apob

Atherosclerosis 206 (2009) 1730

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Atherosclerosis

journa l homepage: www.e lsev ier .com/ locate /a therosc leros is

Review

Apolipoprotein B levels, APOB alleles, and risk of ischemic cardiovascular diseasein the general population, a review

MariannDepartment of

a r t i c l

Article history:Received 4 DeReceived in reAccepted 5 JanAvailable onlin

Keywords:Apolipoprotein BLow density lipoprotein cholesterolAtherosclerosisIschemic heart diseaseIschemic strokeGeneticsEpidemiology

in both genders, and is better than LDL cholesterol in this respect; (2) linkage disequilibrium structure inAPOB is more complex than expected from HapMap data, because a minimal set of tag single nucleotidepolymorphisms capturing the entire variation in APOB cannot be identied, and thus most polymor-phisms must be evaluated separately in association studies; (3) APOB mutations and polymorphisms areassociated with a range of apolipoprotein B and LDL cholesterol levels, although the magnitude of effect

Contents

1. Introd2. Plasm3. APOB4. APOB5. APOB6. Evolu7. APOB8. Discu

AcknoAppenRefere

Abbrehepatic lipasesubtilisin/kexi

Tel.: +45 3E-mail add

0021-9150/$ doi:10.1016/j.asizes of common polymorphisms are modest; (4) both mutations and polymorphisms are associated withLDL metabolism in vivo; (5) association of APOB mutations and polymorphisms with lipid and diseasephenotype 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 ischemiccerebrovascular disease and ischemic stroke, common APOB polymorphisms with modest effect sizes onlipid levels do not predict risk of ischemic heart disease, myocardial infarction, ischemic cerebrovasculardisease, or ischemic stroke in the general population.

2009 Elsevier Ireland Ltd. All rights reserved.

uction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18a apolipoprotein B levels and risk of ischemic cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18mutations and polymorphisms and linkage disequilibrium structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19mutations and polymorphisms and plasma apolipoprotein B levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20mutations and polymorphisms and LDL metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21tionary conservation and predicted effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26mutations and polymorphisms and risk of ischemic cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26ssion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26wledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28dix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28nces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

viations: APOB, apolipoprotein B gene; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor gene; LIPC,gene; LPL, lipoprotein lipase gene; MTP, microsomal triglyceride transfer protein gene; SNP, single nucleotide polymorphism; PCSK9, pro-protein convertasen 9 gene; UTR, un-translated region; VLDL, very low density lipoprotein.5456506; fax: +45 35456500.ress: [email protected].

see front matter 2009 Elsevier Ireland Ltd. All rights reserved.therosclerosis.2009.01.004e Benn

Clinical Biochemistry KB3011, Section for Molecular Genetics, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen , Denmark

e i n f o

cember 2008vised form 5 January 2009uary 2009e 15 January 2009

a b s t r a c t

Apolipoprotein B is a key component in lipid metabolism. Subendothelial retention of apolipoproteinB containing lipoproteins is a necessary initiating event in atherogenesis, and high plasma levels ofapolipoprotein 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 apredictor of ischemic cardiovascular disease, as well as the association of mutations and polymorphismsin 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

18 M. Benn / Atherosclerosis 206 (2009) 1730

1. Introduction

Apolipoprotein B is a non-exchangeable apolipoprotein foundin two forms in humans, apolipoprotein B-48 and apolipoproteinB-100. Apoessential foblood [1,2]triglyceridecle tissue. Aessential fosity lipoproto deliver tsecretion, ttein lipase ttissue. Thenant or intefrom the cirhydrolyzed(LDL). Apolon the LDLapolipoprotized and de

Reducedduction of cand fat-soluing lipoprohomeostasidothelial rea necessary[5]), and hiterol are risapolipoprot

Twin stulevels of aposense mutaonly in sevecardiovascuFrom an ep(minor alleltant risk ofbut their imgle nucleotimay, becausfrom neglig

The presies whichapolipoprotthe effectsapolipoprotcular diseas

2. Plasmacardiovascu

Ischemicand ischemhospitalizatLDL cholestdiovascularscreening tof atheroscber of circuthan to thebeen suggerisk than coA likely exp

ment of the total number of atherogenic particles (including LDL,IDL, VLDL, chylomicrons, and chylomicron remnants), because eachof these contain a single molecule of apolipoprotein B, while LDLcholesterol is an estimate of the mass of cholesterol in the LDL

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n exipolipt isco setwoolesthaslipoprotein B-48 is synthesized in enterocytes and isr the assembly and secretion of chylomicrons to the. The main function of chylomicrons is to transports from the intestine to the liver, adipose, and mus-polipoprotein B-100 is synthesized in the liver and isr the initial lipidation of the nascent very low den-tein (VLDL) particle [1]. The main function of VLDL isriglycerides from the liver to the circulation [2]. Uponhe triglyceride core of VLDL is hydrolyzed by lipopro-hereby delivering free fatty acids to muscle and adiposeresulting triglyceride depleted particle, the VLDL rem-rmediate density lipoprotein (IDL), can either be clearedculation by binding to the hepatic remnant receptor, orfurther by hepatic lipase to form low density lipoproteinipoprotein B is the sole remaining protein componentparticle. LDL is cleared from the blood by binding ofein B to the LDL receptor, and is subsequently internal-graded in the liver [2,3] (Supplementary Fig. 1).secretion of apolipoprotein B results in reduced pro-

hylomicron and VLDL, leading to malabsorption of fatsble vitamins [4]. Although apolipoprotein B contain-

teins are pivotal for lipid absorption and triglycerides, high levels in plasma induce atherosclerosis. Suben-tention of apolipoprotein B-containing lipoproteins is

initiating event of atherogenesis (for a review seegh plasma levels of apolipoprotein B and LDL choles-k factors for atherosclerosis [6], whereas low levels ofein B may provide protection against atherosclerosis.dies suggest that 5060% of the variation in plasmalipoprotein B is genetically determined [79]. Rare mis-

tions in the apolipoprotein B gene (APOB) may result notre hypercholesterolemia and increased risk of ischemiclar disease, but also in hypocholesterolemia [1013].idemiological perspective, rare deleterious mutationse frequency

M. Benn / Atherosclerosis 206 (2009) 1730 19

Fig. 1. Risk of i emic stein B and low ion, th

interventiofollowed bybeing at riskindividuals.College of Cvalidated trwith knowncardiovascu

In summevents in brespect. Prebe improve

3. APOB mdisequilibr

The obsischemic hmakes it imapolipoprotTwo rare mimonogenic144010), wated with arare mutatiand cholestof the variagenetic varipolymorphapolipoprot

The APOand encodmature pro

geimatnony

ant ischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, ischdensity lipoprotein (LDL) cholesterol in women and men from the general populat

n studies in different patient groups and populations,estimation of the number of individuals classied asand the resulting costs of preventive treatment in theseThe American Diabetes Association and the American

of 132approxnon-sy1 variardiology Foundation have recently suggested a similareatment target of 80 mg/dL apolipoprotein B in patients

cardiovascular disease or with diabetes and anotherlar risk factor [34].ary, apolipoprotein B predicts ischemic cardiovascular

oth genders, and is better than LDL cholesterol in thisdiction of future ischemic cardiovascular events couldd by measuring apolipoprotein B.

utations and polymorphisms and linkageium structure

ervation that apolipoprotein B levels predict botheart disease and ischemic cerebrovascular diseaseportant to understand factors that determine plasmaein B levels including genetic variation in APOB itself.ssense mutations, APOB R3500Q and R3500W, cause thedisorder familial defective apolipoprotein B-100 (OMIMhereas another missense mutation R3480P is associ-hypobetalipoproteinemia phenotype, emphasizing thatons in APOB do indeed contribute to apolipoprotein Berol levels [11,13]. Twin studies indicate that 5060%tion in apolipoprotein B levels can be attributed to

ation [79], and in individuals without rare mutations,isms in APOB may play the major role in determiningein B and cholesterol levels.B gene spans 43 kilobases including 28 exons,

es a 27 amino acid signal peptide followed by atein of 4536 amino acids (Fig. 3) [3538]. A total

http://wwwand the larage disequiand can potify geneticgenotypepcorrelated wSNPs), knowreduce thestudy genot

Some smlibrium betresults fromprehensivevariants genr2 and D frodegree of l(D > 0.80),(T2488T, P2these SNPsalleles of P2other r2s a, A591 V,> c, Ivs18 + 1708g > t, T2488Tc > t, and E4154K

tag SNPs (marked in blue text in Fig. 3A and 4)genetic variation in almost the entire APOB genew.hapmap.org/index.html.en): T71I and Ivs4 + 171c > a,oblock covering the N-terminal part of the gene (12,986


Fig. 2. Absolu lic blomen, separate (red)gure legend,

nucleotidesblock covenucleotidesloblocks of,It follows thto HapMapare in the salthough thD > 0.90) nowith N4311T2488Tc > tthat Ivs18 +as a proxyphenotype

In summdisequilibribetween SNSNP separa

4. APOB mapolipopro

Rare mplasma apoR3500Q/Weral populatB of 44% (3[11,12], and(0.93 mmoapolipoprotte 10-year risk of any ischemic cardiovascular event by smoking status, age, systoly. Apolipoprotein B tertile: 1 (green) lower tertile, 2 (orange) middle tertile, and 3

the reader is referred to the web version of the article).

), and Ivs18 + 1708g > t, T2488Tc > t, E4154K anotherring the most C-terminal end of the gene (18,719), while A591 V and Ivs18 + 379a > c cover two hap-respectively, 5585 and 1329 nucleotides in between.at Ivs18 + 1708g > t, T2488Tc > t and E4154K accordingshould tag P2712L, R3611Q, and N4311S, since they

ame haploblock. In the Copenhagen City Heart Study,ese six SNPs are often on the same haplotypes (allne are highly correlated, with the exception of P2712LS (Fig. 4; r2 = 1.0 for P2712L with N4311S, r2 = 0.30 forwith P2712L/N4311S, and all other r2s t, T2488Tc > t and E4154K cannot tag or serve

for P2712L, R3611Q or N4311S, and that the effects onof each of these SNPs must be evaluated separately.ary, several SNPs in APOB are at most in weak linkage

um, and therefore cannot tag each other. AssociationPs and phenotype must therefore be evaluated for each

tely, rather than relying on a few tag SNPs.

utations and polymorphisms and plasmatein B levels

utations in APOB can have profound inuence onlipoprotein B and LDL cholesterol levels (Fig. 5) [11,12].heterozygosity observed in individuals from the gen-ion associated with increases in levels of apolipoprotein7.8 mg/dL) and LDL cholesterol of 80% (1.92 mmol/L)R3480P with decreases of 23% (19.9 mg/dL) and 29%

l/L), respectively [13]. R3531C did not associate withein B or LDL cholesterol levels [11].

Associatlipoproteinstudies an[42,43]. ToSNPs in thein 9185 indprospectiveSix non-synwere locatetion of nasin structuraof VLDL toulating bin[47,48]. In aIvs18 + 379awith T71I, Atag the genof APOB, co

Locationamino acidapolipoprotT71I: 0.33,Ivs18 + 1708E4154K: 0.1

Overall,Ivs18 + 1708A591 V, IvapolipoprotT71I, Ivs18increases inod pressure, and tertiles of apolipoprotein B (apo B) for women andupper tertile [28] (For interpretation of the references to color in thision of non-synonymous SNPs in APOB with lipid andlevels has been examined extensively in several small

d meta-analyses, although, with conicting resultsaddress this further with focus on non-synonymousgeneral population, we genotyped ten SNPs in APOB

ividuals from the Danish general population followedly for 25 years in the Copenhagen City Heart Study [41].onymous SNPs in APOB were selected, because theyd in important functional domains crucial for lipida-

cent apolipoprotein B (T71I, A591 V) [44,45], involvedl changes of apolipoprotein B during the conversionLDL (P2712L) [46], or known or suspected of reg-

ding to the LDL receptor (R3611Q, E4154K, N4311S)ddition, we genotyped four other SNPs (Ivs4 + 171c > a,> c, Ivs18 + 1708g > t, and T2488Tc > t [49]) that together591 V, and E4154K are predicted by HapMap to largely

etic variation in the entire coding and intronic regionsmprising approximately 43 kilobases of genomic DNA.

of the four mutations and ten SNPs relative to thesequence and structural and functional domains of

ein B are shown in Fig. 3. Minor allele frequencies areIvs4 + 171c > a: 0.14, A591 V: 0.47, Ivs18 + 379a > c: 0.30,g > t: 0.45, T2488Tc > t: 0.48, P2712L: 0.21, R3611Q: 0.09,7, and N4311S: 0.21 (Fig. 4).all ten SNPs were associated with either increases (T71I,g > t, T2488Tc > t, R3611Q) or decreases (Ivs4 + 171c > a,

s18 + 379a > c, P2712L, E4154K, N4311S) in plasmaein B (p-values by ANOVA from 0.03 to t, T2488Tc > t, R3611Q were associated with

apolipoprotein B of 3.2%, 3.1%, 2.5%, and 1.9% (2.7,


Fig. 3. (A) Locdomains of apdomain impor-helical domNH2-1-1-the entire APOdiagram of thelipoprotein, ancrosses backwNon-synonymwith apolipop

1.7, 2.1, andcarriers, anincrease in4.7, 5.2, andcarriers. Ivsand N4311Sof 3.7%, 2.9and 2.6 mg/and Ivs4 + 1decreases iand 4.4 mg/In absoluteB as a funin LDL chowhile the m(0.28 mmolversus noncholesterol,or decreasecholesterol,

Includinapolipoprotspectrum owith variatation of ten studied single nucleotide polymorphisms (SNPs) and four mutations in APOBolipoprotein B [41]. Structural studies have suggested a pentapartite secondary structuretant during initial lipidation (aa 1795), two amphiphatic-sheet domains (aa 8272001ains (aa 20452587 and 40174515) that bind lipids reversibly and are suggested to be

2-2-3-COOH conguration [46]. The seven SNPs predicted by HapMap (http://www.B gene (coding regions and introns) are in blue text. Mutations are in red text. aa = amin

structure of apoB-100 on the surface of LDL (adapted from [4648,55]). Apolipoproteid low density lipoprotein (LDL) particles like a belt completing the encirclement by aboutard over the chain between residues 3000 and 3500 [47]. The LDL receptor binding domaous variants are marked in red (associated with increases in apolipoprotein B levels), grrotein B levels) (For interpretation of the references to color in this gure legend, the read

1.6 mg/dL), respectively, in heterozygotes versus non-d T71I, Ivs18 + 1708g > t, and T2488Tc > t also with anapolipoprotein B of 6.5%, 5.5%, 6.2%, and 5.5% (5.5,4.7 mg/dL), respectively, in homozygotes versus non-

4 + 171c > a, A591 V, Ivs18 + 379a > c, P2712L, E4154K,were associated with decreases in apolipoprotein B

%, 1.4%, 3.0%, 2.3%, and 3.0% (3.2, 2.5, 1.7, 2.6, 2.0,dL), respectively, in heterozygotes versus non-carriers,71c > a, A591 V, and E4154K were also associated withn apolipoprotein B of 6.5%, 4.0%, and 5.0% (5.7, 3.5,dL), respectively, in homozygotes versus non-carriers.

values, the maximum increase in apolipoproteinction of SNP genotype was 5.5 mg/dL (0.30 mmol/Llesterol) for T71I homozygotes versus non-carriers,aximum decrease in apolipoprotein B was 5.7 mg/dL

/L in LDL cholesterol) for Ivs4 + 171c > a homozygotes-carriers (Fig. 5). Similar results were found for totalbut none of the ten SNPs were associated with increasein plasma levels of triglycerides, VLDL cholesterol, HDLor apolipoprotein AI [41,49].

g the three previously described mutations inein B (R3480P, R3500Q/W, R3531C) [1113], thef APOB mutations and polymorphisms associated

ion in apolipoprotein B and LDL cholesterol span a

wide rangefor T2488T23% apolipR3500Q, orlevels) (Figpolymorphmost 6%, ctein B levetertile of ap(Figs. 1 and

In summpolymorphtein B and Leffects of th

5. APOB mmetabolism

Differentance [50caused byapolipoprotchanges intor. Metaborelative to the amino acid sequence and the structural and functionalfor apolipoprotein B with one mixed 1 region that forms a globularand 25714032) that bind lipids non-reversibly, and two amphiphatic

involved in export of triglycerides and phospholipids, ordered in ahapmap.org/index.html.en) to tag for the genetic variation in almosto acid, MTP = microsomal triglyceride transfer protein. (B) Schematicn B enwraps the very low density lipoprotein, intermediate densityamino acid residue 4050. The carboxyl terminal end forms a bow thatin of apolipoprotein B has been localized to residues 33593369 [48].een (reductions in apolipoprotein B levels) or yellow (no associationer is referred to the web version of the article).

of allele frequencies (from 0.0004 for R3480P to 0.48c > t) and magnitude of phenotypic effect sizes (fromoprotein B reduction for R3480P to 44% increase forcorresponding 2980%, respectively in LDL cholesterol

. 6). The magnitude of phenotypic effect sizes of theisms on apolipoprotein B levels are fairly modest, atompared to the 31% and 69% increase in apolipopro-l from the lower tertile to the middle and upperolipoprotein B, respectively, in the general population2).ary, these results suggest that several mutations and

isms in APOB contribute to plasma levels of apolipopro-DL cholesterol in the general population, although thee common alleles are modest.

utations and polymorphisms and LDL

t regions in apolipoprotein B are of functional impor-52], and amino acid changes in functional domains

mutations or SNPs may change the structure ofein B and thereby affect LDL metabolism throughthe interaction of apolipoprotein B with the LDL recep-lic studies have shown that ve naturally occurring


Fig. 4. Pairwise linkage disequilibrium between ten single nucleotide polymorphisms (SNPs) examined in the Copenhagen City Heart Study [41]. Seven SNPs predictedby 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 betweenthe observed haplotype frequency (frequency of the two rare alleles occurring together) and the haplotype frequency expected under linkage equilibrium (the product ofthe 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 allelefrequencies and is inversely related to the sample size required to detected genetic association between markers that are in complete linkage disequilibrium. All D valuesare positive, indicating that the rare alleles at each locus segregate together. The color code indicates the degree of linkage disequilibrium between SNPs. MAF = minor allelefrequency (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, theCopenhagen 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 analysisof 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 populationmean values.


Fig. 6. Magnitas a function odiamond denowith a geneticthe Copenhageapolipoproteinstatistically sigline denotes th


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 LDLproduction 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 onLDL cholesterol levels. Middle panel, mean LDL fractional catabolic rate ratio (SE) for R3500Q, T2488T, R3531C, non-carriers, E4154K, R3480P, and R3480W LDL comparedwith fractional catabolic rate ratio for non-carrier LDL determined simultaneously in the same individual. The ratio was calculated to eliminate interindividual variation infractional 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 productionrate ratio was calculated to eliminate interindividual variation in production rate because of other factors than the mutation studied. All variants were studied using thesame 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.


of APOB variants on LDL metabolism. A way to reduce this variationin human in vivo turnover studies is by simultaneous injection ofdifferently labeled autologous and heterologous carrier and non-carrier LDL into the same recipient, so the two different LDLs aremetabolized by the exact same pathways in the same recipient[67]. Results reported above on the R3480P, R3480W, R3500Q, andR3531C mutations [13] and the T2488T [49] and E4154K [67] SNPsfrom the Copenhagen City Heart Study were carried out using this

design (Fig. 7). Stable isotope techniques and other methods study-ing one genotype at a time, have high interindividual variation andtherefore may be less useful to study the relatively modest effectsof APOB SNPs than the simultaneous injection of differently labeledcarrier and non-carrier LDL into the same recipient.

In summary, both mutations and polymorphisms in APOB affectLDL metabolism in vivo. Alleles studied span a magnitude ofeffects 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 withdecreased 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 thehuman APOB sequence and the available stretches of sequence aligned from other species. Shaded boxes indicate evolutionary sequence conservation between alignedsequences. The rare mutations R3480P and R3500Q are associated with, respectively, a substantial reduction of 23% and a substantial increase of 44% in plasma levels ofapolipoprotein B in the general population. The R3531C mutation is not associated with plasma LDL cholesterol levels in the general population, but with a 17% reductionin the LDL fractional catabolic rate [11,13,49]. Sequences are shown for: Homo sapiens (human), Pan troglodytes (chimpanzee), Macaca mulatta (rhesus monkey), Oryctolaguscuniculus (rabbit), Bos taurus (cow), Canis familiaris (dog), Echinops telfairi (hedgehog), Loxodonta Africana (African elephant), Mus musculus (mouse), Rattus norvegicus (rat),Dasypus novem d Stronacid variant on ious (l = unlikely fu eteriou++ = probably d ion ofthe web versiocinctus (nine-banded armadillo), Gallus gallus (chicken), Danio rerio (zebra sh), anprotein function is shown to the right. SIFT version 1 [71]: = tolerated; + = deleter

nctional effect; + = possible deleterious functional effect; ++ = high probability of delamaging. NA = not available, that is, not modeled by the algorithm (For interpretatn of the article). Copyright 2008, The Endocrine Society.gylocentrotus purpur (sea urchin). The predicted effect of each aminoow condence prediction); ++ = deleterious. PANTHER version 6 [72]:s functional effect. PolyPhen [69]: = benign; + = possibly damaging;the references to color in this gure legend, the reader is referred to


for R3500Q to 11% increase for E4154K), and effect sizes on LDLproduction rate (from 29% reduction for R3480P to 21% increasefor T2488T). Mutations have larger effect sizes on LDL fractionalcatabolic rate than polymorphisms (1733% versus 011%).

6. Evolutio

It has be1%) occur wbasepairs thsynonymouprotein funrisk of comptheoreticallSNPs, and oOne challennumber ofphenotype,

The impico by (1) eassessed us[70], becaubetween orimportanceby (2) predthat attempments withand functioamino acidtions, and e

Fig. 8 shsequences ffunctional eof evolutionimpact of eit is suggestional impoPolyPhen, pated with phave a deletMoreover, ois predictedlow probabwith by farphenotypetive apolipothe smallespredicted bto have a de

Taken totors of funclimitationspreviously f

7. APOB mischemic ca

Heterozyprevalent a7.0 (2.222(16388)) [increased rtion (oddshave, consis[11,12] and

heart disease is increased, although it is lower in R3500Q/Wcarriers compared to LDLR carriers (2.68 (1.295.57) versus 8.54(5.2913.80) [74]) [11,75]. R3531C has never been associated withrisk of ischemic heart disease in the general population.

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uals41,4f 1.1oteszygoof apPs alingisea

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emiclipope forentof is

ummriskred tnt stic heiseas

cussi

stuoprots, an

dised frtidebe i

sepaorphL chmonnary conservation and predicted effects

en estimated that SNPs (minor allele frequency aboveith an average density of approximately 1 per 290

roughout the genome [68], that half of these are non-s, and that 20% of the non-synonymous SNPs inuencection and thereby have a high probability of leading tolex disorders [69]. For APOB, spanning 43 kilobases, this

y translates into a total of 148 SNPs, 74 non-synonymousf these 15 SNPs that inuences the protein function.ge of the post-genomic era is to sort through this largeSNPs and identify those that are most likely to affectand ultimately to contribute to disease development.act of SNPs on phenotype can be predicted in sil-volutionary conservation between orthologous genes,ing pairwise or preferably multi-sequence alignmentsse SNPs located in regions or positions conservedthologous genes are more likely to be of functionaland thereby leading to risk of complex diseases [69]; or

iction programs, such as SIFT, PANTHER, and PolyPhent to combine information from multi-sequence align-estimates of impact on the three-dimensional structuren of the protein, derived using current knowledge of thes physiochemical properties, protein structure, interac-volution [69,71,72].ows a multi-sequence alignment of orthologous APOBor mutations and polymorphisms and their predictedffect by SIFT, PANTHER, and PolyPhen [41]. The extentary sequence conservation does not reliably predict theither SNPs or mutations on protein function, althoughted that P2712L, R3531C, and R3500Q may be of func-rtance. Only one computer-based prediction algorithm,redicted one of the six non-synonymous SNPs associ-lasma apolipoprotein B and LDL cholesterol levels toerious effect with high probability (P2712L; ++ in Fig. 8).ne of the three known functional mutations (R3500Q)to be benign by PolyPhen and only deleterious with

ility with SIFT and PANTHER, and this is the mutationthe largest functional effect and the largest effect onin the general population resulting in familial defec-protein B-100 [10,59]. In contrast, the mutation with

t functional effect in the general population (R3531C) isy the two algorithms available, PANTHER and PolyPhen,leterious effect with high a probability.gether, in silico prediction methods are poor predic-tional variation in APOB. This suggests that there areto the usefulness of these methods, as demonstratedor genetic variation in PCSK9 [73].

utations and polymorphisms and risk ofrdiovascular disease

gosity for the R3500Q mutation is undoubtedly moremong patients with ischemic heart disease (odds ratio:)) and familial hypercholesterolemia (odds ratio: 7811] than in controls, but R3500Q also associates withisk of ischemic heart disease in the general popula-ratio: 13 (356)) [11]. Studies of patient populationstent with the ndings of a less severe lipid phenotype

LDL metabolism [57], showed that the risk of ischemic

Replar disassociaanalysreportodds ranalys(0.78the stu

To bwith rdiseaseindividStudy [ratio oerozygheterolevelsten SNcontainheart d(Fig. 9ed inischem

E41of ischdencestrokeHowevstudy,nine Sof isch

Inapolipintervadecreathe chaof ischon apoincreasagreemon risk

In stion incompapendeischemcular d

8. Dis

Theapolipgenderlinkageexpectnucleocannotuatedpolymand LDof comon SNPs in APOB and risk of ischemic cardiovascu-have been even more contradictory than results onwith lipid levels, as exemplied by two recent meta-E4154K and risk of ischemic heart disease: one studyincreased risk of ischemic heart disease with an overallf 1.32 (1.141.54; n = 3870) [42], while the other meta-orted no increase in risk and an overall odds ratio of 1.15n = 2014) [43], despite a considerable overlap betweenincluded.r assess this, we studied association of ten APOB SNPsf ischemic heart disease and ischemic cerebrovascular

prospective general population study including 9185with 30 years follow-up, the Copenhagen City Heart

9,67]. In this study, we had 80% power to detect a hazard3 or above for risk of ischemic heart disease in het-versus non-carriers for the least common SNP (R3611Qte frequency: 0.17). Despite the effect sizes on plasmaolipoprotein B and LDL cholesterol (Fig. 5), neither theone, nor the estimated nine most common haplotypes,seven tag SNPs from HapMap, predicted risk of ischemicse or myocardial infarction in the general population]. Results on risk of ischemic heart disease were veri-econtrol study including 944 independent cases withart disease and 7664 controls [41].K homozygosity associated with a 60% reduction in riskcerebrovascular disease (hazard ratio: 0.4 (95% con-

val 0.20.9)) and an 80% reduction in risk of ischemic(0.10.7)), compared with non-carriers (Fig. 9) [67].ese ndings need to be conrmed in an independent

use it could be spurious ndings. None of the otheralone or as combined haplotypes associated with riskcerebrovascular disease or ischemic stroke.openhagen City Heart Study, a 5 mg/dL increase inein B predicts a hazard ratio of 1.06 (95% condence041.07) for ischemic heart disease, and a 5 mg/dLazard ratio of 0.94 (0.930.96) if the risk is attributed to

in apolipoprotein B alone. Thus, the association with riskheart disease predicted by the largest observed effectsrotein B for the SNPs and haplotypes studied (5.5 mg/dLT71I) would correspond to a hazard ratio close to 1.0, in

with the lack of association of both SNPs and haplotypeschemic heart disease seen in this study.ary, E4154K KK homozygosity predicts a 6080% reduc-

of ischemic cerebrovascular disease and ischemic strokeo non-carriers which needs to be conrmed in an inde-udy, however, none of the other SNPs predict risk ofart disease, myocardial infarction, ischemic cerebrovas-e, or ischemic stroke in the general population.

on and perspectives

dies reviewed can be summarized as follows: (1)ein B predicts ischemic cardiovascular events in bothd is better than LDL cholesterol in this respect; (2)

equilibrium structure in APOB is more complex thanom HapMap data, because a minimal set of tag singlepolymorphisms capturing the entire variation in APOBdentied, and thus most polymorphisms must be eval-rately in association studies; (3) APOB mutations andisms are associated with a range of apolipoprotein Bolesterol levels, although the magnitude of effect sizes

polymorphisms are modest; (4) both mutations and


Fig. 9. Risk ofIvs18+1708g >

polymorphassociationdisease pheconservatiofor the E415risk of ischemon APOB pdo not predischemic cepopulation.

Althoughels of apolipheart diseaphenotypetion with rraises threeischemic heart disease, myocardial infarction, ischemic cerebrovascular disease, andt, T2488Tc > t, P2712L, R3611Q, E4154K, and N4311S genotypes in the general population,

isms are associated with LDL metabolism in vivo; (5)of APOB mutations and polymorphisms with lipid andnotype cannot be predicted in silico using evolutionaryn or existing prediction programs; and nally, (6) except4K polymorphism that possibly predicts a reduction inmic cerebrovascular disease and ischemic stroke, com-olymorphisms with modest effect sizes on lipid levels

ict risk of ischemic heart disease, myocardial infarction,rebrovascular disease, or ischemic stroke in the general

mutations in APOB can have profound inuence on lev-oprotein B and LDL cholesterol and on risk of ischemic

se, the nding of relatively modest effect sizes on lipidassociated with APOB polymorphisms, and no associa-isk of ischemic heart disease is quite disappointing. Itimportant questions.

First, areor decreasefunction ofcies from 9changes inincrease inity, and theB levels froand upperreported froon almost 1effect sizes[41,49]. Chatude as thosimilar allebe expectedischemic stroke by APOB T71I, Ivs4+171c > a, A591 V, Ivs18+379a > c,the Copenhagen City Heart Study [41,67]. CI = condence interval.

these ndings true? Fig. 6 shows the percent increasein apolipoprotein B levels for each genetic variant as a

rare allele frequency. The common SNPs (allele frequen-% to 48%) are indeed associated with modest (26%)plasma apolipoprotein B level, compared to the 44%

apolipoprotein B associated with R3500Q heterozygos-31% and 69% increase, respectively, in apolipoproteinm the lower apolipoprotein B tertile to the middletertiles of apolipoprotein B, respectively. The resultsm the Copenhagen City Heart Study (Fig. 6), are based0,000 individuals from a general population study andare consistent over time, indicating their robustnessnges in LDL cholesterol levels are of the same magni-

se observed for SNPs in the LDLR and PCSK9 genes withle frequencies [76,77], suggesting that this is what can

in general by common SNPs in genes involved in the


VLDL-IDL-LDL pathway. The observed lack of association betweenSNPs and risk of ischemic heart disease is also in accordance withwhat one would expect from the modest changes in apolipoproteinB and LDL cholesterol levels. In the Copenhagen City Heart Study,a lifelong inhazard ratiois attributedin agreemethe approxidisease, anfrom the lolarger incre

Second,variation intwin studiein APOB, hocould be duapolipoprot(MTP), LDLisin/kexin 9or some of tEach of thecontribute land togethegesting thaAPOB, whic

Third, armost to lipSNPs havequency of 0importanceis explainedregion of APuals is a relascreened spals, or seleccapture a limscreened), alow numbeSNPs (frequ

The straof 0.10.5 wplex diseasby the comgesting thatprimarily bmodest effeently has novariation rethat either thypothesis,and complecommon dthat suscepalleles withindividuallypopulationas a functioesis for varipurifying setribution toand R3480Pwith this hrare SNPs iaccording toshould be id

APOB alleles do not account for the 5060% variation in apolipopro-tein B attributed to genetic variation, that we may have used thewrong approach to identify functional SNPs in APOB, and poten-tially have overlooked relatively rare SNPs collectively contributing

obsem the

SNPpro

f intB. Anhis reviducenton didirecen toationtelycinges oas b

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ted wsity licrease in apolipoprotein B levels of 5 mg/dL predicts aof 1.06 (1.041.07) for ischemic heart disease, if the riskto the increase in apolipoprotein B levels alone. This is

nt with what is observed, and also in accordance withmately 2-fold increase in relative risk of ischemic heartd 1115% increase in absolute 10-years risk observedwer to the upper apolipoprotein B tertile, for a muchase of 69% or 45 mg/dL in apolipoprotein B level.do the APOB SNPs studied so far account for the 5060%apolipoprotein B levels attributed to genetic variation ins? The majority of this variation could be due to variationwever, we do not know how much of the variation thate to other genes encoding proteins that may inuenceein B levels, i.e. microsomal triglyceride transfer proteinreceptor protein (LDLR), pro-protein convertase subtil-(PCSK9), lipoprotein lipase (LPL), hepatic lipase (LIPC),

he many genes involved in reverse cholesterol transport.ten SNPs studied in the Copenhagen City Heart Studyess than 1% to variability in apolipoprotein B levels [49],r as combined genotypes with approximately 1%, sug-

t there are many other functionally important SNPs inh have not yet been identied or studied.e the common APOB SNPs those that contribute the

id phenotype and risk of disease? All studies of APOBfocused on associations of SNPs with a rare allele fre-.10.5 and located in regions known to be of functional. This limitation of screening strategies applied so far

by the huge size of APOB. Re-sequencing the codingOB of 14,121 basepairs in a sufcient number of individ-tively costly undertaking, and most studies have eitherecic parts of the gene in a limited number of individu-

ted SNPs reported in databases. Such a strategy will onlyited spectrum of alleles (limited by the gene fragments

nd predominantly the common variants (limited by ther of individuals screened), explaining why so few rareencies of 0.010.1) have been identied.tegy of studying APOB SNPs with a rare allele frequency

ith the aim of estimating the contribution to a com-e such as ischemic cardiovascular disease is justiedmon diseasecommon variant hypothesis (Fig. 6), sug-genetic susceptibility to common diseases is conferred

y alleles that are common in the population, but havects in the individual [7880]. But as this approach appar-t captured all the variation in APOB, or at least not the

levant for predicting risk of disease, one must concludehe study design, the common diseasecommon variantor both have failed. An alternative hypothesis on SNPsx disease has been posed by Pritchard and named the

iseaserare variant hypothesis [81] (Fig. 6). It suggeststibility to common diseases is the result of multiple rarea large magnitude of effect on phenotype, and althoughrare, these alleles may be collectively common in the

[81,82]. The expected contribution to genetic variancen of allele frequency as predicted by Pritchards hypoth-ants under a mild (dashed line) and moderate (solid line)lection is shown in blue in Fig. 6 [81]. The results on con-phenotype observed for the APOB mutations, R3500Q, and SNPs with a rare allele frequency of 0.10.5, t

ypothesis. Unfortunately, we do not yet have data onn the frequency spectrum from 0.01 to 0.08, which

this hypothesis is where the variation of signicanceentied. The conclusion is therefore so far that known

to theFro

the tagdictionareas oof APOtrue, tof indieral recommtested

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[13] Benciadenrved genetic variation in apolipoprotein B levels.results summarized in this review on the usefulness ofapproach, evolutionary conservation, and in silico pre-

grams, there is no doubt that we cannot select relevanterest, but must re-sequence the entire coding sequenced if the common diseaserare variant hypothesis holds-sequencing should be carried out in a large number

als to be able to capture rare variants. There are sev-re-sequencing and association studies that support theseaserare variant hypothesis, although, it has not beently [77,8385].gether, these data suggest that the contribution of APOBin apolipoprotein B and LDL cholesterol levels is notdescribed by the present studies. A strategy of re-genes of interest in a large number of individuals at the

f the population distribution of LDL and HDL cholesteroleen useful in several studies to identify the full spectrum

these genes, and particularly rare variants with largehenotype [77,83,84]. A similar approach could readily

in future studies of APOB.

gements

ebted to the staff and participants of the Copenhagentudy for their important contributions and to Drs. Annensen and Brge G. Nordestgaard for helpful discussions.

by a grant (07-D-BR63-A1916-B415-A-22435) from thert Foundation.

. Supplementary data

entary data associated with this article can be found, inersion, at doi:10.1016/j.atherosclerosis.2009.01.004.

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[64] SerteinBio

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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.

Apolipoprotein B levels, APOB alleles, and risk of ischemic cardiovascular disease in the general population, a reviewIntroductionPlasma apolipoprotein B levels and risk of ischemic cardiovascular diseaseAPOB mutations and polymorphisms and linkage disequilibrium structureAPOB mutations and polymorphisms and plasma apolipoprotein B levelsAPOB mutations and polymorphisms and LDL metabolismEvolutionary conservation and predicted effectsAPOB mutations and polymorphisms and risk of ischemic cardiovascular diseaseDiscussion and perspectivesAcknowledgementsSupplementary dataReferencesFurther reading

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