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Atherosclerosis 206 (2009) 1730

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Review

Apolipoprotein B levels, APOB alleles, and risk of ischemic cardiovascular disease


in the general population, a review
Marianne Benn
Department of Clinical Biochemistry KB3011, Section for Molecular Genetics, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen , Denmark

a r t i c l e

i n f o

Article history:
Received 4 December 2008
Received in revised form 5 January 2009
Accepted 5 January 2009
Available online 15 January 2009
Keywords:
Apolipoprotein B
Low density lipoprotein cholesterol
Atherosclerosis
Ischemic heart disease
Ischemic stroke
Genetics
Epidemiology

a b s t r a c t
Apolipoprotein B is a key component in lipid metabolism. Subendothelial retention of apolipoprotein
B containing lipoproteins is a necessary initiating event in atherogenesis, and high plasma levels of
apolipoprotein B is a risk factor for atherosclerosis, whereas low levels may provide protection.
The present review examines, with focus on general population studies, apolipoprotein B levels as a
predictor of ischemic cardiovascular disease, as well as the association of mutations and polymorphisms
in APOB with plasma apolipoprotein B levels, and risk of ischemic cardiovascular disease.
The studies can be summarized as follows: (1) apolipoprotein B predicts ischemic cardiovascular events
in both genders, and is better than LDL cholesterol in this respect; (2) linkage disequilibrium structure in
APOB is more complex than expected from HapMap data, because a minimal set of tag single nucleotide
polymorphisms capturing the entire variation in APOB cannot be identied, and thus most polymorphisms must be evaluated separately in association studies; (3) APOB mutations and polymorphisms are
associated with a range of apolipoprotein B and LDL cholesterol levels, although the magnitude of effect
sizes of common polymorphisms are modest; (4) both mutations and polymorphisms are associated with
LDL metabolism in vivo; (5) association of APOB mutations and polymorphisms with lipid and disease
phenotype cannot be predicted in silico using evolutionary conservation or existing prediction programs;
and nally, (6) except for the E4154K polymorphism that possibly predicts a reduction in risk of ischemic
cerebrovascular disease and ischemic stroke, common APOB polymorphisms with modest effect sizes on
lipid levels do not predict risk of ischemic heart disease, myocardial infarction, ischemic cerebrovascular
disease, or ischemic stroke in the general population.
2009 Elsevier Ireland Ltd. All rights reserved.

Contents
1.
2.
3.
4.
5.
6.
7.
8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plasma apolipoprotein B levels and risk of ischemic cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
APOB mutations and polymorphisms and linkage disequilibrium structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
APOB mutations and polymorphisms and plasma apolipoprotein B levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
APOB mutations and polymorphisms and LDL metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evolutionary conservation and predicted effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
APOB mutations and polymorphisms and risk of ischemic cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Discussion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: APOB, apolipoprotein B gene; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor gene; LIPC,
hepatic lipase gene; LPL, lipoprotein lipase gene; MTP, microsomal triglyceride transfer protein gene; SNP, single nucleotide polymorphism; PCSK9, pro-protein convertase
subtilisin/kexin 9 gene; UTR, un-translated region; VLDL, very low density lipoprotein.
Tel.: +45 35456506; fax: +45 35456500.
E-mail address: marianne@benn.dk.
0021-9150/$ see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2009.01.004

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M. Benn / Atherosclerosis 206 (2009) 1730

1. Introduction
Apolipoprotein B is a non-exchangeable apolipoprotein found
in two forms in humans, apolipoprotein B-48 and apolipoprotein
B-100. Apolipoprotein B-48 is synthesized in enterocytes and is
essential for the assembly and secretion of chylomicrons to the
blood [1,2]. The main function of chylomicrons is to transport
triglycerides from the intestine to the liver, adipose, and muscle tissue. Apolipoprotein B-100 is synthesized in the liver and is
essential for the initial lipidation of the nascent very low density lipoprotein (VLDL) particle [1]. The main function of VLDL is
to deliver triglycerides from the liver to the circulation [2]. Upon
secretion, the triglyceride core of VLDL is hydrolyzed by lipoprotein lipase thereby delivering free fatty acids to muscle and adipose
tissue. The resulting triglyceride depleted particle, the VLDL remnant or intermediate density lipoprotein (IDL), can either be cleared
from the circulation by binding to the hepatic remnant receptor, or
hydrolyzed further by hepatic lipase to form low density lipoprotein
(LDL). Apolipoprotein B is the sole remaining protein component
on the LDL particle. LDL is cleared from the blood by binding of
apolipoprotein B to the LDL receptor, and is subsequently internalized and degraded in the liver [2,3] (Supplementary Fig. 1).
Reduced secretion of apolipoprotein B results in reduced production of chylomicron and VLDL, leading to malabsorption of fats
and fat-soluble vitamins [4]. Although apolipoprotein B containing lipoproteins are pivotal for lipid absorption and triglyceride
homeostasis, high levels in plasma induce atherosclerosis. Subendothelial retention of apolipoprotein B-containing lipoproteins is
a necessary initiating event of atherogenesis (for a review see
[5]), and high plasma levels of apolipoprotein B and LDL cholesterol are risk factors for atherosclerosis [6], whereas low levels of
apolipoprotein B may provide protection against atherosclerosis.
Twin studies suggest that 5060% of the variation in plasma
levels of apolipoprotein B is genetically determined [79]. Rare missense mutations in the apolipoprotein B gene (APOB) may result not
only in severe hypercholesterolemia and increased risk of ischemic
cardiovascular disease, but also in hypocholesterolemia [1013].
From an epidemiological perspective, rare deleterious mutations
(minor allele frequency <1%; e.g. APOB R3500Q/W) confer an important risk of ischemic cardiovascular disease in mutation carriers,
but their impact at the population level is minimal. Conversely, single nucleotide polymorphisms (SNPs; minor allele frequency 1%)
may, because they are frequent, have a population impact that is far
from negligible, despite a weak effect at the individual level.
The present review summarizes recent general population studies which have provided insight into the predictive value of
apolipoprotein B levels for ischemic cardiovascular disease, and
the effects of rare and common APOB alleles on plasma levels of
apolipoprotein B, lipid metabolism, and risk of ischemic cardiovascular disease.
2. Plasma apolipoprotein B levels and risk of ischemic
cardiovascular disease
Ischemic cardiovascular disease, that is ischemic heart disease
and ischemic cerebrovascular disease, is one of the major causes of
hospitalization and death in afuent societies [6]. Because elevated
LDL cholesterol levels cause atherosclerosis and thus ischemic cardiovascular disease, levels of LDL cholesterol are widely used for
screening to identify individuals at risk of this disease. Since risk
of atherosclerosis may be more directly related to the total number of circulating atherogenic particles that enter the arterial wall,
than to the concentration of cholesterol in LDL particles only, it has
been suggested that apolipoprotein B could be a better predictor of
risk than concentrations of cholesterol in the LDL fraction [1419].
A likely explanation for this is that apolipoprotein B is a measure-

ment of the total number of atherogenic particles (including LDL,


IDL, VLDL, chylomicrons, and chylomicron remnants), because each
of these contain a single molecule of apolipoprotein B, while LDL
cholesterol is an estimate of the mass of cholesterol in the LDL
fraction only [20].
Several studies have shown that plasma levels of apolipoprotein B predict risk of ischemic cardiovascular disease and ischemic
heart disease in the general population, as well as in patients
with a previous myocardial infarction or the metabolic syndrome
[1419,2124]. Apolipoprotein B as a predictor of ischemic cerebrovascular disease is not so well established, but results from the
AMORIS study and the Chin-Shan Community Cohort have shown
that apolipoprotein B predicts risk of fatal ischemic stroke in the
general population, and in patients with a previous myocardial
infarction [2527].
The Copenhagen City Heart Study is the rst prospective study
to simultaneously examine prediction of ischemic heart disease,
myocardial infarction, ischemic cerebrovascular disease, ischemic
stroke, and any ischemic cardiovascular disease risk in the same
general population study including both women and men, and
to compare apolipoprotein B and LDL cholesterol levels as predictors of these events [28]. Results from this study showed that
women with apolipoprotein B in the upper versus lower tertile
had hazard ratios for ischemic heart disease of 1.8 (95% condence
interval: 1.22.5), for myocardial infarction of 2.6 (1.44.7), and for
any ischemic cardiovascular event of 1.8 (1.32.3) (Fig. 1). Men had
hazard ratios for ischemic heart disease of 1.9 (1.52.6), for myocardial infarction of 2.4 (1.53.6), and for any ischemic cardiovascular
event of 1.6 (1.32.1). Apolipoprotein B predicted risk of ischemic
cerebrovascular disease and ischemic stroke in women with hazard ratios of, respectively, 1.9 (1.22.9) and 1.6 (1.02.8), but did not
predict risk in men [28].
In the same study, apolipoprotein B was superior to LDL cholesterol in the prediction of ischemic heart disease, myocardial
infarction, any ischemic cardiovascular event, and any non-fatal
ischemic cardiovascular event in both genders (P = 0.03 to <0.001)
[28]. Similar results have been found with respect to prediction of
ischemic heart disease in several studies [14,17,22,23,26,28,29].
Most studies have solely reported data on the relative risk of
ischemic cardiovascular disease by apolipoprotein B levels, like
those shown in Fig. 1, however, information on absolute 10-year risk
of any cardiovascular event may be a more useful tool in the primary
prevention of such events, and can be used to identify patients with
asymptomatic or subclinical disease who could potentially benet
from intensive preventive efforts. Absolute 10-year risk of any cardiovascular event estimated in the Copenhagen City Heart Study
showed that in smokers older than 60 years of age with systolic
blood pressure above 160 mmHg, apolipoprotein B contributed 11%
in women and 15% in men to the increase in absolute 10-year risk
from the lower to the upper apolipoprotein B tertile (Fig. 2).
Although data from all the above studies indicate that
apolipoprotein B can contribute further information to better dene
cardiovascular disease risk, not only in men but also in women, neither the American Heart Associations recommendations for risk
assessment in asymptomatic individuals based on the Framingham Study [3032], nor the recommendations of the First Joint
Task Force of the European Societies on Coronary Prevention based
on the SCORE project have included apolipoprotein B in their risk
assessments [33]. A reason for the reluctance to include apolipoprotein B in existing guidelines is that there is a large overlap in levels of
both apolipoprotein B and LDL cholesterol in individuals with and
without ischemic cardiovascular events [28], and it is therefore difcult to select clinically relevant cut-points to separate between
these two diagnostic groups using levels of apolipoprotein B or
LDL cholesterol alone. A consensus diagnostic cut-point/treatment
target has been established for LDL cholesterol through years of

M. Benn / Atherosclerosis 206 (2009) 1730

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Fig. 1. Risk of ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, ischemic stroke, and any ischemic cardiovascular event by tertiles of apolipoprotein B and low density lipoprotein (LDL) cholesterol in women and men from the general population, the Copenhagen City Heart Study [28]. CI = condence interval.

intervention studies in different patient groups and populations,


followed by estimation of the number of individuals classied as
being at risk and the resulting costs of preventive treatment in these
individuals. The American Diabetes Association and the American
College of Cardiology Foundation have recently suggested a similar
validated treatment target of 80 mg/dL apolipoprotein B in patients
with known cardiovascular disease or with diabetes and another
cardiovascular risk factor [34].
In summary, apolipoprotein B predicts ischemic cardiovascular
events in both genders, and is better than LDL cholesterol in this
respect. Prediction of future ischemic cardiovascular events could
be improved by measuring apolipoprotein B.
3. APOB mutations and polymorphisms and linkage
disequilibrium structure
The observation that apolipoprotein B levels predict both
ischemic heart disease and ischemic cerebrovascular disease
makes it important to understand factors that determine plasma
apolipoprotein B levels including genetic variation in APOB itself.
Two rare missense mutations, APOB R3500Q and R3500W, cause the
monogenic disorder familial defective apolipoprotein B-100 (OMIM
144010), whereas another missense mutation R3480P is associated with a hypobetalipoproteinemia phenotype, emphasizing that
rare mutations in APOB do indeed contribute to apolipoprotein B
and cholesterol levels [11,13]. Twin studies indicate that 5060%
of the variation in apolipoprotein B levels can be attributed to
genetic variation [79], and in individuals without rare mutations,
polymorphisms in APOB may play the major role in determining
apolipoprotein B and cholesterol levels.
The APOB gene spans 43 kilobases including 28 exons,
and encodes a 27 amino acid signal peptide followed by a
mature protein of 4536 amino acids (Fig. 3) [3538]. A total

of 132 genetic variants have been reported in APOB: one


approximately 2000 basepairs upstream, one in the 5 UTR, 63
non-synonymous coding, 22 synonymous coding, 44 intronic, and
1 variant in the 3 UTR (http://www.hapmap.org/index.html.en,
http://www.ensembl.org/index.html). Due to the large size of APOB
and the large number of common genetic variants reported, linkage disequilibrium structure between polymorphisms is of interest
and can potentially provide information on how best to identify genetic variants with phenotypic effects, and help interpret
genotypephenotype associations. Because polymorphisms highly
correlated with each other may serve as a proxy for each other (tag
SNPs), knowledge of the linkage disequilibrium structure may also
reduce the number of genetic variants needed to be genotyped to
study genotypephenotype associations.
Some smaller studies have reported results on linkage disequilibrium between a limited number of APOB SNPs [39,40], but the
results from the Copenhagen City Heart Study are the most comprehensive with respect to sample size (n = 9185) and number of
variants genotyped (ten SNPs). Results on linkage disequilibrium as
r2 and D from this study are shown in Fig. 4 [41]. Generally, a high
degree of linkage disequilibrium is present throughout the gene
(D > 0.80), but especially between the ve most C-terminal SNPs
(T2488T, P2712L, R3611Q, E4154K, and N4311S), indicating that
these SNPs are on the same haplotypes. However, only the minor
alleles of P2712L and N4311S are also highly correlated (r2 = 1.0, all
other r2 s <0.70), and can therefore tag or serve as proxy for the other
SNP.
According to HapMap, T71I, Ivs4 + 171c > a, A591 V,
Ivs18 + 379a > c, Ivs18 + 1708g > t, T2488Tc > t, and E4154K
are seven tag SNPs (marked in blue text in Fig. 3A and 4)
capturing genetic variation in almost the entire APOB gene
(http://www.hapmap.org/index.html.en): T71I and Ivs4 + 171c > a,
form a haploblock covering the N-terminal part of the gene (12,986

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M. Benn / Atherosclerosis 206 (2009) 1730

Fig. 2. Absolute 10-year risk of any ischemic cardiovascular event by smoking status, age, systolic blood pressure, and tertiles of apolipoprotein B (apo B) for women and
men, separately. Apolipoprotein B tertile: 1 (green) lower tertile, 2 (orange) middle tertile, and 3 (red) upper tertile [28] (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of the article).

nucleotides), and Ivs18 + 1708g > t, T2488Tc > t, E4154K another


block covering the most C-terminal end of the gene (18,719
nucleotides), while A591 V and Ivs18 + 379a > c cover two haploblocks of, respectively, 5585 and 1329 nucleotides in between.
It follows that Ivs18 + 1708g > t, T2488Tc > t and E4154K according
to HapMap should tag P2712L, R3611Q, and N4311S, since they
are in the same haploblock. In the Copenhagen City Heart Study,
although these six SNPs are often on the same haplotypes (all
D > 0.90) none are highly correlated, with the exception of P2712L
with N4311S (Fig. 4; r2 = 1.0 for P2712L with N4311S, r2 = 0.30 for
T2488Tc > t with P2712L/N4311S, and all other r2 s <0.11), indicating
that Ivs18 + 1708g > t, T2488Tc > t and E4154K cannot tag or serve
as a proxy for P2712L, R3611Q or N4311S, and that the effects on
phenotype of each of these SNPs must be evaluated separately.
In summary, several SNPs in APOB are at most in weak linkage
disequilibrium, and therefore cannot tag each other. Association
between SNPs and phenotype must therefore be evaluated for each
SNP separately, rather than relying on a few tag SNPs.
4. APOB mutations and polymorphisms and plasma
apolipoprotein B levels
Rare mutations in APOB can have profound inuence on
plasma apolipoprotein B and LDL cholesterol levels (Fig. 5) [11,12].
R3500Q/W heterozygosity observed in individuals from the general population associated with increases in levels of apolipoprotein
B of 44% (37.8 mg/dL) and LDL cholesterol of 80% (1.92 mmol/L)
[11,12], and R3480P with decreases of 23% (19.9 mg/dL) and 29%
(0.93 mmol/L), respectively [13]. R3531C did not associate with
apolipoprotein B or LDL cholesterol levels [11].

Association of non-synonymous SNPs in APOB with lipid and


lipoprotein levels has been examined extensively in several small
studies and meta-analyses, although, with conicting results
[42,43]. To address this further with focus on non-synonymous
SNPs in the general population, we genotyped ten SNPs in APOB
in 9185 individuals from the Danish general population followed
prospectively for 25 years in the Copenhagen City Heart Study [41].
Six non-synonymous SNPs in APOB were selected, because they
were located in important functional domains crucial for lipidation of nascent apolipoprotein B (T71I, A591 V) [44,45], involved
in structural changes of apolipoprotein B during the conversion
of VLDL to LDL (P2712L) [46], or known or suspected of regulating binding to the LDL receptor (R3611Q, E4154K, N4311S)
[47,48]. In addition, we genotyped four other SNPs (Ivs4 + 171c > a,
Ivs18 + 379a > c, Ivs18 + 1708g > t, and T2488Tc > t [49]) that together
with T71I, A591 V, and E4154K are predicted by HapMap to largely
tag the genetic variation in the entire coding and intronic regions
of APOB, comprising approximately 43 kilobases of genomic DNA.
Location of the four mutations and ten SNPs relative to the
amino acid sequence and structural and functional domains of
apolipoprotein B are shown in Fig. 3. Minor allele frequencies are
T71I: 0.33, Ivs4 + 171c > a: 0.14, A591 V: 0.47, Ivs18 + 379a > c: 0.30,
Ivs18 + 1708g > t: 0.45, T2488Tc > t: 0.48, P2712L: 0.21, R3611Q: 0.09,
E4154K: 0.17, and N4311S: 0.21 (Fig. 4).
Overall, all ten SNPs were associated with either increases (T71I,
Ivs18 + 1708g > t, T2488Tc > t, R3611Q) or decreases (Ivs4 + 171c > a,
A591 V, Ivs18 + 379a > c, P2712L, E4154K, N4311S) in plasma
apolipoprotein B (p-values by ANOVA from 0.03 to <0.001) (Fig. 5).
T71I, Ivs18 + 1708g > t, T2488Tc > t, R3611Q were associated with
increases in apolipoprotein B of 3.2%, 3.1%, 2.5%, and 1.9% (2.7,

M. Benn / Atherosclerosis 206 (2009) 1730

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Fig. 3. (A) Location of ten studied single nucleotide polymorphisms (SNPs) and four mutations in APOB relative to the amino acid sequence and the structural and functional
domains of apolipoprotein B [41]. Structural studies have suggested a pentapartite secondary structure for apolipoprotein B with one mixed 1 region that forms a globular
domain important during initial lipidation (aa 1795), two amphiphatic -sheet domains (aa 8272001 and 25714032) that bind lipids non-reversibly, and two amphiphatic
-helical domains (aa 20452587 and 40174515) that bind lipids reversibly and are suggested to be involved in export of triglycerides and phospholipids, ordered in a
NH2-1 -1 -2 -2 -3 -COOH conguration [46]. The seven SNPs predicted by HapMap (http://www.hapmap.org/index.html.en) to tag for the genetic variation in almost
the entire APOB gene (coding regions and introns) are in blue text. Mutations are in red text. aa = amino acid, MTP = microsomal triglyceride transfer protein. (B) Schematic
diagram of the structure of apoB-100 on the surface of LDL (adapted from [4648,55]). Apolipoprotein B enwraps the very low density lipoprotein, intermediate density
lipoprotein, and low density lipoprotein (LDL) particles like a belt completing the encirclement by about amino acid residue 4050. The carboxyl terminal end forms a bow that
crosses backward over the chain between residues 3000 and 3500 [47]. The LDL receptor binding domain of apolipoprotein B has been localized to residues 33593369 [48].
Non-synonymous variants are marked in red (associated with increases in apolipoprotein B levels), green (reductions in apolipoprotein B levels) or yellow (no association
with apolipoprotein B levels) (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article).

1.7, 2.1, and 1.6 mg/dL), respectively, in heterozygotes versus noncarriers, and T71I, Ivs18 + 1708g > t, and T2488Tc > t also with an
increase in apolipoprotein B of 6.5%, 5.5%, 6.2%, and 5.5% (5.5,
4.7, 5.2, and 4.7 mg/dL), respectively, in homozygotes versus noncarriers. Ivs4 + 171c > a, A591 V, Ivs18 + 379a > c, P2712L, E4154K,
and N4311S were associated with decreases in apolipoprotein B
of 3.7%, 2.9%, 1.4%, 3.0%, 2.3%, and 3.0% (3.2, 2.5, 1.7, 2.6, 2.0,
and 2.6 mg/dL), respectively, in heterozygotes versus non-carriers,
and Ivs4 + 171c > a, A591 V, and E4154K were also associated with
decreases in apolipoprotein B of 6.5%, 4.0%, and 5.0% (5.7, 3.5,
and 4.4 mg/dL), respectively, in homozygotes versus non-carriers.
In absolute values, the maximum increase in apolipoprotein
B as a function of SNP genotype was 5.5 mg/dL (0.30 mmol/L
in LDL cholesterol) for T71I homozygotes versus non-carriers,
while the maximum decrease in apolipoprotein B was 5.7 mg/dL
(0.28 mmol/L in LDL cholesterol) for Ivs4 + 171c > a homozygotes
versus non-carriers (Fig. 5). Similar results were found for total
cholesterol, but none of the ten SNPs were associated with increase
or decrease in plasma levels of triglycerides, VLDL cholesterol, HDL
cholesterol, or apolipoprotein AI [41,49].
Including the three previously described mutations in
apolipoprotein B (R3480P, R3500Q/W, R3531C) [1113], the
spectrum of APOB mutations and polymorphisms associated
with variation in apolipoprotein B and LDL cholesterol span a

wide range of allele frequencies (from 0.0004 for R3480P to 0.48


for T2488Tc > t) and magnitude of phenotypic effect sizes (from
23% apolipoprotein B reduction for R3480P to 44% increase for
R3500Q, or corresponding 2980%, respectively in LDL cholesterol
levels) (Fig. 6). The magnitude of phenotypic effect sizes of the
polymorphisms on apolipoprotein B levels are fairly modest, at
most 6%, compared to the 31% and 69% increase in apolipoprotein B level from the lower tertile to the middle and upper
tertile of apolipoprotein B, respectively, in the general population
(Figs. 1 and 2).
In summary, these results suggest that several mutations and
polymorphisms in APOB contribute to plasma levels of apolipoprotein B and LDL cholesterol in the general population, although the
effects of the common alleles are modest.
5. APOB mutations and polymorphisms and LDL
metabolism
Different regions in apolipoprotein B are of functional importance [5052], and amino acid changes in functional domains
caused by mutations or SNPs may change the structure of
apolipoprotein B and thereby affect LDL metabolism through
changes in the interaction of apolipoprotein B with the LDL receptor. Metabolic studies have shown that ve naturally occurring

22

M. Benn / Atherosclerosis 206 (2009) 1730

Fig. 4. Pairwise linkage disequilibrium between ten single nucleotide polymorphisms (SNPs) examined in the Copenhagen City Heart Study [41]. Seven SNPs predicted
by HapMap (http://www.hapmap.org/index.html.en) to tag for the genetic variation in almost the entire APOB gene (coding regions and introns) are marked in blue text.
Disequilibrium statistics reported as exact values of D , ranging from 1 to +1, below the diagonal, and of r2 above the diagonal. D is a measure of the difference between
the observed haplotype frequency (frequency of the two rare alleles occurring together) and the haplotype frequency expected under linkage equilibrium (the product of
the two allele frequencies). D values of or +1 indicate absence of recombination events between the two loci. r2 is D squared and divided by the product of the four allele
frequencies and is inversely related to the sample size required to detected genetic association between markers that are in complete linkage disequilibrium. All D values
are positive, indicating that the rare alleles at each locus segregate together. The color code indicates the degree of linkage disequilibrium between SNPs. MAF = minor allele
frequency (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article). Copyright 2008, The Endocrine Society.

Fig. 5. Plasma levels of apolipoprotein B and low density lipoprotein (LDL) cholesterol as a function of APOB mutations and polymorphisms in the general population, the
Copenhagen City Heart Study [11,13,41]. Values are means standard errors. Number of individuals in each genotype is given below the bars. P-values above bars by analysis
of variance or t-test (2-group comparisons). Post hoc tests by t-test: * P < 0.05, P < 0.01, P < 0.001. Bonferroni corrected signicance level P < 0.008. Dashed lines are population
mean values.

M. Benn / Atherosclerosis 206 (2009) 1730

Fig. 6. Magnitude of observed phenotypic associations with apolipoprotein B levels


as a function of the allele frequency of mutations and polymorphisms APOB. Each
diamond denotes the percent increase or decrease in apoliporotein B level associated
with a genetic variant plotted against the frequency of the variant. All values are from
the Copenhagen City Heart Study [11,13,41]. Variants are marked in red (increasing
apolipoprotein B levels), green (decreasing apolipoprotein B levels) or yellow (no
statistically signicant association with apolipoprotein B levels). The vertical dashed
line denotes the one percent threshold separating mutations (minor allele frequency
<1%) and polymorphisms (1%). The observed magnitude of association of variants
(diamonds) are compared with the blue curves that correspond to what is predicted
by the common diseaserare variant hypothesis for variants under a mild (dashed
line) and moderate (solid line) purifying selection (adapted from original [81]) (For
interpretation of the references to color in this gure legend, the reader is referred
to the web version of the article).

APOB mutations are associated with varying degrees of defective LDL receptor binding: R3500Q [50], R3500W [53], R3531C
[54], R3480W [55], and R3480P [13], but result in very different
lipid phenotypes. Results on human in vivo LDL turnover studies for these genotypes (except R3500W) together with studies
on the common T2488T and E4154K polymorphisms identied
in the general population, the Copenhagen City Heart Study, are
summarized in Fig. 7. These studies were all carried out using
a design with simultaneously injection of labeled carrier LDL
and differently labeled non-carrier LDL into the same individual,
thus reducing interindividual variation, because carrier and noncarrier LDL is metabolized at the same time under exact the same
conditions.
The R3500Q/W mutations are known to cause familial defective apolipoprotein B-100 (FDB, OMIM 144010) characterized by
severe hypercholesterolemia (Fig. 7) [11,50,56]. Both mutations
have reduced LDL fractional catabolic rate by 3370% in human in
vivo turnover studies [13,57,58], and similarly reduced LDL receptor
afnity by 75% in in vitro broblast binding studies [10,51,54,55,59].
The severity of the phenotype in humans may be modulated by a
reduction in LDL production rate [57,58], which we, however, could
not conrm in our studies (Fig. 7).
The R3531C mutation was initially identied in a patient with
increased lipid levels [54], and in in vitro broblast binding studies R3531C LDL had a reduced afnity for the LDL receptor by
2851%, suggesting that R3531C might cause hypercholesterolemia
[51,54,60]. However, a later study showed that R3531C was not associated with variation in lipid levels when identied in the general
population, the Copenhagen City Heart Study [11], and a recent in
vivo turnover study in humans found that R3531C LDL associated
with a mere 17% reduction in LDL fractional catabolic rate in carriers identied in the general population, explaining the normal lipid
phenotype in these individuals (Fig. 7) [13].

23

The R3480W mutation was also rst identied in a patient with


hypercholesterolemia [61] and R3480W LDL associated with a 73%
reduction in afnity for the LDL receptor in vitro [55]. Interestingly
therefore, when screening a general population cohort, the Copenhagen City Heart Study, heterozygosity for R3480W was identied
in one individual, while four individuals were heterozygous for a
new mutation in the same codon: R3480P. Unexpectedly, all ve
heterozygotes had a hypobetalipoproteinemia phenotype with on
average 68% lower LDL cholesterol levels than non-carriers (Fig. 7)
[13]. In human in vivo turnover studies R3480P/W LDL had reduced
fractional catabolic rate by 23%, a 29% reduction in LDL production
rate, and an approximately 70% reduction in amount of VLDL converted to LDL, resulting in the observed hypobetalipoproteinemia
phenotype [13].
These ve naturally occurring mutations are all associated with
varying degrees of defective LDL receptor binding (1233% reduction in fractional catabolic rates), but result in phenotypes ranging
from hypobetalipoproteinemia to hypercholesterolemia. The explanation for this apparent contradiction appears to be a considerable
variation in clearance of LDL precursor particles via the LDL and
apolipoprotein E receptors, and in the amount of VLDL and IDL converted to LDL. These effects could not have been predicted in silico
from protein structure or function, or from in vitro studies alone,
emphasizing the importance of combining in vitro studies with both
human in vivo and population-based studies.
Several other naturally occurring mutations located around
the receptor binding region have been studied (S3252G, V3396M,
E3405Q, S3455R, and N3516K), but none have been associated with
lipid levels, changes in LDL receptor binding afnity in vitro, or LDL
metabolism in vivo [51,62].
Two common SNPs in APOB, T2488T and E4154K, have been
shown to affect LDL metabolism (Fig. 7) [13]. In in vivo turnover
studies T2488T tt LDL has unaltered fractional catabolic rate, but
a 21% increase in LDL production rate compared to non-carriers,
explaining the 14% increase in plasma LDL cholesterol level associated with this genotype [49]. T2488T tt homozygosity also
associates with a 6% reduction in the number of plasma apolipoprotein E molecules per VLDL particle compared to non-carriers in the
general population, which may be a possible explanation for the
larger amount of IDL converted to LDL in homozygotes [49]. The
results on LDL fractional catabolic rate reported above are in agreement with another human in vivo turnover study [63], and with in
vitro studies in human broblasts [64], but in conict with two other
studies [65,66] reporting decreased LDL fractional catabolic rates
in tt homozygotes. These differences could be due to differences in
study design (see below).
The E4154K SNP is located in the carboxyl terminal tail of
apolipoprotein B suggested to regulate the binding of apolipoprotein B to the LDL receptor. In human in vivo turnover studies, E4154K
heterozygotes had 11% higher LDL fractional catabolic rate and unaltered LDL production rate compared to non-carriers, which may
explain the reduction in plasma LDL cholesterol in these individuals
[63,65,67]. The R3611Q SNP has also been studied in vivo, but has not
been associated with changes in LDL metabolism [63]. Importantly,
however, because all polymorphisms in APOB at most have a modest
effect on apolipoprotein B and LDL cholesterol levels, such polymorphisms could still have a small effect on LDL fractional catabolic
rate or LDL production rates not detected in the published studies [13,63,65,66], simply because of insufcient statistical power,
and/or lack of sensitivity of the methods used.
Metabolism of apolipoprotein B containing particles can be
dependent upon multiple factors other than APOB genotype, i.e.
gender, age, APOE genotype, LDLR mutations, estrogen levels, diabetes, lipoprotein lipase activity and hepatic lipase activity. There is
a large interindividual variation in LDL metabolism that in some
study designs could easily obscure the relatively modest effects

24

M. Benn / Atherosclerosis 206 (2009) 1730

Fig. 7. LDL metabolism in carriers of mutations and polymorphisms in APOB compared with non-carriers. LDL cholesterol ratio, LDL fractional catabolic rate ratio, and LDL
production rate ratio as a function of APOB genotype are shown. Upper panel, mean LDL cholesterol ratio and standard error (SE) for R3500Q, T2488T, R3531C, non-carriers,
E4154K, R3480P, and R3480W heterozygotes compared with apoE 33 non-carriers in the Copenhagen City Heart Study. Variants are ordered according to effect size on
LDL cholesterol levels. Middle panel, mean LDL fractional catabolic rate ratio (SE) for R3500Q, T2488T, R3531C, non-carriers, E4154K, R3480P, and R3480W LDL compared
with fractional catabolic rate ratio for non-carrier LDL determined simultaneously in the same individual. The ratio was calculated to eliminate interindividual variation in
fractional catabolic rate due to other factors than the mutation or polymorphism studied. Lower panel, mean LDL production rate ratio (SE) for R3500Q, T2488T, R3531C,
non-carriers, E4154K, R3480P, and R3480W LDL compared with production rate ratio for non-carrier LDL determined simultaneously in the same individual. The production
rate ratio was calculated to eliminate interindividual variation in production rate because of other factors than the mutation studied. All variants were studied using the
same study design and values of fractional catabolic rate and production rate are comparable between mutations and polymorphisms. t-Test versus non-carriers: * P 0.05;
**
P 0.01; and *** P 0.001.

M. Benn / Atherosclerosis 206 (2009) 1730

of APOB variants on LDL metabolism. A way to reduce this variation


in human in vivo turnover studies is by simultaneous injection of
differently labeled autologous and heterologous carrier and noncarrier LDL into the same recipient, so the two different LDLs are
metabolized by the exact same pathways in the same recipient
[67]. Results reported above on the R3480P, R3480W, R3500Q, and
R3531C mutations [13] and the T2488T [49] and E4154K [67] SNPs
from the Copenhagen City Heart Study were carried out using this

25

design (Fig. 7). Stable isotope techniques and other methods studying one genotype at a time, have high interindividual variation and
therefore may be less useful to study the relatively modest effects
of APOB SNPs than the simultaneous injection of differently labeled
carrier and non-carrier LDL into the same recipient.
In summary, both mutations and polymorphisms in APOB affect
LDL metabolism in vivo. Alleles studied span a magnitude of
effects sizes on LDL fractional catabolic rate (from 33% reduction

Fig. 8. Evolutionary sequence conservation and predicted functional importance of common non-synonymous SNPs and rare mutations(*) in APOB. Variants associated with
decreased apolipoprotein B levels in the general population (green), variants without statistical signicant association with apolipoprotein B levels in the general population
(yellow), and variants associated with increased apolipoprotein B levels in the general population (red) [11,13,49]. Similarity percent is the percent similarity between the
human APOB sequence and the available stretches of sequence aligned from other species. Shaded boxes indicate evolutionary sequence conservation between aligned
sequences. The rare mutations R3480P and R3500Q are associated with, respectively, a substantial reduction of 23% and a substantial increase of 44% in plasma levels of
apolipoprotein B in the general population. The R3531C mutation is not associated with plasma LDL cholesterol levels in the general population, but with a 17% reduction
in the LDL fractional catabolic rate [11,13,49]. Sequences are shown for: Homo sapiens (human), Pan troglodytes (chimpanzee), Macaca mulatta (rhesus monkey), Oryctolagus
cuniculus (rabbit), Bos taurus (cow), Canis familiaris (dog), Echinops telfairi (hedgehog), Loxodonta Africana (African elephant), Mus musculus (mouse), Rattus norvegicus (rat),
Dasypus novemcinctus (nine-banded armadillo), Gallus gallus (chicken), Danio rerio (zebra sh), and Strongylocentrotus purpur (sea urchin). The predicted effect of each amino
acid variant on protein function is shown to the right. SIFT version 1 [71]: = tolerated; + = deleterious (low condence prediction); ++ = deleterious. PANTHER version 6 [72]:
= unlikely functional effect; + = possible deleterious functional effect; ++ = high probability of deleterious functional effect. PolyPhen [69]: = benign; + = possibly damaging;
++ = probably damaging. NA = not available, that is, not modeled by the algorithm (For interpretation of the references to color in this gure legend, the reader is referred to
the web version of the article). Copyright 2008, The Endocrine Society.

26

M. Benn / Atherosclerosis 206 (2009) 1730

for R3500Q to 11% increase for E4154K), and effect sizes on LDL
production rate (from 29% reduction for R3480P to 21% increase
for T2488T). Mutations have larger effect sizes on LDL fractional
catabolic rate than polymorphisms (1733% versus 011%).
6. Evolutionary conservation and predicted effects
It has been estimated that SNPs (minor allele frequency above
1%) occur with an average density of approximately 1 per 290
basepairs throughout the genome [68], that half of these are nonsynonymous, and that 20% of the non-synonymous SNPs inuence
protein function and thereby have a high probability of leading to
risk of complex disorders [69]. For APOB, spanning 43 kilobases, this
theoretically translates into a total of 148 SNPs, 74 non-synonymous
SNPs, and of these 15 SNPs that inuences the protein function.
One challenge of the post-genomic era is to sort through this large
number of SNPs and identify those that are most likely to affect
phenotype, and ultimately to contribute to disease development.
The impact of SNPs on phenotype can be predicted in silico by (1) evolutionary conservation between orthologous genes,
assessed using pairwise or preferably multi-sequence alignments
[70], because SNPs located in regions or positions conserved
between orthologous genes are more likely to be of functional
importance and thereby leading to risk of complex diseases [69]; or
by (2) prediction programs, such as SIFT, PANTHER, and PolyPhen
that attempt to combine information from multi-sequence alignments with estimates of impact on the three-dimensional structure
and function of the protein, derived using current knowledge of the
amino acids physiochemical properties, protein structure, interactions, and evolution [69,71,72].
Fig. 8 shows a multi-sequence alignment of orthologous APOB
sequences for mutations and polymorphisms and their predicted
functional effect by SIFT, PANTHER, and PolyPhen [41]. The extent
of evolutionary sequence conservation does not reliably predict the
impact of either SNPs or mutations on protein function, although
it is suggested that P2712L, R3531C, and R3500Q may be of functional importance. Only one computer-based prediction algorithm,
PolyPhen, predicted one of the six non-synonymous SNPs associated with plasma apolipoprotein B and LDL cholesterol levels to
have a deleterious effect with high probability (P2712L; ++ in Fig. 8).
Moreover, one of the three known functional mutations (R3500Q)
is predicted to be benign by PolyPhen and only deleterious with
low probability with SIFT and PANTHER, and this is the mutation
with by far the largest functional effect and the largest effect on
phenotype in the general population resulting in familial defective apolipoprotein B-100 [10,59]. In contrast, the mutation with
the smallest functional effect in the general population (R3531C) is
predicted by the two algorithms available, PANTHER and PolyPhen,
to have a deleterious effect with high a probability.
Taken together, in silico prediction methods are poor predictors of functional variation in APOB. This suggests that there are
limitations to the usefulness of these methods, as demonstrated
previously for genetic variation in PCSK9 [73].
7. APOB mutations and polymorphisms and risk of
ischemic cardiovascular disease
Heterozygosity for the R3500Q mutation is undoubtedly more
prevalent among patients with ischemic heart disease (odds ratio:
7.0 (2.222)) and familial hypercholesterolemia (odds ratio: 78
(16388)) [11] than in controls, but R3500Q also associates with
increased risk of ischemic heart disease in the general population (odds ratio: 13 (356)) [11]. Studies of patient populations
have, consistent with the ndings of a less severe lipid phenotype
[11,12] and LDL metabolism [57], showed that the risk of ischemic

heart disease is increased, although it is lower in R3500Q/W


carriers compared to LDLR carriers (2.68 (1.295.57) versus 8.54
(5.2913.80) [74]) [11,75]. R3531C has never been associated with
risk of ischemic heart disease in the general population.
Reports on SNPs in APOB and risk of ischemic cardiovascular disease have been even more contradictory than results on
association with lipid levels, as exemplied by two recent metaanalyses on E4154K and risk of ischemic heart disease: one study
reported an increased risk of ischemic heart disease with an overall
odds ratio of 1.32 (1.141.54; n = 3870) [42], while the other metaanalysis reported no increase in risk and an overall odds ratio of 1.15
(0.781.70; n = 2014) [43], despite a considerable overlap between
the studies included.
To better assess this, we studied association of ten APOB SNPs
with risk of ischemic heart disease and ischemic cerebrovascular
disease in a prospective general population study including 9185
individuals with 30 years follow-up, the Copenhagen City Heart
Study [41,49,67]. In this study, we had 80% power to detect a hazard
ratio of 1.13 or above for risk of ischemic heart disease in heterozygotes versus non-carriers for the least common SNP (R3611Q
heterozygote frequency: 0.17). Despite the effect sizes on plasma
levels of apolipoprotein B and LDL cholesterol (Fig. 5), neither the
ten SNPs alone, nor the estimated nine most common haplotypes,
containing seven tag SNPs from HapMap, predicted risk of ischemic
heart disease or myocardial infarction in the general population
(Fig. 9) [41]. Results on risk of ischemic heart disease were veried in a casecontrol study including 944 independent cases with
ischemic heart disease and 7664 controls [41].
E4154K KK homozygosity associated with a 60% reduction in risk
of ischemic cerebrovascular disease (hazard ratio: 0.4 (95% condence interval 0.20.9)) and an 80% reduction in risk of ischemic
stroke (0.2 (0.10.7)), compared with non-carriers (Fig. 9) [67].
However, these ndings need to be conrmed in an independent
study, because it could be spurious ndings. None of the other
nine SNPs alone or as combined haplotypes associated with risk
of ischemic cerebrovascular disease or ischemic stroke.
In the Copenhagen City Heart Study, a 5 mg/dL increase in
apolipoprotein B predicts a hazard ratio of 1.06 (95% condence
interval: 1.041.07) for ischemic heart disease, and a 5 mg/dL
decrease a hazard ratio of 0.94 (0.930.96) if the risk is attributed to
the change in apolipoprotein B alone. Thus, the association with risk
of ischemic heart disease predicted by the largest observed effects
on apolipoprotein B for the SNPs and haplotypes studied (5.5 mg/dL
increase for T71I) would correspond to a hazard ratio close to 1.0, in
agreement with the lack of association of both SNPs and haplotypes
on risk of ischemic heart disease seen in this study.
In summary, E4154K KK homozygosity predicts a 6080% reduction in risk of ischemic cerebrovascular disease and ischemic stroke
compared to non-carriers which needs to be conrmed in an independent study, however, none of the other SNPs predict risk of
ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, or ischemic stroke in the general population.
8. Discussion and perspectives
The studies reviewed can be summarized as follows: (1)
apolipoprotein B predicts ischemic cardiovascular events in both
genders, and is better than LDL cholesterol in this respect; (2)
linkage disequilibrium structure in APOB is more complex than
expected from HapMap data, because a minimal set of tag single
nucleotide polymorphisms capturing the entire variation in APOB
cannot be identied, and thus most polymorphisms must be evaluated separately in association studies; (3) APOB mutations and
polymorphisms are associated with a range of apolipoprotein B
and LDL cholesterol levels, although the magnitude of effect sizes
of common polymorphisms are modest; (4) both mutations and

M. Benn / Atherosclerosis 206 (2009) 1730

27

Fig. 9. Risk of ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, and ischemic stroke by APOB T71I, Ivs4+171c > a, A591 V, Ivs18+379a > c,
Ivs18+1708g > t, T2488Tc > t, P2712L, R3611Q, E4154K, and N4311S genotypes in the general population, the Copenhagen City Heart Study [41,67]. CI = condence interval.

polymorphisms are associated with LDL metabolism in vivo; (5)


association of APOB mutations and polymorphisms with lipid and
disease phenotype cannot be predicted in silico using evolutionary
conservation or existing prediction programs; and nally, (6) except
for the E4154K polymorphism that possibly predicts a reduction in
risk of ischemic cerebrovascular disease and ischemic stroke, common APOB polymorphisms with modest effect sizes on lipid levels
do not predict risk of ischemic heart disease, myocardial infarction,
ischemic cerebrovascular disease, or ischemic stroke in the general
population.
Although mutations in APOB can have profound inuence on levels of apolipoprotein B and LDL cholesterol and on risk of ischemic
heart disease, the nding of relatively modest effect sizes on lipid
phenotype associated with APOB polymorphisms, and no association with risk of ischemic heart disease is quite disappointing. It
raises three important questions.

First, are these ndings true? Fig. 6 shows the percent increase
or decrease in apolipoprotein B levels for each genetic variant as a
function of rare allele frequency. The common SNPs (allele frequencies from 9% to 48%) are indeed associated with modest (26%)
changes in plasma apolipoprotein B level, compared to the 44%
increase in apolipoprotein B associated with R3500Q heterozygosity, and the 31% and 69% increase, respectively, in apolipoprotein
B levels from the lower apolipoprotein B tertile to the middle
and upper tertiles of apolipoprotein B, respectively. The results
reported from the Copenhagen City Heart Study (Fig. 6), are based
on almost 10,000 individuals from a general population study and
effect sizes are consistent over time, indicating their robustness
[41,49]. Changes in LDL cholesterol levels are of the same magnitude as those observed for SNPs in the LDLR and PCSK9 genes with
similar allele frequencies [76,77], suggesting that this is what can
be expected in general by common SNPs in genes involved in the

28

M. Benn / Atherosclerosis 206 (2009) 1730

VLDL-IDL-LDL pathway. The observed lack of association between


SNPs and risk of ischemic heart disease is also in accordance with
what one would expect from the modest changes in apolipoprotein
B and LDL cholesterol levels. In the Copenhagen City Heart Study,
a lifelong increase in apolipoprotein B levels of 5 mg/dL predicts a
hazard ratio of 1.06 (1.041.07) for ischemic heart disease, if the risk
is attributed to the increase in apolipoprotein B levels alone. This is
in agreement with what is observed, and also in accordance with
the approximately 2-fold increase in relative risk of ischemic heart
disease, and 1115% increase in absolute 10-years risk observed
from the lower to the upper apolipoprotein B tertile, for a much
larger increase of 69% or 45 mg/dL in apolipoprotein B level.
Second, do the APOB SNPs studied so far account for the 5060%
variation in apolipoprotein B levels attributed to genetic variation in
twin studies? The majority of this variation could be due to variation
in APOB, however, we do not know how much of the variation that
could be due to other genes encoding proteins that may inuence
apolipoprotein B levels, i.e. microsomal triglyceride transfer protein
(MTP), LDL receptor protein (LDLR), pro-protein convertase subtilisin/kexin 9 (PCSK9), lipoprotein lipase (LPL), hepatic lipase (LIPC),
or some of the many genes involved in reverse cholesterol transport.
Each of the ten SNPs studied in the Copenhagen City Heart Study
contribute less than 1% to variability in apolipoprotein B levels [49],
and together as combined genotypes with approximately 1%, suggesting that there are many other functionally important SNPs in
APOB, which have not yet been identied or studied.
Third, are the common APOB SNPs those that contribute the
most to lipid phenotype and risk of disease? All studies of APOB
SNPs have focused on associations of SNPs with a rare allele frequency of 0.10.5 and located in regions known to be of functional
importance. This limitation of screening strategies applied so far
is explained by the huge size of APOB. Re-sequencing the coding
region of APOB of 14,121 basepairs in a sufcient number of individuals is a relatively costly undertaking, and most studies have either
screened specic parts of the gene in a limited number of individuals, or selected SNPs reported in databases. Such a strategy will only
capture a limited spectrum of alleles (limited by the gene fragments
screened), and predominantly the common variants (limited by the
low number of individuals screened), explaining why so few rare
SNPs (frequencies of 0.010.1) have been identied.
The strategy of studying APOB SNPs with a rare allele frequency
of 0.10.5 with the aim of estimating the contribution to a complex disease such as ischemic cardiovascular disease is justied
by the common diseasecommon variant hypothesis (Fig. 6), suggesting that genetic susceptibility to common diseases is conferred
primarily by alleles that are common in the population, but have
modest effects in the individual [7880]. But as this approach apparently has not captured all the variation in APOB, or at least not the
variation relevant for predicting risk of disease, one must conclude
that either the study design, the common diseasecommon variant
hypothesis, or both have failed. An alternative hypothesis on SNPs
and complex disease has been posed by Pritchard and named the
common diseaserare variant hypothesis [81] (Fig. 6). It suggests
that susceptibility to common diseases is the result of multiple rare
alleles with a large magnitude of effect on phenotype, and although
individually rare, these alleles may be collectively common in the
population [81,82]. The expected contribution to genetic variance
as a function of allele frequency as predicted by Pritchards hypothesis for variants under a mild (dashed line) and moderate (solid line)
purifying selection is shown in blue in Fig. 6 [81]. The results on contribution to phenotype observed for the APOB mutations, R3500Q
and R3480P, and SNPs with a rare allele frequency of 0.10.5, t
with this hypothesis. Unfortunately, we do not yet have data on
rare SNPs in the frequency spectrum from 0.01 to 0.08, which
according to this hypothesis is where the variation of signicance
should be identied. The conclusion is therefore so far that known

APOB alleles do not account for the 5060% variation in apolipoprotein B attributed to genetic variation, that we may have used the
wrong approach to identify functional SNPs in APOB, and potentially have overlooked relatively rare SNPs collectively contributing
to the observed genetic variation in apolipoprotein B levels.
From the results summarized in this review on the usefulness of
the tag SNP approach, evolutionary conservation, and in silico prediction programs, there is no doubt that we cannot select relevant
areas of interest, but must re-sequence the entire coding sequence
of APOB. And if the common diseaserare variant hypothesis holds
true, this re-sequencing should be carried out in a large number
of individuals to be able to capture rare variants. There are several recent re-sequencing and association studies that support the
common diseaserare variant hypothesis, although, it has not been
tested directly [77,8385].
Taken together, these data suggest that the contribution of APOB
to variation in apolipoprotein B and LDL cholesterol levels is not
adequately described by the present studies. A strategy of resequencing genes of interest in a large number of individuals at the
extremes of the population distribution of LDL and HDL cholesterol
levels has been useful in several studies to identify the full spectrum
of alleles in these genes, and particularly rare variants with large
effects on phenotype [77,83,84]. A similar approach could readily
be applied in future studies of APOB.
Acknowledgements
I am indebted to the staff and participants of the Copenhagen
City Heart Study for their important contributions and to Drs. Anne
Tybjrg-Hansen and Brge G. Nordestgaard for helpful discussions.
Supported by a grant (07-D-BR63-A1916-B415-A-22435) from the
Danish Heart Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.atherosclerosis.2009.01.004.
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Further reading
Web resources: The URLs for data presented herein are as follows:
Ensemble Genome Browser, http://www.ensembl.org/index.html.
International HapMap Project, http://www.hapmap.org/index.
html.en.
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.
nlm.nih.gov/sites/entrez?db=OMIM.
PANTHER Classication System, http://www.pantherdb.org/.
PolyPhen, http://genetics.bwh.harvard.edu/pph/.
Sorting Intolerant From Tolerant (SIFT), http://blocks.fhcrc.
org/sift/SIFT.html.
Westgard QC, http://www.westgard.com/biodatabase1.html.