Identification of Soat1 as a Quantitative Trait Locus Gene on Mouse Chromosome 1 Contributing to Hyperlipidemia
et al. (2011) Identification of Soat1 as a Quantitative Trait Locus Gene on Mouse Chromosome 1 Contributing to
Hyperlipidemia. PLoS ONE 6(10): e25344. doi:10.1371/journal.pone.0025344
Identification of Soat1 as a Quantitative Trait Locus Gene on Mouse Chromosome 1 Contributing to Hyperlipidemia
Zongji Lu 0
Zuobiao Yuan 0
Toru Miyoshi 0
Qian Wang 0
Zhiguang Su 0
Catherine C. Chang 0
Heribert Schunkert, Universitatsklinikum Schleswig-Holstein - Campus Luebeck, Germany
0 1 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, Virginia, United States of America, 2 Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, United States of America, 3 Department of Biochemistry, Dartmouth Medical School , Hanover, New Hampshire , United States of America
We previously identified two closely linked quantitative trait loci (QTL) on distal chromosome 1 contributing to major variations in plasma cholesterol and triglyceride levels in an intercross derived from C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE2/2) mice. Soat1, encoding sterol o-acyltransferase 1, is a functional candidate gene located underneath the proximal linkage peak. We sequenced the coding region of Soat1 and identified four single nucleotide polymorphisms (SNPs) between B6 and C3H mice. Two of the SNPs resulted in amino-acid substitutions (Ile147Val and His205Tyr). Functional assay revealed an increased enzyme activity of Soat1 in peritoneal macrophages of C3H mice relative to those of B6 mice despite comparable protein expression levels. Allelic variants of Soat1 were associated with variations in plasma cholesterol and triglyceride levels in an intercross between B6.apoE2/2 and C3H.apoE2/2 mice. Inheritance of the C3H allele resulted in significantly higher plasma lipid levels than inheritance of the B6 allele. Soat1 variants were also significantly linked to major variations in plasma esterified cholesterol levels but not with free cholesterol levels. Trangenic expression of C3H Soat1 in B6.apoE2/2 mice resulted in elevations of plasma cholesterol and triglyceride levels. These results indicate that Soat1 is a QTL gene contributing to hyperlipidemia.
. These authors contributed equally to this work.
Hyperlipidemia, comprising elevated levels of plasma
cholesterol, triglyceride, or both, is a major risk factor for atherosclerotic
cardiovascular disease . Although a small subset of
hyperlipidemia cases are caused by rare mutants that result in Mendelian
traits segregating in families, the common forms of hyperlipidemia
involve multiple genes and exhibit significant gene-environment
interactions . Recent genome-wide association studies (GWAS)
have been remarkably successful in identifying novel genetic loci
contributing to lipid metabolism , although it is challenging
to establish causality between a genetic variant and trait in
humans due to small gene effect, complex genetic structure, and
One effective approach to the identification of complex trait
genes is the use of mouse models. Apolipoprotein E-deficient
(apoE2/2) mice develop spontaneous hyperlipidemia and
atherosclerosis even when fed a low-fat diet ,. Using intercrosses
derived from apoE2/2 mouse strains, we and others have
identified distal chromosome 1 as a major region contributing to
hyperlipidemia ,,. QTL analysis of an intercross derived
from C57BL/6 (B6) and C3H/HeJ (C3H) apoE2/2 mice has
suggested that two closely linked loci in the distal chromosome 1
region account for major variations in plasma HDL, non-HDL
cholesterol, and triglyceride levels . The distal locus corresponds
to Hdlq5, a HDL QTL identified in advanced intercross lines derived
from B6 and NZB/B mice . Subsequent studies have identified
Apoa2 as the causative gene of Hdlq5 . The proximal locus
overlaps with Cq1 (158.6 Mbp), a locus identified in a B66KK-Ay
intercross for plasma cholesterol concentrations . Soat1 is a
functional candidate gene close to the linkage peak of the proximal
QTL. It encodes an enzyme in the endoplasmic reticulum that
catalyzes the formation of cholesteryl esters from cholesterol and
fatty acyl coenzyme A . In mammals, two Soat genes have been
identified: Soat1 is ubiquitously expressed and is responsible for
cholesteryl ester formation in the brain, adrenal glands,
macrophages, kidneys, and the liver, and Soat2 is expressed in the liver and
intestines. Soat1 deficiency results in a significant reduction in
nonHDL levels of apoE2/2 and LDLR2/2 mice fed a chow or Western
diet . Human genetic studies indicate that Soat1 variants are
associated with elevations in plasma concentrations of HDL
cholesterol and apoA-I among subjects with hyperlipidemia .
In the present study, we tested whether Soat1 was a QTL gene
contributing to naturally occurring variation in plasma lipid levels,
especially under the circumstances of hyperlipidemia, in mice.
Soat1 sequence variation
As part of an effort to find causal genes for the proximal lipid
QTL on distal chromosome 1, all genes within the confidence
interval (154.9,172.8 Mb) were perused for sequence differences in
coding or promoter regions between B6 and C3H mice by querying
public accessible databases (http://www.ncbi.nlm.nih.gov/SNP/
MouseSNP.cgi, http://cgd.jax.org/tools/diversityarray.shtml, and
www.ensembl.org). 16 likely candidate genes whose sequence
variations may lead to changes in either the structure or quantity
of a gene product were identified, which included Qsox1, Cep350,
Tor1aip1, Tdrd5, Nphs2, Soat1, Tor3a, sec16b, Lztr2, Tnr, AI848100,
LOC100040571, 1700015E13Rik, Nos1ap, Olfml2b, and Atf6. Among
them, Soat1 is a gene located close to the linkage peak and is also
involved in lipid metabolism. We sequenced the coding region of
Soat1 by using cDNA as template and found four SNPs between B6
and C3H mice (Figure 1) (the accession number of the C3H/HeJ
Soat1 gene sequence in the NCBI GenBank is bankit838062
DQ903181). Two SNPs, A/G at position 439 and C/T at 613, led
to amino-acid substitutions with isoleucine (Ile) to valine (Val) at
amino acid residue 147 (Ile147Val) and histidine (His) to tyrosine
(Tyr) at residue 205 (His205Tyr), respectively. The other two SNPs,
A/C at 421 and C/T at 454, were synonymous base changes.
We compared Soat1 peptide sequences of five mammal species,
including the mouse, and found that 205His is conserved in
humans, chimpanzees, dogs, rats, and B6 mice (Figure 2), suggesting
that 205Tyr is a mutation in C3H mice. The nonsynonymous SNP
at 439 also represents a conservative change from leucine to
isoleucine in B6 or to valine in C3H.
Enhanced enzyme activity
Soat1 activity was determined in vitro using cell homogenates
prepared from peritoneal macrophages of B6.apoE2/2 and
C3H.apoE2/2 mice. The enzyme activity was optimally
solubilized with the zwitterionic detergent CHAPS at concentrations of
13% (Figure 3A). At all the concentrations used, the Soat
specificity activity was nearly twice as high in C3H as in B6
(P,0.05). The expression of Soat1 in peritoneal macrophages was
examined by western blot analysis (Figure 3B). Densitometry of
Soat1 bands was comparable between the two strains [442660 vs.
4216115 (optical density); P = 0.87].
Association with variation in plasma lipid levels
We then examined whether Soat1 variants were associated with
variations in plasma HDL, non-HDL cholesterol, and triglyceride
levels in female F2 mice derived from B6.apoE2/2 and
C3H.apoE2/2 mice. As shown in Table 1, inheritance of two
copies of the C3H allele (CC genotype) resulted in significantly
higher triglyceride, HDL, and non-HDL cholesterol levels than
inheritance of two copies of the B6 allele (BB genotype) at the Soat1
locus (P,0.05 for each trait).
Linkage to plasma esterified cholesterol
Because Soat1 is an enzyme that catalyzes free cholesterol to
cholesterol esters, we determined whether the Soat1 locus was
linked to variations in plasma esterified and free cholesterol levels
in the BXH cross. QTL analysis of F2 mice revealed that loci on
chromosome 1 were responsible for major variations in plasma
esterified cholesterol and free cholesterol levels (Figure 4). The
interval mapping graph for chromosome 1 showed that the
proximal peak of linkage curves for esterified cholesterol
overlapped precisely with the Soat1 locus (Figure 5), which had a
LOD score of 4.2 and explained 8% of the variance (Table 2). The
distal peak of linkage curves appeared near marker D1Mit206
(174.9 Mbp), which had a LOD score of 4.4 and accounted for
9% of the variance. In contrast, free cholesterol was controlled by
two significant QTLs on chromosome 1, near markers D1Mit45
(94.9 Mbp) and D1Mit270 (172.7 Mbp), respectively, and a
suggestive locus near marker D9Mit297 (33.8 Mbp) on
chromosome 9. The QTL near marker D1Mit45 had a significant LOD
Figure 2. Comparison of Soat1 protein sequences in five mammal species. Amino acid residues that are different between B6 and C3H mice
are highlighted. GenBank accession No. are shown following the names of the species.
score of 4.6 and accounted for 9% of the variance, and the QTL
near marker D1Mit270 had a LOD score of 3.4 and explained 7%
of the variance. The suggestive QTL near marker D9Mit297
(33.9 Mbp) for free cholesterol had a LOD score of 3.3 and
accounted for 6% of the variance, and this QTL overlaps with Cq4
and Cq5 mapped in B66KK-A F2 and KK6RR F2 crosses .
Analysis of transgenic mice
To directly evaluate the role of Soat1 in hyperlipidemia, we
constructed transgenic mice that expressed C3H Soat1 and crossed
the mice with B6.apoE2/2 mice for more than six generations.
The expression of Soat1 protein in transgenic mice was analyzed by
western blotting, and it was found in the liver, kidney, spleen, and
the lung but not in the aorta, heart, or skeletal muscle (Figure 6).
Real-time PCR analysis revealed that Soat1 mRNA expression
levels in the liver were 2-fold as high in transgenic mice as in
nontransgenic littermates (6.5761.04 vs. 3.4760.44 per 10,000 copies
of GAPDH; P = 0.029). The mRNA expression level of GAPDH
was comparable between transgenic and non-transgenic mice with
respect to real-time PCR cycle threshold values (18.1760.32 vs.
18.3160.28). Soat1 protein in the liver was higher in transgenic
mice than non-transgenic littermates (optical density:
617526261338918 vs. 427065461017286), although the
difference was not statistically significant (P = 0.295). Compared to
nontransgenic littermates, transgenic mice had significantly elevated
plasma levels of non-HDL cholesterol (308.6618.5 vs.
250.668.4 mg/dl; P = 0.029) and HDL cholesterol (68.966.1 vs.
37.262.3 mg/dl; P = 0.0048) (Figure 7). Plasma triglyceride levels
were also higher in transgenic mice (78.165.1 vs. 67.065.5 mg/
dl), although the difference was not statistically significant
(P = 0.17).
QTLs for plasma HDL on mouse distal chromosome 1 have
been reported many times in numerous crosses . QTLs for
plasma triglyceride and non-HDL on distal chromosome 1 have
also been reported in several crosses, including three BXH crosses
,,,. In the intercross between B6.apoE2/2 and
C3H.apoE2/2 mice, we have observed that QTLs for HDL
coincide with QTLs for triglyceride and non-HDL on distal
chromosome 1 , suggesting that these plasma lipid phenotypes
are controlled by the same genes. We have also observed two
distinct peaks of the linkage curves for plasma triglyceride or
nonHDL with the distal peak near marker D1Mit270 (172.7 Mbp) and
the proximal peak near marker D1Mit425 (158.6 Mbp) ,
suggesting the existence of two QTLs for plasma lipids in the
distal chromosome 1 region. Two other studies have also suggested
the existence of a lipid QTL near 81.6 cM on mouse chromosome
1 , .
The distal QTL overlaps with Hdlq5 , and there is conclusive
BB (n = 61) BC (n = 112) CC (n = 45) Variance (%) P value
Measurements are presented as means 6 SD. The unit for these measurements
is mg/dl. Variance (%) accounted for by the Soat1 locus is expressed as the
percentage of the total phenotypic variance detected in the F2 cohort. BB,
homozygous for C57BL/6 alleles; CC, homozygous for C3H alleles; BC,
heterozygous for C57BL/6 and C3H alleles. The percentage of variance
explained by Soat1 genotype and likelihood ratio P values are shown.
evidence supporting Apoa2 (92.6 cM) to be the causal gene for the
QTL. Indeed, sequence analysis of the Apoa2 coding region in
many mouse strains has revealed a number of nucleotide
differences , . Apoa2 variants are associated with variation
in HDL cholesterol levels of mice ,. Transgenic
overexpression of Apoa2 elevates plasma HDL, non-HDL cholesterol, and
triglyceride levels , and Apoa2 deficiency reduces plasma HDL,
non-HDL, and triglyceride levels in mice .
The present study strongly suggests that Soat1 is the causal gene
for the proximal QTL. Multiple polymorphisms have been found
in the coding region of this gene between the two parental strains
that had been used in our previous studies to map the lipid QTL,
and two of the polymorphisms lead to amino acid substitutions in
the protein product. These polymorphisms led to changes in the
function of Soat1 enzyme. As macrophages express only Soat1, we
measured its activity in these cells. The present finding that C3H
Figure 5. LOD score plots for esterified and free cholesterol levels on chromosome 1. The x-axis depicts the marker positions in
centimorgans, and the y-axis depicts the LOD score. The microsatellite markers typed are listed below the x-axis, corresponding to their map
locations on the chromosome.
Chromosome marker (cM)a
Model of inheritancef
aFrom Mouse Genome Informatics database at http://www.informatics.jax.org.
bSuggestive QTL and significant QTL were 2.4 and 3.4, respectively, for free cholesterol and 2.4 and 3.3, respectively, for esterified cholesterol as defined by 1000
cSupport intervals (SI) were defined by a 1-unit decrease in LOD score on either side of the peak marker.
dVariance (%) indicates the percentage of the phenotypic variance at the peak marker.
ePemp, empirically determined P-value for the whole genome, was calculated using the permutation test function of MapManager QTXb20.
fModel of inheritance was determined using the MapManager QT program. C3H was the high allele at all the markers, contributing to elevated free or esterified
had significantly higher Soat enzyme activity than B6 despite
comparable protein expression in macrophages indicates that
these SNPs have resulted in increases in Soat activity. This study
has also provided several lines of other evidence supporting Soat1
to be a QTL gene affecting plasma lipid levels. First, the parental
strain C3H, which has higher Soat1 enzyme activity, exhibits
higher plasma cholesterol and triglyceride levels than the B6
strain, which has lower enzyme activity . Second, F2 mice with
the C3H allele at the Soat1 locus had higher plasma cholesterol
and triglyceride levels than those with the B6 allele. Third, the
QTL for esterified cholesterol coincided with the QTLs for
triglyceride, HDL, and non-HDL at the Soat1 locus, suggesting
that a gene involved in cholesterol esterification affected plasma
HDL and non-HDL cholesterol levels. Soat1 is such a gene that
synthesizes cholesterol esters and potentially affects lipoprotein
assembling. Finally, the direct evidence is that transgenic mice
expressing C3H Soat1 had significantly elevated plasma levels of
HDL and non-HDL cholesterol.
A previous study also showed that Soat1 deficiency reduces
plasma total cholesterol levels of apoE2/2 mice . However,
the previous knockout mice were generated using embryonic stem
cells derived from 129/SvJ mice and then backcrossed onto the B6
background. Linkage would cause the retention of a significant
segment of 129/SvJ chromosome harboring the targeted gene in
recipient mice. Several genes adjacent to Soat1, including Apoa2,
are polymorphic between B6 and 129/SvJ and could contribute to
variations in plasma lipid levels. The present study of transgenic
mice has excluded a possible interference from Apoa2 and thus
provided more definite evidence on the role of Soat1 in modifying
plasma lipid levels. In this study, we found that transgenic mice
had an increased level of HDL cholesterol. In contrast, the effect of
Soat1 on plasma HDL cholesterol was not observed in the
knockout mice. An explanation for the discrepant results is that
apoE2/2 mice have an extremely low HDL cholesterol level, and
thus it would be harder for a gene to exert an effect to further
reduce than to elevate it. In humans, a missense variant (R526G)
and a variant in the 59 untranslated region (277G.A) have been
found in the Soat1 gene, and individuals with 277G.A variant
have significantly higher plasma HDL concentrations than those
without the variant among hyperlipidemic subjects .
Nevertheless, genome-wide association studies (GWAS) to date have
failed to detect any association with lipid traits in humans. One
probable explanation for this outcome is that the gene is large
(64.89 kb) and includes many variants (794 SNPs in the NCBI
dbSNP database). Only a small number of markers in Soat1 have
been typed, and these markers may not have the strongest
In summary, we have provided reasonable evidence to support
Soat1 to be a QTL gene, although further studies are needed to
prove which SNP affects Soat1 function. Soat enzyme catalyzes
the formation of cholesteryl esters from free cholesterol and fatty
acyl coenzyme A. Because of its potential role in foam cell
formation, this enzyme has been a target for developing
therapeutic drugs for the past two decades. Theoretically,
inhibition of the enzyme should block the esterification of
cholesterol and prevents the transformation of macrophages into
foam cells. However, some animal studies show that Soat
inhibitors exert hypolipidemic effects and reduce atherosclerosis
,, while some studies show that inhibition of Soat
activity promotes atherosclerosis . In humans, administration
of Soat inhibitors failed to reduce, rather increase, plaque volume
,. Studies with more specific inhibitors of Soat1 activity
have also shown aggravation rather than alleviation of
atherosclerosis in rabbits and mice ,. As Soat1 elevates plasma levels
of both good (HDL) and bad (non-HDL) cholesterol, the present
results may explain why Soat1 inhibitors are not clinically effective
as expected and many have failed in clinical stages probably due to
its effect on HDL cholesterol. Moreover, continued inhibition of
Soat activity increases intracellular free cholesterol and limits the
efflux of free cholesterol, which may induce cytotoxic effects within
Materials and Methods
All procedures were carried out in accordance with current
National Institutes of Health guidelines and approved by the
University of Virginia Animal Care and Use Committee
(Assurance #A3245-01, Animal Protocol #3109).
B6.apoE2/2 mice were purchased from the Jackson
Laboratory, and C3H.apoE2/2 mice were generated in our laboratory.
The generation of F2 mice from B6.apoE2/2 and C3H.apoE2/2
mice was reported previously . At 6 weeks of age, female F2
mice were started on a Western-type diet containing 21%
butterfat, 34% sucrose, and 0.2% cholesterol (TD 88137, Harlan
Laboratories) and maintained on the diet for 12 weeks. To
generate transgenic mice, a clone containing the Soat1 gene in the
pTARBAC2.1 vector was picked from the CHORI-34 Mouse
C3H/HeJ BAC library constructed by the Pieter De Jongs
Laboratory at Childrens Hospital Oakland Research Institute.
This clone contained the entire Soat1 gene, 142,565 bp upstream
and 14,893 bp downstream from the 59 and 39 ends, respectively.
The integrity of Soat1 was confirmed through partial sequencing
and restriction enzyme digestion before the purified BAC DNA
was microinjected into B6D2 F1 fertilized eggs at a
concentration of 1 mg/ml. Transgenic founders were identified by PCR
amplifications of both forward and reverse fragments of the BAC
DNA with primers
59AAGTCAGAACTGTGGCTTGT-39/59-CAGCACTGGTTTAAT GTCC-39. Positive transgenic mice were backcrossed with
B6.apoE2/2 mice for more than 6 generations and maintained in
a heterozygous condition for the transgene.
Plasma lipid measurements
Plasma total cholesterol, HDL cholesterol, and triglyceride were
measured as reported previously . Non-HDL cholesterol was
calculated by subtracting the HDL cholesterol levels from the
total. Free cholesterol levels were determined using a kit from
Wako (Richmond, VA). Briefly, 6 ml of plasma samples, lipid
standards, and controls were loaded in a 96-well plate and then
mixed with 150 ml of free cholesterol reagent. After a 5-min
incubation at 37uC, the absorbance at 600 nm was read on a
Molecular Devices (Menlo Park, CA) plate reader. Esterified
cholesterol levels were calculated by subtracting free cholesterol
levels from total cholesterol levels.
Soat1 cDNA sequencing and genotyping
Total RNA isolated from the liver of B6 mice and C3H mice
was reverse transcribed to cDNA with use of the Superscript
PCR kit (Invitrogen). The PCR primers used for amplification of
Soat1 cDNA in both directions were as follows:
59-GCAAAGATCCACTACCCACAG-39/59-AACACGTACCGACAAGTCCAG-39. After purification with a QIAquick
PCR purification kit, PCR products were sequenced on an ABI
Prism Cycle sequencer 310 (Applied Biosystems). The Soat1
polymorphism at T454C was used to screen the BXH intercross
with the PCR-restriction fragment length polymorphism
(PCRRFLP)-based method. PCR amplification on genomic DNA was
performed using primers
59-AGATTGTCCCTCTAAGGCGCCAA-39 and 59-AGCCTAGCTGGACCACTTTCTCAA-39, and
the 712 bp amplicon was then digested with ClaI restriction
enzyme (New England BioLabs, Hertfordshire, UK) according to
the manufacturers instruction. The PCR product from the B6
allele but not that from the C3H allele was digested by ClaI to
511 bp and 201 bp fragments. Thus, the BB allele should exhibit
two bands, the CC allele one band, and the BC alleles three bands
on an agarose gel.
Soat1 activity assay in macrophages
Peritoneal macrophages were prepared as we previously
described . Following a brief culture as a monolayer in RPMI
medium containing 10% fetal bovine serum, macrophages were
harvested by hypotonic shock and scraping, and the resultant cell
homogenate was kept in a buffer (50 mM Tris, 1 mM EDTA at
pH 7.8 with protease inhibitors) at concentrations of 24 mg/ml.
To solubilize the enzyme, 1 M KCl and various concentrations of
CHAPS were added. The enzyme activity of Soat1 was measured
in duplicate in taurochoate/cholesterol/PC mixed micelles as
described by Chang et al .
Western blot analysis
The presence of Soat1 in various tissues of transgenic mice and
in peritoneal macrophages of B6.apoE2/2 and C3H.apoE2/2
mice was determined by western blot analysis. Proteins were
prepared as we previously described , separated by
electrophoresis on 412% Tris-polyacrylamide gels, and
electrophoretically transferred to nitrocellulose membranes. The membrane was
probed with a rabbit polyclonal antibody against Soat1 (H-125,
Santa Cruz), and signals were detected by the chemiluminescence
method (Invitrogen). The density of the bands was quantified with
a densitometer (Molecular Devices, CA).
Real-time PCR analysis of Soat1 expression
Total RNA extracted from the liver of transgenic mice was
treated with DNase I, and then reverse transcribed to cDNA as
described above. cDNA was mixed with SYBR Green supermix
reagent (Bio-Rad) and specific primers to assess expression of Soat1
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by
real-time PCR. Primers used for Soat1 amplification were
59TGTGCATCAGAAAGGTACCACGGA-39/59-GTTGCCAGGAAACCACCAAAGTGA-39 and for GAPDH were
TTTGGCCGTATT-39/59-GGCCTTGACTGTGCCGTTGAATTT-39. Real-time PCR on each sample was run in
triplicate on an iCycler iQ5 machine (Bio-Rad) under the condition of
50uC for 2 minutes, 95uC for 2 minutes, then 95uC 30 seconds, 60uC
30 seconds, and 72uC 30 seconds for 40 cycles as reported . The
expression level of Soat1 was expressed as mRNA copy number relative
to 10,000 copies of GAPDH mRNA.
QTL analysis was performed as we previously described
, ANOVA was used for determining if the mean phenotype
values of progeny with different genotypes at a specific marker
were significantly different. The Student t test was used when only
two means were compared. Differences were considered
statistically significant at P,0.05.
Conceived and designed the experiments: ZL ZY WS. Performed the
experiments: ZL ZY TM QW ZS CCC WS. Analyzed the data: ZL ZY
QW CCC WS. Contributed reagents/materials/analysis tools: WS. Wrote
the paper: ZY WS. Obtained grants for support of this project: WS.
1. National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) . ( 2002 ) Third report of the national cholesterol education program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III) final report . Circulation 106 ( 25 ): 3143 - 3421 .
2. Wierzbicki AS , Graham CA , Young IS , Nicholls DP ( 2008 ) Familial combined hyperlipidaemia: Under - defined and under - diagnosed? Curr Vasc Pharmacol 6 ( 1 ): 13 - 22 .
3. Teslovich TM , Musunuru K , Smith AV , Edmondson AC , Stylianou IM , et al. ( 2010 ) Biological, clinical and population relevance of 95 loci for blood lipids . Nature 466 ( 7307 ): 707 - 713 .
4. Plump AS , Smith JD , Hayek T , Aalto-Setala K , Walsh A , et al. ( 1992 ) Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells . Cell 71 ( 2 ): 343 - 353 .
5. Zhang SH , Reddick RL , Piedrahita JA , Maeda N ( 1992 ) Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258 ( 5081 ): 468 - 471 .
6. Dansky HM , Shu P , Donavan M , Montagno J , Nagle DL , et al. ( 2002 ) A phenotype-sensitizing apoe-deficient genetic background reveals novel atherosclerosis predisposition loci in the mouse . Genetics 160 ( 4 ): 1599 - 1608 .
7. Su Z , Li Y , James JC , McDuffie M , Matsumoto AH , et al. ( 2006 ) Quantitative trait locus analysis of atherosclerosis in an intercross between C57BL/6 and C3H mice carrying the mutant apolipoprotein E gene . Genetics 172 ( 3 ): 1799 - 1807 .
8. Wang SS , Shi W , Wang X , Velky L , Greenlee S , et al. ( 2007 ) Mapping, genetic isolation, and characterization of genetic loci that determine resistance to atherosclerosis in C3H mice . Arterioscler Thromb Vasc Biol 27 ( 12 ): 2671 - 2676 .
9. Wang X , Le Roy I , Nicodeme E , Li R , Wagner R , et al. ( 2003 ) Using advanced intercross lines for high-resolution mapping of HDL cholesterol quantitative trait loci . Genome Res 13 ( 7 ): 1654 - 1664 .
10. Wang X , Korstanje R , Higgins D , Paigen B ( 2004 ) Haplotype analysis in multiple crosses to identify a QTL gene . Genome Res 14 ( 9 ): 1767 - 1772 .
11. Suto J , Matsuura S , Yamanaka H , Sekikawa K ( 1999 ) Quantitative trait loci that regulate plasma lipid concentration in hereditary obese KK and KK-ay mice . Biochim Biophys Acta 1453 ( 3 ): 385 - 395 .
12. Chang TY , Li BL , Chang CC , Urano Y ( 2009 ) Acyl-coenzyme A:Cholesterol acyltransferases . Am J Physiol Endocrinol Metab 297 ( 1 ): E1 - 9 .
13. Yagyu H , Kitamine T , Osuga J , Tozawa R , Chen Z , et al. ( 2000 ) Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia . J Biol Chem 275 ( 28 ): 21324 - 21330 .
14. Ohta T , Takata K , Katsuren K , Fukuyama S ( 2004 ) The influence of the acylCoA:Cholesterol acyltransferase-1 gene (277GRA) polymorphisms on plasma lipid and apolipoprotein levels in normolipidemic and hyperlipidemic subjects . Biochim Biophys Acta 1682 ( 1-3 ): 56 - 62 .
15. Wang X , Paigen B ( 2005 ) Genetics of variation in HDL cholesterol in humans and mice . Circ Res 96 ( 1 ): 27 - 42 .
16. Machleder D , Ivandic B , Welch C , Castellani L , Reue K , et al. ( 1997 ) Complex genetic control of HDL levels in mice in response to an atherogenic diet. coordinate regulation of HDL levels and bile acid metabolism . J Clin Invest 99 ( 6 ): 1406 - 1419 .
17. Doolittle MH , LeBoeuf RC , Warden CH , Bee LM , Lusis AJ ( 1990 ) A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition . J Biol Chem 265 ( 27 ): 16380 - 16388 .
18. LeBoeuf RC , Doolittle MH , Montcalm A , Martin DC , Reue K , et al. ( 1990 ) Phenotypic characterization of the ath-1 gene controlling high density lipoprotein levels and susceptibility to atherosclerosis . J Lipid Res 31 ( 1 ): 91 - 101 .
19. Hedrick CC , Castellani LW , Warden CH , Puppione DL , Lusis AJ ( 1993 ) Influence of mouse apolipoprotein A-II on plasma lipoproteins in transgenic mice . J Biol Chem 268 ( 27 ): 20676 - 20682 .
20. Weng W , Breslow JL ( 1996 ) Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility . Proc Natl Acad Sci U S A 93(25) : 14788 - 14794 .
21. Shi W , Wang NJ , Shih DM , Sun VZ , Wang X , et al. ( 2000 ) Determinants of atherosclerosis susceptibility in the C3H and C57BL/6 mouse model: Evidence for involvement of endothelial cells but not blood cells or cholesterol metabolism . Circ Res 86 ( 10 ): 1078 - 1084 .
22. Bocan TM , Krause BR , Rosebury WS , Lu X , Dagle C , et al. ( 2001 ) The combined effect of inhibiting both ACAT and HMG-CoA reductase may directly induce atherosclerotic lesion regression . Atherosclerosis 157 ( 1 ): 97 - 105 .
23. Bocan TM , Krause BR , Rosebury WS , Mueller SB , Lu X , et al. ( 2000 ) The ACAT inhibitor avasimibe reduces macrophages and matrix metalloproteinase expression in atherosclerotic lesions of hypercholesterolemic rabbits . Arterioscler Thromb Vasc Biol 20 ( 1 ): 70 - 79 .
24. Delsing DJ , Offerman EH , van Duyvenvoorde W , van Der Boom H , de Wit EC , et al. ( 2001 ) Acyl-CoA:Cholesterol acyltransferase inhibitor avasimibe reduces atherosclerosis in addition to its cholesterol-lowering effect in ApoE*3-leiden mice . Circulation 103 ( 13 ): 1778 - 1786 .
25. Perrey S , Legendre C , Matsuura A , Guffroy C , Binet J , et al. ( 2001 ) Preferential pharmacological inhibition of macrophage ACAT increases plaque formation in mouse and rabbit models of atherogenesis . Atherosclerosis 155 ( 2 ): 359 - 370 .
26. Nissen SE , Tuzcu EM , Brewer HB , Sipahi I , Nicholls SJ , et al. ( 2006 ) Effect of ACAT inhibition on the progression of coronary atherosclerosis . N Engl J Med 354 ( 12 ): 1253 - 1263 .
27. Tardif JC , Heinonen T ( 2006 ) Can ACAT inhibition limit the progression of atherosclerosis in patients with coronary artery disease ? Nat Clin Pract Cardiovasc Med 3 ( 7 ): 366 - 367 .
28. Su YR , Dove DE , Major AS , Hasty AH , Boone B , et al. ( 2005 ) Reduced ABCA1-mediated cholesterol efflux and accelerated atherosclerosis in apolipoprotein E-deficient mice lacking macrophage-derived ACAT1 . Circulation 111 ( 18 ): 2373 - 2381 .
29. Tian J , Pei H , James JC , Li Y , Matsumoto AH , et al. ( 2005 ) Circulating adhesion molecules in apoE-deficient mouse strains with different atherosclerosis susceptibility . Biochem Biophys Res Commun 329 ( 3 ): 1102 - 1107 .
30. Shi W , Brown MD , Wang X , Wong J , Kallmes DF , et al. ( 2003 ) Genetic backgrounds but not sizes of atherosclerotic lesions determine medial destruction in the aortic root of apolipoprotein E-deficient mice . Arterioscler Thromb Vasc Biol 23 ( 10 ): 1901 - 1906 .
31. Chang CC , Sakashita N , Ornvold K , Lee O , Chang ET , et al. ( 2000 ) Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine . J Biol Chem 275 ( 36 ): 28083 - 28092 .
32. Shi W , Wang X , Shih DM , Laubach VE , Navab M , et al. ( 2002 ) Paradoxical reduction of fatty streak formation in mice lacking endothelial nitric oxide synthase . Circulation 105 ( 17 ): 2078 - 2082 .
33. Yuan Z , Pei H , Roberts DJ , Zhang Z , Rowlan JS , et al. ( 2009 ) Quantitative trait locus analysis of neointimal formation in an intercross between C57BL/6 and C3H/HeJ apolipoprotein E-deficient mice . Circ Cardiovasc Genet 2 ( 3 ): 220 - 228 .