© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Severe Hyperlipidemia in Apolipoprotein E2 Homozygotes Due to a Combined Effect of Hyperinsulinemia and an SstI Polymorphism
From the Department of General Internal Medicine, Medical Faculty (E.J.G.S., A.E.M., A.H.M.S.), and the MGC–Department of Human Genetics (M.J.V.H., R.R.F., P.D.K.), Leiden University Medical Center, and TNO-PG, Gaubius Laboratory (L.M.H.), Leiden, Netherlands.
Correspondence to E.J.G. Sijbrands, Department of General Internal Medicine, Academic Medical Center, Amsterdam, c/o Overtoom 49, 1054 HB Amsterdam, The Netherlands. E-mail nrexpert{at}euronet.nl
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Abstract |
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Abstract—More than 90% of patients with type III hyperlipoproteinemia are homozygous carriers of the apolipoprotein (apo) E*2 allele. The great majority of these apoE2(Arg158
Cys) homozygotes in the general
population, however, are normolipidemic. Apparently, expression of the
hyperlipidemic state requires additional genetic and/or
environmental factors, suggesting a multifactorial etiology. To
elucidate these additional risk factors, we analyzed
normolipidemic and hyperlipidemic apoE2 homozygotes.
Hyperinsulinemia was observed in 27 of 49 apoE2
homozygotes and associated with elevated lipid levels:
hyperinsulinemic apoE2 homozygotes had type III
hyperlipoproteinemia 6 times more often than
apoE2 homozygotes with normal insulin levels (odds ratio 6.2,
P=0.02). We screened the normolipidemic and
hyperlipidemic apoE2 homozygotes for common variants in
candidate genes involved in lipolysis—the APOA1-C3-A4 gene cluster,
lipoprotein lipase, and hepatic lipase—and analyzed for
associations with the expression of hyperlipidemia. In
the hyperinsulinemic group, the 7 carriers of the
SstI polymorphism (S2) in the APOC3 gene displayed
severely elevated VLDL cholesterol
(Pinsulin by SstI<0.001) and
VLDL triglyceride
(Pinsulin by SstI<0.01)
and low levels of HDL
(Pinsulin by SstI<0.02).
In the normoinsulinemic group, no such relation of the
SstI polymorphism with
hyperlipidemia was observed. These data provide the
first evidence for a combined effect of
hyperinsulinemia and the SstI
polymorphism on the expression of hyperlipidemia in
apoE2 homozygotes.
Key Words: apolipoprotein E • hyperlipoproteinemia type III • hyperinsulinemia • xanthomas
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Introduction |
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Atherosclerosis has a complex etiology in which hyperlipidemia accelerates the deposition of lipids in the artery wall.1 2 One of the inherited lipid disorders, type III hyperlipoproteinemia (HLP), is characterized by dysfunctional apolipoprotein (apo) E and by increased serum concentrations of both cholesterol and triglyceride.3 Biochemically, the disorder is characterized by the presence of ß-VLDL particles, which are cholesterol-enriched chylomicron and VLDL remnants. More than 90% of the patients with type III HLP are homozygous for a specific isoform of apoE: apoE2(Arg158
Cys).4 Compared
with the other common isoforms, apoE3 and E4, apoE2 has <2% binding
activity for hepatic lipoprotein receptors,5 6 which
renders apoE2 homozygotes susceptible to accumulation of circulating
remnant lipoprotein particles. However, the majority of apoE2
homozygotes in the general population are normolipidemic or even
hypolipidemic.7 8 The latter is due to low plasma LDL
levels as a result of (1) compensatory upregulated expression of LDL
receptors on the surface of hepatocytes to maintain a
normal intrahepatocellular cholesterol concentration and
(2) a delayed conversion of IDL into LDL in plasma.9 The
expression of overt hyperlipidemia in apoE2 homozygotes
occurs despite compensatory mechanisms, when additional genetic and/or
environmental factors result in large amounts of circulating
remnants.4 For the majority of the apoE2 homozygotes with type III HLP, the additional factors causing hyperlipidemia are not known. It has been reported that uncontrolled diabetes mellitus10 11 and hypothyroidism12 occasionally contribute to the expression of type III HLP in apoE2 homozygotes. Obesity associates with type III HLP, which suggests an influence of insulin resistance.13 14 Even though insulin resistance is a frequent disorder of metabolism that is consistently associated with increased levels of triglyceride-rich lipoproteins, its contribution to hyperlipidemia in apoE2 homozygotes is unknown.15
Common variants of other proteins involved in lipolytic conversion, such as lipoprotein lipase (LPL), hepatic lipase (HL), and apoC3, may contribute to the accumulation of triglyceride-rich particles in the circulation and lead to the expression of type III HLP in apoE2 homozygotes. This hypothesis is supported by findings of others showing that(1) LPL gene mutations aggregate in hyperlipidemic apoE2 carriers,16 and several LPL gene mutations associate with dyslipidemia17 18 19 20 ; (2) HL activity is much less stimulated by apoE2 than by apoE321 ; (3) apoC3 inhibits LPL22 and reduces the hepatic uptake of triglyceride-rich remnant particles23 24 ; (4) in mice, apoC3 was shown to decrease the lipolysis at the cell surfaces25 ; and (5) in various human populations, the SstI polymorphism (S2) of the APOC3 gene associates with hypertriglyceridemia and coronary artery disease.26 27 28 29 30 The SstI polymorphism is located in the 3' noncoding region of the APOC3 gene and does not lead to a change in the amino acid sequence of the protein. Probably, the SstI polymorphism is in linkage disequilibrium with hitherto unknown mutation(s) causing hypertriglyceridemia.24 28 31 32 33
The availability for research of both normolipidemic and hyperlipidemic apoE2 homozygotes enabled us to investigate additional risk factors required for the overt expression of type III HLP. Here, we report the association of (1) fasting insulin levels and (2) common polymorphisms in 3 candidate genes—LPL, HL, and the APOA1-C3-A4 gene cluster—with the expression of type III HLP in apoE2 homozygotes.
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Methods |
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ApoE2(Arg158
Cys) HomozygotesApoE phenotypes were determined according to a standard procedure34 and were confirmed by APOE genotyping as previously described by others.35 A total of 49 homozygous carriers of apoE2(Arg158
Cys) were identified. Ten unrelated carriers
were detected during a population-based study among 2018 randomly
selected 35-year-old men36 and were analyzed in
1985 at age 35 years and again in 1995 (present study) at age 45
years. Between July 1988 and December 1990, 895 probands, who were
unrelated up to the third degree, with inherited HLP were referred to
our Lipid Clinic. Among them, a total of 25 probands were identified
with homozygosity for apoE2. Routine measurement of the lipids by
general practitioners yielded 13 of these 25 patients; a
further 6 were identified after having onset of symptoms of
coronary artery disease; another 6 patients were identified by
lipidologists/internists of other hospitals in our region, and those
patients were referred to our university outpatient Lipid Clinic for
diagnostic reasons (ultracentrifugation and
apoE genotyping). Eight more men >34 years old and 6 women >38 years old were found by screening of 5 families of Lipid Clinic patients and 3 families of apoE2 homozygotes detected during the population-based study.
This combination of selection methods allowed us to include normolipoproteinemic and hyperlipoproteinemic apoE2 homozygotes in the study. The diagnosis of type III HLP among our apoE2 homozygotes was based on a ratio of VLDL cholesterol to serum triglyceride >0.689. In apoE2 homozygotes, high ratios reflect the presence of ß-VLDL, which is indicative for the expression of type III HLP.3
With the exception of the sampling of 8 normolipidemic 35-year-old men in 1985, all blood samples were obtained after an overnight fast. The insulin concentration was determined by a radioimmunoassay (Ins-Ria-100, Medgenix) in which the antibody cross-reacts with proinsulin (40%) but not with C-peptide. Fasting insulin levels measured with a similar radioimmunoassay were shown to correlate consistently with insulin resistance in normoglycemic subjects.37 The other methods of the clinical chemical analyses have been described previously.38 Informed consent was given by each participant, and the study was approved by the Ethics Committee of our hospital.
Molecular Analyses of Candidate Genes
Identification of the mutations in the LPL and HL genes was
performed with polymerase chain reaction followed by specific
restriction digestion as previously described by others:
LPL(Asp9Asn),17 LPL(Asn291Ser),19
LPL(Ser447Ter),39 and HL(Val73Met).40
Carriers of the SstI polymorphism in exon 4 of the APOC3
gene were detected by PCR and restriction enzyme analysis:
homozygosity for the wild-type allele was designated S1S1, and
heterozygosity for the polymorphic variant at the restriction site
was designated S1S2.24 The positions -482 and -455
of the promoter region of the APOC3 gene were screened for 2 frequently
occurring point mutations.41 Two MspI sites at
positions -75 and +83 in the APOA1 gene,42 which is
located downstream of the SstI polymorphism, were
screened to perform haplotyping.
Statistical Analyses
Fisher exact tests were applied to compare allele
frequencies between groups. The frequencies and the probabilities of
allele-specific haplotypes were estimated by the expectation
maximization resampling method and maximum-likelihood
statistics.43 All lipid and lipoprotein values were
presented as mean±SEM. Analyses were adjusted for age,
sex, and fasting glucose concentration with both multiple linear
regression and 2-way ANCOVA. The findings of the regression
analyses were identical to those of 2-way ANCOVA.
Statistical significance was assessed at the 5% level of probability.
Statistical analyses of serum triglycerides and
VLDL triglycerides were performed after logarithmic
transformation.
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Results |
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General Characteristics
The total of 49 apoE2 homozygotes comprised 32 men and 17 women. Their median ages were 44 years (range 27 to 83 years) and 54 years (range 25 to 75 years), respectively. A ratio of VLDL cholesterol to serum triglyceride >0.689 was detected in 17 men and 11 women. The women with and without type III HLP had a similar mean age (52±2 [mean±SEM] versus 55±7 years, P=0.7). Ten men had previously been analyzed for lipid parameters in a population-based study in 1985. A comparison between the lipid profiles of these individuals obtained in 1985 and 1995 (present study) revealed no significant changes (Figure 1
).
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symbols
indicate apoE2 homozygotes that were not treated with
cholesterol-lowering drugs during follow-up.
A total of 12 persons had palmar or tuberous xanthomas. The presence of
these xanthomas was associated with high VLDL levels and high fasting
insulin concentrations (Table 1).
Adjusted for age and sex, apoE2 homozygotes with a ratio of VLDL
cholesterol to triglycerides >0.689 had 14
times more frequent occurrence of xanthomas than those with a ratio
0.689 (odds ratio 14.3, 95% CI 1.6 to 126.6, P=0.02).
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The 49 persons had neither diabetes mellitus (fasting serum glucose concentrations ranged from 3.7 to 6.9 mmol/L) nor thyroid disorders.
Influence of Hyperinsulinemia
Among the 49 apoE2 homozygotes, fasting insulin levels varied from
22 to 258 pmol/L, with a median of 100 pmol/L. Persons with fasting
insulin values 
100 pmol/L were assigned to the high-insulin group and
were compared with the remaining persons, who had values <100 pmol/L.
In our general population, a fasting insulin concentration 90 pmol/L
is considered normal for both women and men. The results were similar
when this value was used as a cutoff point (data not shown). The 15 men
and 7 women in the high-insulin group were older than the 17 men and 10
women in the low-insulin group (54±2 versus 43±2 years,
P=0.001). The mean body mass index was 26.1±0.6
kg/m2 in the high-insulin group and 25.1±0.9
kg/m2 in the low-insulin group
(P=0.4). The mean fasting glucose concentrations of the
high- and low-insulin groups were 5.19±0.13 and 5.02±0.12 mmol/L
(P=0.1), respectively, and the corresponding mean fasting
insulin levels were 145±8 and 51±5 pmol/L. The high-insulin group had
significantly higher mean levels of total cholesterol,
triglycerides, LDL and IDL cholesterol, VLDL
cholesterol, and VLDL triglycerides and a lower
mean HDL cholesterol than the other group (Table 2). The contribution of high insulin
levels to the expression of type III HLP in apoE2 homozygotes was
analyzed adjusted for age, sex, fasting glucose concentration,
body mass index, and cigarette smoking. These covariables did not
contribute to type III HLP (data not shown). The persons with insulin
levels 100 pmol/L had a ratio of VLDL cholesterol to
serum triglyceride >0.689 six times more often (odds ratio
6.2, 95% CI 1.3 to 28.7, P=0.02).
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LPL Gene and HL Gene
All individuals were screened for frequently occurring
mutations in the LPL and HL genes: the LPL(Asp9Asn) mutation was not
detected, but 3 heterozygous carriers of the LPL(Asn291Ser)
mutation, 2 carriers of the LPL(Ser447Ter) mutation, and 6 carriers
of the HL(Val73Met) mutation were identified (Table 3). The carriers of these specific
mutations were unrelated. Severe hyperlipidemia was
observed only in hyperinsulinemic patients.
Hyperlipidemia among carriers of the LPL(Asn291Ser)
and the HL(Val73Met) mutation was not unequivocally related to
hyperinsulinemia or the SstI
polymorphic site (S1S2). The mean fasting insulin concentration of
9 LPL(Asn291Ser) and HL(Val73Met) carriers was 81±18 pmol/L, and
that of 40 persons without such a lipase mutation was 108±9 pmol/L
(P=0.2). The LPL(Asn291Ser) and the HL(Val73Met)
mutations were expected to associate with
hyperlipidemia; however, these lipase mutations were
equally distributed among normolipidemic and
hyperlipidemic apoE2 homozygotes: 4 (17±8%) and 5
(20±8%) carriers of a lipase gene mutation, respectively
(P=0.8). The mean serum concentrations of VLDL
cholesterol and triglycerides did not differ
between 9 persons with and 40 without the said lipase gene mutations
either in the total group or in both insulin subgroups (data not
shown). Adjustment for age, sex, and fasting glucose did not change
these results. Although the LPL(Ser447Ter) mutation was expected to
associate with a favorable lipid profile, 1 of 2 carriers was
hyperlipidemic. The mean HDL cholesterol
level of the 10 heterozygous lipase gene carriers was 1.46±0.17
mmol/L, compared with 1.08±0.06 mmol/L in the 39 persons without
lipase gene mutations (P=0.01).
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Influence of APOA1-C3-A4 Gene Cluster
A total of 15 heterozygous carriers of the SstI
polymorphism (S1S2) in exon 4 of the APOC3 gene were identified. We
did not observe any effect of entering relatives into the study: 2
pairs of sisters were carriers of the polymorphic SstI
site of the APOC3 gene, of which 1 proband was a
hyperlipidemic patient whose sister was normolipidemic
(family 142 in Figure 2). The other pair
was normolipidemic (family 144 in Figure 2), and all other
carriers of the SstI polymorphism were unrelated. The
persons with an S2 allele tended to have higher concentrations of
VLDL cholesterol than those with S1S1 (6.93±2.04 versus
3.96±0.56 mmol/L, P=0.06). Separate analyses
in the high- and low-fasting-insulin groups showed that this trend is
based on an effect of the S2 allele in
hyperinsulinemic patients. In the low-fasting-insulin
group, no significant effect of the SstI polymorphism
was observed (Table 4). In the
high-fasting-insulin group, however, the 7 unrelated subjects with S1S2
had a much more deleterious lipid profile than the 20 persons with
S1S1. The mean insulin level was 172±14 pmol/L in the 7 S2 allele
carriers and 136±9 pmol/L in the remaining 20 persons
(P=0.05). The highly significant insulin by SstI
interaction terms (Pinsulin by
SstI) shown in Table 4 indicate a
combined effect of hyperinsulinemia and the
polymorphism on VLDL metabolism.
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Because the SstI polymorphism is located in the 3'
noncoding region of the apoC3 gene, it could be argued that the APOC3
effect is not caused by the SstI polymorphism but rather
is due to a closely linked functional mutation. The recently described
common genetic variants in the insulin/phorbol ester response element
at positions -482 and -455 of the promoter region of APOC3 could be
candidates.41 We subsequently screened all
individuals for these 2 point mutations and performed haplotype
analysis. In both insulin groups, significant linkage
disequilibrium was observed between the SstI
polymorphism (S2) and the sequence variations in the promoter
region (allele P2, P=0.001, Table 5). Therefore, we performed subgroup
analyses with the 3 alleles of the promoter
polymorphism instead of the SstI polymorphism.
However, no differences were detected in lipid levels between carriers
of the 3 alleles P1, P2, and P3 (data not shown). This indicates
that the effect of the SstI polymorphism or a closely
linked mutation is contained within a P2S2 haplotype.
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Ascertainment Through Family Studies
The 8 family trees that were studied to ascertain additional apoE2
homozygotes are shown in Figure 2
. Analyses of these
kindreds were inconclusive with regard to the expression of type III
HLP: hyperinsulinemia, the SstI
polymorphism (S1S2), and the HL(Val73Met) mutation occurred with
and without hyperlipidemia. Nonetheless, these family
trees illustrate that additional familial factors are needed to express
type III HLP: apoE2 homozygotes ascertained through 3 probands from a
population-based study (families 126, 129, and 145) did not express
HLP, whereas >50% of such relatives of the
hyperlipidemic probands had type III HLP (56%, 95% CI
21% to 86%).
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Discussion |
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The present study was initiated to identify additional factors that contribute to type III HLP in apoE2 homozygotes. Type III HLP in apoE2 homozygotes is an autosomal recessive disorder with a reduced penetrance and variable expression. In cross-sectional analyses, late onset of hyperlipidemia could explain the observation of such incomplete expression. With regard to apoE2 homozygotes, it is often stated that in men, hyperlipidemia manifests in the third decade of life and in women, after menopause. Our follow-up of 10 male apoE2 homozygotes, however, suggests that essentially no change could be observed between the lipid profiles obtained at age 35 years and those obtained at age 45 years (Figure 1
). Thus, at least during this
period of their life, in which most inherited lipid disorders are
assumed to become manifest in men, age was not the factor leading to
the expression of type III HLP. Although the sample was small, the
findings suggest that we did not introduce an important bias by
studying the expression of type III HLP cross-sectionally. A
population-based study to investigate the expression of type III HLP is
unfeasible, because the frequency of apoE2 homozygotes in the general
population is low (0.5%), and only a small percentage is
hyperlipidemic (
2%). Therefore, we recruited apoE2
homozygotes from our Lipid Clinic. Additional apoE2 homozygotes were
ascertained by screening of families. In contrast to the normolipidemic
men from the follow-up sample, inclusion of family members in the
normolipidemic group may have involved differences in exposure time to
additional familial risk factors. This could have diminished the
contrast between the hyperlipidemic and the
normolipidemic groups; however, we included only male relatives >34
years old and mostly postmenopausal women. The kindreds show a clear
difference between the families that were ascertained through
normolipidemic and hyperlipidemic probands. This
confirms that additional familial factors are required for the
expression of type III HLP. However, more families need to be studied
to analyze the specific interacting factors in such kindreds
properly. Hyperinsulinemia is an independent risk factor for atherosclerosis44 and ischemic heart disease45 and associates with increased levels of triglyceride-rich lipoproteins, mainly VLDL.46 Hyperinsulinemia stimulates the production and secretion of VLDL, and subsequently the clearance capacity for these particles may become overwhelmed in apoE2 homozygotes.47 48 49 In humans, short-term infusion of long-chain triglycerides in turn enhances hyperinsulinemia by substrate competition50 ; however, chronic endogenous hypertriglyceridemia is unlikely to produce hyperinsulinemia. In fact, hypertriglyceridemic transgenic mice carrying the human APOC3 gene were neither hyperinsulinemic nor insulin resistant.51 In our apoE2 homozygotes, hyperinsulinemia was associated with high ratios of VLDL cholesterol to serum triglycerides, which reflects the presence of ß-VLDL and is indicative for the expression of type III HLP.3 Furthermore, the hyperinsulinemic apoE2 homozygotes also had low levels of HDL, which may further increase susceptibility for coronary artery disease. These effects of hyperinsulinemia on the expression of type III HLP and HDL levels were analyzed irrespective of the cause of the high insulin levels.
Heterozygosity for mutations in genes that are involved in the
lipolytic conversion of lipoproteins could also contribute to
accumulation of VLDL, as has been shown for the LPL(Asn291Ser)
mutation.18 19 Although such mutations do not always lead
to metabolic disorders, hyperlipidemia may
develop when other factors stress the biochemical
pathway.19 However, we did not detect a contribution to
hyperlipidemia in our apoE2 homozygotes of the
LPL(Asp9Asn), LPL(Asn291Ser), and HL(Val73Met) mutations. Our
data suggest that apoE2 homozygotes with lipase gene mutations may have
a more favorable lipid profile because of relatively high levels of HDL
cholesterol. Such an advantage has been described
previously in carriers of the LPL(Ser447Ter)
mutation20 52 and in carriers of a promoter
polymorphism (G-250A) of the HL gene. The heterozygous
and homozygous carriers of the latter mutation were shown to have high
levels of HDL2
cholesterol.53 Our heterozygous carriers of HL
gene mutations probably have a more favorable lipid profile as a result
of a decreased activity of HL.53 54 55 56 57 58 59 Recently, variation
in the HL gene was shown to associate with HL activity and with HDL
triglycerides but not with VLDL
triglycerides.60 In agreement with the latter
study, we also did not observe an influence on VLDL
triglyceride levels. Zhang et al16 observed a
contribution of the LPL(Asn291Ser) sequence variation to type III
HLP: the mutation was observed in 4 of 17 apoE2 homozygotes with type
III HLP, which was significantly more than the 2 carriers in 230 Dutch
control subjects.16 In the present study, 2 carriers
of the LPL(Asn291Ser) mutation were observed among 25 patients with
type III HLP. The allele frequency (4.0%) in patients with type
III HLP in the present study did not differ significantly from the
allele frequency (11.8%) in apoE2 homozygotes of the study by
Zhang et al and the allele frequency (0.7%) of the Dutch control
subjects. Moreover, we also detected an apoE2 homozygote with the
LPL(Asn291Ser) sequence variation but without
hyperlipidemia. Nevertheless, meaningful conclusions
about this mutation cannot be drawn because of the small numbers in the
2 studies, and our findings do not exclude a contribution of other
mutations in lipase genes.
It has been shown previously that the presence of an SstI restriction site in exon 4 of the APOC3 gene is associated with hypertriglyceridemia.26 27 28 29 30 Therefore, we screened our study population for this polymorphism. Although the S2 allele itself was not associated with hypertriglyceridemia in our total study group, our data show a strong interaction between the polymorphism and hyperinsulinemia: we observed expression of severe type III HLP in hyperinsulinemic apoE2 homozygotes who had the SstI restriction site (S2). An increased production of VLDL due to insulin resistance may lead to hypertriglyceridemia when remnant metabolism fails to compensate. A decreased remnant clearance in apoE2 homozygotes, who have an increased production of apoC3, may then result in severe type III HLP. This susceptibility to accumulation of remnant particles in apoE2 homozygotes may prove to be a sensitive model for identifying additional genetic factors that are generally contributory to hyperlipidemia. In addition to endogenous hypertriglyceridemia without apoE2 homozygosity, these "second factors" may also contribute to the atherogenic lipid profile of familial combined hyperlipidemia, as suggested by the low levels of HDL in the hyperinsulinemic S2 carriers. We probably need to know the effect of such additional factors in the first place to allow analyses of the main causes of familial combined hyperlipidemia.61 62 63 Directly identifying additional factors in patients with endogenous hypertriglyceridemia or familial combined hyperlipidemia is much more complex than in apoE2 homozygotes because of the lack of normolipidemic controls within the affected individuals.
Dammerman et al24 found a susceptibility to
hypertriglyceridemia among carriers of the
SstI polymorphism (S2). Previously, we also observed
this relation with severe
hypertriglyceridemia.30
The study by Dammerman et al was probably performed in a
hypertriglyceridemic population that, in
addition to 11 hyperlipidemic apoE2 homozygotes,
consisted of a large number of patients with endogenous
hypertriglyceridemia. This effect of the
polymorphic site in patients with severe
hypertriglyceridemia may also be based on
an interaction with hyperinsulinemia. In the
present study, the SstI polymorphism was associated
with hypertriglyceridemia exclusively when
patients had hyperinsulinemia. Notably, the
carriers of this polymorphic site, who had low levels of fasting
insulin, tended to have even lower triglyceride
concentrations than the apoE2 homozygotes with the wild-type variant
(Table 4).
Because the SstI polymorphism is located in the 3' noncoding region of the APOC3 gene and thus does not lead to a change in the amino acid sequence of the protein, the possibility cannot be excluded that the effect found for the SstI polymorphism is due to another hitherto unknown mutation in the APOC3 gene or in another gene near the restriction site. Extended haplotyping and subgroup analysis with 2 MspI polymorphisms in the APOA1 gene, downstream, did not identify haplotypes associated with a higher risk of expression of type III HLP (data not shown).42
The combined effect of hyperinsulinemia and
an APOC3 haplotype may suggest that insulin influences the expression
of apoC3. Recently, 2 common variations of an insulin/phorbol ester
response element of the promoter region of the APOC3 gene were
described. In vitro studies showed increased expression of apoC3 for
both promoter variants due to a defective response to insulin at the
DNA level.41 In transgenic mice, such increased expression
of apoC3 causes
hypertriglyceridemia.64 In the
present study, the SstI polymorphism was found
almost exclusively in combination with the presence of the 2 promoter
variants. Such a strong linkage disequilibrium between the
SstI polymorphism and the 2 promoter variants is in
agreement with the findings of others.24 It could
therefore be argued that the observed effect of the SstI
polymorphism is due to 1 or both promoter variants. However, we
observed no effect of the 3 promoter alleles on the lipid levels.
This can be explained by the fact that although the S2 allele was
strictly associated with the P2 allele in the
hyperinsulinemic group, an even higher number of P2
alleles were found to be linked with the S1 allele (Table 5). This is in agreement with observations in the ARIC
population33 and in Italian school
children.32
In conclusion, the combination of hyperinsulinemia and the presence of the SstI polymorphism in the APOC3 gene results in the expression of severe type III HLP in apoE2 homozygotes. This finding enables a screening strategy to identify those apoE2 homozygotes at risk for developing type III HLP and subsequently premature coronary artery disease.
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Acknowledgments |
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This work was funded in part by the Praeventiefonds (#2822300).
Received September 12, 1998; accepted August 4, 1999.
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