients,
63 normolipidemic relatives and 14 spouses) carried one or two
rare alleles for the S19W SNP and wildtype alleles for the
−1131T>C SNP. A rare allele for both the −1131T>C SNP
and the S19W SNP were present in 18 subjects (9 FCH
patients, 9 normolipidemic relatives and 0 spouses), referred to
as “compound heterozygotes”.
3.3. Linkage analyses of the APOA5 region
Two-point linkage analyses performed utilizing the −1131T>
C and S19W SNPs in the APOA5 gene did not provide evidence
of linkage for FCH (−1131T>C; lod score=0.04 and S19W; lod
S19W SNP, whereas a lod score of 0.05 was found for the
−1131T>C SNP. For all other phenotypes, including apoB, TC,
RLPc and HDLc levels, and the presence of sdLDL, no linkage
was present (all two-point lod scores <0.5).
3.4. Association analyses of APOA5 variants with FCH and
The associations of the −1131T>C and the S19W SNPs,
individually and as haplotypes, with FCH and its related
phenotypes are presented in Table 2A. These associations are
corrected for age, gender and multiple testing. The APOA5*2
haplotype was not associated with FCH, while the APOA5*3
haplotype was overrepresented in patients with FCH (p=0.014).
Haplotype analyses showed that the APOA5 haplotypes were
of FCH for subjects (n=119) with the rare allele for the S19W
SNP (1.62 [95% C.I.: 1.03, 2.55]; p=0.026) and for the
compound heterozygotes (n=19) carrying a rare allele for the
0.008). The APOA5*3 haplotype was also associated with
individual lipid and lipoprotein levels, including TC (p=0.042),
TG (p<0.001), apoB (p=0.024), HDLc (p=0.019), K-value
(p=0.024) and RLPc (p=0.001). No associations were found for
the obesity (BMI) and insulin resistance parameters (HOMA-
associated with any of the characteristics of FCH. Comparable
the possible presence of linkage (−e option) were taken into
account (data not shown).
Association of rare alleles of the −1131T>C and the S19W SNPs and
haplotypes in the APOA5 gene with familial combined hyperlipidemia and its
−1131C 19W APOA5*2APOA5*3 Overall
Apo B (mg/L)
RLP-c (mmol/L)§559 0.031
p-values for the individual SNPs (FBAT) and haplotypes (HBAT) corrected for
age, gender and multiple testing are presented; §, Ln-transformed variable; N,
number of subjects with available data for the relevant phenotype; APOA5*2,
haplotype represented by the rare allele of −1131T>C; APOA5*3, haplotype
represented by the rare allele of S19W; Overall haplotype, the p-value obtained
for the model including all haplotypes; FCH, familial combined hyperlipidemia
was present in 157 subjects; BMI, body mass index; TC, total cholesterol; TG,
triglycerides; Apo B, apolipoprotein B; LDL-c, LDL-cholesterol; HDL-c, HDL-
cholesterol; K-value, a value <−0.1 represents the presence of small dense
LDL; RLP-c, remnant-like particle cholesterol; HOMA, homeostasis model
assessment-index;CVD,cardiovascular diseasewaspresent in56subjects;IMT,
mean common carotid artery intima-media thickness data were present in 61
FCH patients and 155 normolipidemic relatives.
84 G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
3.5. Explained variance in FCH by the APOA5 gene
By measured genotype analyses, it was estimated that 2% of
the genetic variance in FCH is attributable to the S19W SNP
(p<0.001). The S19W SNP also explained 3% of the variation
in TG levels (p<0.001), 1% of the variance in HDLc levels
(p=0.005) and 1% of the variation in K-value (p=0.005). The
−1131T>C SNP did not contribute to the variation in FCH or
any of the related phenotypes (TC, TG, Apo B, LDL-c, HDL-c,
K-value, RLP-c and HOMA-index); in fact, the point estimates
for the combined effects of both genotypes, S19W and
−1131T>C, were identical to the point estimate obtained for
3.6. Association analyses of the APOA5 gene with IMT and
The associations of the −1131T>C and the S19W SNPs in
APOA5, and their haplotypes, with IMT values and CVD
prevalence, are presented in Table 2B. These associations are
corrected for age, gender and multiple testing. The APOA5*3
haplotype was not associated with IMTor CVD. The APOA5*2
haplotype did show a trend towards significant association with
CVD (p=0.07), but no association with IMT was present.
Overall, no association of the haplotypes with IMT values or
CVD prevalence was evident. Further adjustment of IMT values
for obesity did not change these results.
3.7. Effect of APOA5 variants on lipid and lipoprotein levels,
IMT and prevalance of CVD
To determine the effect of the genetic variation in the APOA5
gene on lipid levels in the general population, we combined the
normolipidemic relatives and spouses in one control group,
which was allowed because no difference in anthropometric and
biochemical parameters, based on genotypes, were observed
between relatives and spouses (data not shown). The results
were standardized for age and gender and presented in Table 3.
Compared to the wildtypes, individuals carrying a rare allele of
the S19W SNP had increased TG levels (+22%) apoB levels
(+5%) and decreased HDLc levels (−7%). Furthermore, in
subjects with a 19Wallele, a K-value below −0.1 was present in
26% of the subjects, which was significantly higher than in
wildtypes (16%; p=0.046). Additionally, in carriers of the
−1131T>C rare allele, sdLDL was present in more individuals
(29%; p=0.035) compared to wildtypes. The compounds had
approximately 15% lower HDLc levels and sdLDL was present
in more individuals. Compared to the wildtypes, individuals
carrying rare alleles for either or both SNPs had no increased
IMT or increased prevalance of CVD (Table 3).
The effect of APOA5 on lipid levels among FCH patients
were comparable (data not shown).
The results from this study demonstrate that the S19W
variant in the APOA5 gene is associated with FCH. We show
that the APOA5*3 haplotype is over-represented in patients
with FCH. Furthermore, we provide evidence for the associa-
tion of the APOA5*3 haplotype with a more atherogenic lipid
profile. However, no association of the −1131T>C and S19W
variants in the APOA5 gene with higher IMT values or an
increased prevalence of CVD in patients with FCH was evident.
The 19W allele was previously associated with an increased
risk of FCH and elevated levels of TG and apoB [11,22]. In the
present study, we performed family-based haplotype analyses
with the FBAT program, corrected either for multiple testing or
for the presence of linkage in this region. We confirmed the
independent association of the 19W allele with FCH and
elevated TG levels. Additionally, we showed that the 19Wallele
is associated with an atherogenic lipid profile including elevated
apoB levels, decreased HDLc levels and the presence of sdLDL.
This is in agreement with previously documented associations
in general populations [11,13,20,21]. From the literature, it is
also suggested that the haplotype containing the 19W is
associated with the highest TG levels [19,34].
In the present study, no independent association for the
−1131C allele was found with FCH or any of the related lipid
phenotypes whereas the −1131C allele was previously
associated with an increased risk of FCH, and elevated levels
of TG and sdLDL [11,22,23]. In the present study we defined
FCH based on our nomogram .To exclude that the lack of
association of the −1131T>C SNP with FCH in our population
is due to different diagnostic criteria of FCH, we repeated the
analyses, now applying the traditional diagnostic criteria of
Differences in phenotypic parameters in the control group including
normolipidemic relatives and spouses with a rare allele for the −1131T>C or
the S19W SNP in the APOA5 gene compared to wildtype individuals
CVD (n (%))
HDL-c (mmol/L) 1.24 (1.20–1.28) −2.4%
RLP-c (mmol/L) 0.25 (0.24–0.26) +0.0%
0.74 (0.72–0.76) −1.4%
4.99 (4.86–5.11) +3.0%
1.07 (1.01–1.14) +8.4%
975 (949–1001) +5.3%
5 (8.9%) 6 (7.8%)
48 (15.9%) 16 (28.6%)* 20 (26.0%)* 4 (44.4%)*
Presented are the date of the combined group of normolipidemic relatives and
spouses. For the subjects wildtype for both SNPs, dichotomous variables are
presented as number (%) and the continuous variables, standardized for age and
gender, are presented as mean (95% CI); For the other groups, dichotomous
variables are presented as number (%) and the continuous variables as percent
of difference compared to the wildtype individuals; Rare allele −1131T>C,
subjects carrying either 1 or 2 rare alleles for the −1131T>C SNP; Rare allele
S19W, subjects carrying either 1 or 2 rare alleles for the S19W SNP;
Compounds, subjects carrying 1 rare allele for both SNPs; CVD, cardiovas-
cular disease; IMT, common carotid artery intima-media thickness; TC, total
cholesterol; TG, triglycerides; ApoB, apolipoprotein B; HDL-c, HDL
cholesterol; K-value, a value<−0.1 represents the presence of small dense
LDL; RLP-c, remnant-like particle cholesterol; *p<0.05 compared to
85G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
FCH, based on TC and/or TG levels above the 90th
percentile. No association of the −1131T>C SNP with
FCH, based on the traditional diagnostic criteria, was found
(Supplementary Table 3). Most likely, the small number of
subjects carrying one or two C alleles of the −1131T>C SNP
contribute to the lack of involvement of this SNP to FCH. In
contrast, compound heterozygotes did show a significant
increased risk on FCH, despite the low number of subjects.
However, as the lipid and lipoprotein levels among subjects
who were compound heterozygotes did not differ from
wildtype carriers, it may well be possible that the observed
increased risk of FCH in compound heterozygotes is a false
positive finding and therefore additional studies are required
for confirmation of our observed increased risk on FCH in
Previously, two studies investigated the association of
variants in the APOA5 gene and RLPc levels. In the Framing
Heart Study population, both the 19Wand −1131C alleles were
associated with RLPc levels, while no association was found in
the MONICA study population [20,43]. We report no associa-
tion of the rare alleles of the −1131T>C and the S19W SNPs in
the APOA5 gene with RLPc levels in our FCH study population,
which is in agreement with the findings of the MONICA study
Since the variants in the APOA5 are suggested to be
associated with plasma lipid levels, it is likely that it plays a
role in the development of CVD. Several studies investigated
possible associations between variants in the APOA5 gene and
the prevalence of CVD in Caucasians, however, these led to
contradictory results [20,25–29,44]. The LOCAT study
showed a trend toward increased progression of atherogenesis
in male carriers of the rare allele of the S19W SNP; in
contrast, no effect was found for the −1131C allele . In
the Framingham Heart Study, an almost 2-fold increased risk
of CVD was observed in females carrying the rare allele of the
−1131T>C SNP, but there was no effect in males. For the
19W allele, no effect was found in either gender . In
contrast, in the same Framingham Heart Study, the 19W allele
was associated with IMT values in both genders. In obese
subjects, this relationship with IMT was also observed for the
rare allele of the −1131T>C SNP .
The association of variants in the APOA5 gene with CVD
and IMT values was not previously studied in FCH patients,
who are known to have thicker IMT and an increased risk of
CVD . In the present study, we show that, although the rare
allele of the S19W variant in the APOA5 gene was associated
with FCH and a disturbed lipid profile, no association with
CVD or IMT was evident. This could be a result of the limited
number of individuals with CVD in our study population, and
the availability of IMT data in only a sub-set of the study
population, resulting in limited power for studying these
associations. Possible explanations for the contradictory results
previously found for CVD could be that the association of
variants in the APOA5 gene with CVD is independent of TG
levels, suggesting different mechanisms, or that the influence of
APOA5gene variants on TG is relatively small and therefore not
translated into differences in CVD [25,27,29].
In the present study, the measured genotype method and
FBAT were used to analyze the association of variants in the
APOA5 gene with FCH and its related phenotypes. The
measured genotype method is more powerful, uses the whole
pedigree and provides an effect estimate for the tested variables,
but is not robust in the presence of population stratification.
FBAT, however, is robust to stratification, but is less powerful
and uses incomplete pedigree information. The results of both
statistical analyses were highly concordant, thereby providing
strong evidence for the role of the S19W SNP of the APOA5
gene in FCH patients.
In the general population, the influence of variations in the
APOA5 gene on lipid levels is still controversial. Some studies
show that the association of variations in the APOA5 gene with
increased TG levels is limited or not present in the general
population [11,22,23]. Based on these studies, there is
conjecture that APOA5 gene variants modulate TG levels only
when there is an altered genetic and metabolic background. In
the present study, we show that the influence of the rare allele of
the S19W SNP on lipid levels was not restricted to FCH
patients, but also present in normolipidemic relatives and
In conclusion, in the present study of Dutch FCH families,
the rare allele of the S19W SNP in the APOA5 gene is
associated with FCH and a more atherogenic lipid profile.
Philippa J Talmud is supported by the British Heart
foundation. We thank Dr. M. den Heijer for the statistical
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bbadis.2006.10.012.
 P.N. Hopkins, G. Heiss, R.C. Ellison, M.A. Province, J.S. Pankow, J.H.
Eckfeldt, S.C. Hunt, Coronary artery disease risk in familial combined
hyperlipidemia and familial hypertriglyceridemia: a case-control compar-
ison from the National Heart, Lung, and Blood Institute Family Heart
Study, Circulation 108 (2003) 519–523.
 J.L. Goldstein, H.G. Schrott, W.R. Hazzard, E.L. Bierman, A.G. Motulsky,
Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid
levels in 176 families and delineation of a new inherited disorder,
combined hyperlipidemia, J. Clin. Invest. 52 (1973) 1544–1568.
 J. de Graaf, G. van der Vleuten, A.F. Stalenhoef, Diagnostic criteria in
relation to the pathogenesis of familial combined hyperlipidemia, Semin.
Vasc. Med. 4 (2004) 229–240.
 B.E. Aouizerat, H. Allayee, R.M. Cantor, R.C. Davis, C.D. Lanning, P.Z.
Wen, G.M. Dalinga-Thie, T.W. de Bruin, J.I. Rotter, A.J. Lusis, A genome
scan for familial combined hyperlipidemia reveals evidence of linkage
with a locus on chromosome 11, Am. J. Hum. Genet. 65 (1999) 397–412.
 H. Coon, R.H. Myers, I.B. Borecki, D.K. Arnett, S.C. Hunt, M.A.
Province, L. Djousse, M.F. Leppert, Replication of linkage of familial
combined hyperlipidemia to chromosome 1q with additional heteroge-
neous effect of apolipoprotein A-I/C-III/A-IV locus. The NHLBI Family
Heart Study, Arterioscler, Thromb. Vasc. Biol. 20 (2000) 2275–2280.
86 G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
 A. Huertas-Vazquez, C. guilar-Salinas, A.J. Lusis, R.M. Cantor, S.
Canizales-Quinteros, J.C. Lee, L. Mariana-Nunez, R.M. Laura Riba-
Ramirez, A. Jokiaho, T. Tusie-Luna, P. Pajukanta, Familial combined
hyperlipidemia in Mexicans: association with upstream transcription factor
1 and linkage on chromosome 16q24.1, Arterioscler. Thromb. Vasc. Biol.
25 (2005) 1985–1991.
E.L. Jones, J. Amey, S. Colilla, C.K. Neuwirth, R. Allotey, M. Seed, D.J.
Betteridge, D.J. Galton, N.J. Cox, G.I. Bell, J. Scott, C.C. Shoulders,
Confirmed locus on chromosome 11p and candidate loci on 6q and 8p for
the triglyceride and cholesterol traits of combined hyperlipidemia,
Arterioscler. Thromb. Vasc. Biol. 23 (2003) 2070–2077.
 P. Pajukanta, I. Nuotio, J.D. Terwilliger, K.V. Porkka, K. Ylitalo, J.
Pihlajamaki, A.J. Suomalainen, A.C. Syvanen, T. Lehtimaki, J.S.
Viikari, M. Laakso, M.R. Taskinen, C. Ehnholm, L. Peltonen, Linkage
of familial combined hyperlipidaemia to chromosome 1q21–q23, Nat.
Genet. 18 (1998) 369–373.
 W. Pei, H. Baron, B. Muller-Myhsok, H. Knoblauch, S.A. Al-Yahyaee, R.
Hui, X. Wu, L. Liu, A. Busjahn, F.C. Luft, H. Schuster, Support for linkage
of familial combined hyperlipidemia to chromosome1q21–q23 in Chinese
and German families, Clin. Genet. 57 (2000) 29–34.
 B.E. Aouizerat, H. Allayee, R.M. Cantor, G.M. Dalinga-Thie, C.D.
Lanning,T.W. de Bruin, A.J.Lusis, J.I. Rotter, Linkage of a candidate gene
locus to familial combined hyperlipidemia: lecithin:cholesterol acyltrans-
ferase on 16q, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 2730–2736.
 S. Eichenbaum-Voline, M. Olivier, E.L. Jones, R.P. Naoumova, B.
Jones, B. Gau, H.N. Patel, M. Seed, D.J. Betteridge, D.J. Galton, E.M.
Rubin, J. Scott, C.C. Shoulders, L.A. Pennacchio, Linkage and
association between distinct variants of the APOA1/C3/A4/A5 gene
cluster and familial combined hyperlipidemia, Arterioscler. Thromb.
Vasc. Biol. 24 (2004) 167–174.
 A.P. Wojciechowski, M. Farrall, P. Cullen, T.M. Wilson, J.D. Bayliss, B.
Farren, B.A. Griffin, M.J. Caslake, C.J. Packard, J. Shepherd, Familial
combined hyperlipidaemia linked to the apolipoprotein AI–CII–AIV gene
cluster on chromosome 11q23–q24, Nature 349 (1991) 161–164.
 L.A. Pennacchio, M. Olivier, J.A. Hubacek, J.C. Cohen, D.R. Cox, J.C.
Fruchart, R.M. Krauss, E.M. Rubin, An apolipoprotein influencing
triglycerides in humans and mice revealed by comparative sequencing,
Science 294 (2001) 169–173.
 P.J. O'Brien, W.E. Alborn, J.H. Sloan, M. Ulmer, A. Boodhoo, M.D.
Knierman, A.E. Schultze, R.J. Konrad, The novel apolipoprotein A5 is
present in human serum, is associated with VLDL, HDL, and
chylomicrons, and circulates at very low concentrations compared with
other apolipoproteins, Clin. Chem. 51 (2005) 351–359.
 M. Ishihara, T. Kujiraoka, T. Iwasaki, M. Nagano, M. Takano, J. Ishii,
M. Tsuji, H. Ide, I.P. Miller, N.E. Miller, H. Hattori, A sandwich
enzyme-linked immunosorbent assay for human plasma apolipoprotein
A–V concentration, J. Lipid Res. 46 (2005) 2015–2022.
 L.A. Pennacchio, E.M. Rubin, Apolipoprotein A5, a newly identified gene
that affects plasma triglyceride levels in humans and mice, Arterioscler.
Thromb. Vasc. Biol. 23 (2003) 529–534.
 M. Merkel,B. Loeffler, M.Kluger, N.Fabig,G.Geppert,L.A.Pennacchio,
A. Laatsch, J. Heeren, Apolipoprotein AVaccelerates plasma hydrolysis of
triglyceride-rich lipoproteins by interaction with proteoglycan-bound
lipoprotein lipase, J. Biol. Chem. 280 (2005) 21553–21560.
 F.G. Schaap, P.C. Rensen, P.J. Voshol, C. Vrins, H.N. van der Vliet, R.A.
Chamuleau, L.M. Havekes, A.K. Groen, K.W. van Dijk, ApoAV reduces
plasma triglycerides by inhibiting very low density lipoprotein-triglyceride
(VLDL-TG) production and stimulating lipoprotein lipase-mediated
VLDL-TG hydrolysis, J. Biol. Chem. 279 (2004) 27941–27947.
 L.A. Pennacchio, M. Olivier, J.A. Hubacek, R.M. Krauss, E.M. Rubin,
J.C. Cohen, Two independent apolipoprotein A5 haplotypes influence
human plasma triglyceride levels, Hum. Mol. Genet. 11 (2002)
 C.Q. Lai, S. Demissie, L.A. Cupples, Y. Zhu, X. Adiconis, L.D. Parnell,
D. Corella, J.M. Ordovas, Influence of the APOA5 locus on plasma
triglyceride, lipoprotein subclasses, and CVD risk in the Framingham
Heart Study, J. Lipid Res. 45 (2004) 2096–2105.
 S. Martin, V. Nicaud, S.E. Humphries, P.J. Talmud, Contribution of
APOA5 gene variants to plasma triglyceride determination and to the
response to both fat and glucose tolerance challenges, Biochim. Biophys.
Acta 1637 (2003) 217–225.
 R. Mar, P. Pajukanta, H. Allayee, M. Groenendijk, G.M. Dalinga-Thie,
R.M. Krauss, J.S. Sinsheimer, R.M. Cantor, T.W. de Bruin, A.J. Lusis,
Association of the APOLIPOPROTEIN A1/C3/A4/A5 gene cluster with
triglyceride levels and LDL particle size in familial combined
hyperlipidemia, Circ. Res. 94 (2004) 993–999.
 J. Ribalta, L. Figuera, J. Fernandez-Ballart, E. Vilella, C.M. Castro,
L. Masana, J. Joven, Newly identified apolipoprotein AV gene
predisposesto high plasma
hyperlipidemia, Clin. Chem. 48 (2002) 1597–1600.
 J.E. Hokanson, M.A. Austin, Plasma triglyceride level is a risk factor for
cardiovascular disease independent of high-density lipoprotein cholesterol
level: a meta-analysis of population-based prospective studies,
J. Cardiovasc. Risk 3 (1996) 213–219.
 R. Elosua, J.M. Ordovas, L.A. Cupples, C.Q. Lai, S. Demissie, C.S. Fox,
J.F. Polak, P.A. Wolf, R.A. D'Agostino, C.J. O'donnell, Variability at the
APOA5 locus is associated with carotid atherosclerosis with a modifying
effect of obesity: the Framingham Heart Study, J. Lipid Res. 47 (2006)
 J.A. Hubacek, Z. Skodova, V. Adamkova, V. Lanska, R. Poledne, The
influence of APOAV polymorphisms (T-1131>C and S19>W) on plasma
triglyceride levels and risk of myocardial infarction, Clin. Genet. 65 (2004)
 K.W. Lee, A.F. Ayyobi, J.J. Frohlich, J.S. Hill, APOA5 gene polymor-
phism modulates levels of triglyceride, HDL cholesterol and FERHDL but
is not a risk factor for coronary artery disease, Atherosclerosis 176 (2004)
 C. Szalai, M. Keszei, J. Duba, Z. Prohaszka, G.T. Kozma, A. Csaszar, S.
Balogh, Z. Almassy, G. Fust, A. Czinner, Polymorphism in the promoter
region of the apolipoprotein A5 gene is associated with an increased
susceptibility for coronary artery disease, Atherosclerosis 173 (2004)
 P.J. Talmud, S. Martin, M.R. Taskinen, M.H. Frick, M.S. Nieminen, Y.A.
Kesaniemi, A. Pasternack, S.E. Humphries, M. Syvanne, APOA5 gene
variants, lipoprotein particle distribution, and progression of coronary
heart disease: results from the LOCAT study, J. Lipid Res. 45 (2004)
 G. Assmann, H. Schulte, Relation of high-density lipoprotein cholesterol
and triglycerides to incidence of atherosclerotic coronary artery disease
(the PROCAM experience). Prospective Cardiovascular Munster study,
Am. J. Cardiol. 70 (1992) 733–737.
 M.J. Veerkamp, J. de Graaf, J.C. Hendriks, P.N. Demacker, A.F.
Stalenhoef, Nomogram to diagnose familial combined hyperlipidemia on
the basis of results of a 5-year follow-up study, Circulation 109 (2004)
 K. Nakajima, T. Saito, A. Tamura, M. Suzuki, T. Nakano, M. Adachi,
A. Tanaka, N. Tada, H. Nakamura, E. Campos, Cholesterol in remnant-
like lipoproteins in human serum using monoclonal anti apo B-100 and
anti apo A-I immunoaffinity mixed gels, Clin. Chim. Acta 223 (1993)
 S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for
extracting DNA from human nucleated cells, Nucleic Acids Res. 16 (1988)
 P.J. Talmud, E. Hawe, S. Martin, M. Olivier, G.J. Miller, E.M. Rubin, L.A.
Pennacchio, S.E. Humphries, Relative contribution of variation within the
APOC3/A4/A5 gene cluster in determining plasma triglycerides, Hum.
Mol. Genet. 11 (2002) 3039–3046.
 E.ter Avest, S. Holewijn, S.J.H. Bredie, A.F.H. Stalenhoef, J.de Graaf,
Remnant particles are the major determinant of an increased intima
mediathickness in patients with familial combined hyperlipidemia (FCH),
Atherosclerosis (in press).
 P.J. Touboul, P. Prati, P.Y. Scarabin, V. Adrai, E. Thibout, P.
Ducimetiere, Use of monitoring software to improve the measurement
of carotid wall thickness by B-mode imaging, J. Hypertens (Suppl. 10)
triglycerides in familialcombined
87 G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88
 J.R. O'Connell, D.E. Weeks, PedCheck: a program for identification of Download full-text
genotype incompatibilities in linkage analysis, Am. J. Hum. Genet. 63
 J.C. Barrett, B. Fry, J. Maller, M.J. Daly, Haploview: analysis and visuali-
zation of LD and haplotype maps, Bioinformatics 21 (2005) 263–265.
 S.B. Gabriel, S.F. Schaffner, H. Nguyen, J.M. Moore, J. Roy, B.
Blumenstiel, J. Higgins, M. DeFelice, A. Lochner, M. Faggart, S.N. Liu-
Cordero, C. Rotimi, A. Adeyemo, R. Cooper, R. Ward, E.S. Lander, M.J.
Daly, D. Altshuler, The structure of haplotype blocks in the human
genome, Science 296 (2002) 2225–2229.
 N.J. Risch, Searching for genetic determinants in the new millennium,
Nature 405 (2000) 847–856.
 S. Horvath, X. Xu, N.M. Laird, The family based association test method:
strategies for studying general genotype–phenotype associations, Eur. J.
Hum. Genet. 9 (2001) 301–306.
 L. Almasy, J. Blangero, Multipoint quantitative-trait linkage analysis in
general pedigrees, Am. J. Hum. Genet. 62 (1998) 1198–1211.
 J.A. Hubacek, J. Kovar, Z. Skodova, J. Pit'ha, V. Lanska, R. Poledne,
Genetic analysis of APOAV polymorphisms (T-1131/C, Ser19/Trp and
Val153/Met): no effect on plasma remnant particles concentrations, Clin.
Chim. Acta 348 (2004) 171–175.
 E.A. Ruiz-Narvaez, Y. Yang, Y. Nakanishi, J. Kirchdorfer, H. Campos,
APOC3/A5 haplotypes, lipid levels, and risk of myocardial infarction in
the Central Valley of Costa Rica, J. Lipid Res. 46 (2005) 2605–2613.
88 G.M. van der Vleuten et al. / Biochimica et Biophysica Acta 1772 (2007) 81–88