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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
Review
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
18
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-
19
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.
20
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).
21
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
22
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.
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
24
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.
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
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
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.
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
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.