© 1997 American Heart Association, Inc.
Articles |
The Role of a Triplet Repeat Sequence of the Very Low Density Lipoprotein Receptor Gene in Plasma Lipid and Lipoprotein Level Variability in Humans
From the Service d'Epidémiologie et de Santé Publique—INSERM CJF 95-05, Institut Pasteur de Lille (N.H., V.C., P.A.); INSERM U 325, Institut Pasteur de Lille (J.D., J.-C.F.), Lille France; Laboratoire d'Epidémiologie et de Santé Publique, Strasbourg France (D.A.); Département d'Epidémiologie, Faculté de Médecine, Toulouse (J.-B.R.) France; Department of Epidemiology and Public Health, The Queen's University of Belfast, Belfast, Northern Ireland (A.E.); INSERM SC7, Paris, France (F.C.)
Correspondence to Pr Philippe Amouyel, Service d'Epidémiologie et de Santé Publique et INSERM, CJF 95-05, Institut Pasteur de Lille, 1 rue du Professeur Calmette, 59019 Lille Cedex, France.
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Abstract |
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Abstract The biological role of the very low density lipoprotein receptor (VLDL-R) in humans is not yet elucidated. This cellular receptor binds apolipoprotein E (apoE)-containing lipoparticles and is mainly expressed in peripheral tissues. The VLDL-R gene contains a polymorphic triplet (CGG) repeat located 19 bp upstream of the initiation codon. We explored the allelic distribution of this repeat in 1384 subjects of European Caucasian origin, 609 of them surviving a myocardial infarction. Six alleles corresponding to 5, 6, 7, 8, 9, and 11 repeats were detected in this population. The alleles 5, 8, and 9 were the most frequent, with frequencies of 0.413, 0.275, and 0.292, respectively. No association was found between the VLDL-R polymorphism and myocardial infarction. In controls without lipid lowering treatment, a statistically significant interaction between VLDL-R genotype and apoE phenotype was found for plasma triglycerides (P<.04), suggesting a gene-gene interaction. There was also a main effect of the VLDL-R polymorphism on LpE:B and LpA-I. The VLDL-R 9 allele was associated with lower levels of plasma LpE:B (P<.05) and higher concentrations of plasma LpA-I (P<.01) than the other alleles. These results suggest that VLDL-R has a modest influence on circulating lipoproteins in humans.
Key Words: VLDL receptor • triglyceride-rich lipoproteins • polymorphism • lipoprotein metabolism
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The VLDL-receptor (VLDL-R)1 belongs to the family of protein receptors that includes the LDL receptor,2 the LDL receptor-related protein/
2-macroglobulin receptor (LRP),3 the
kidney glycoprotein 330,4 a putative
G-protein-coupled receptor in Lymnaea
stagnalis,5 and the apoE receptor
2.6 Among these proteins, the VLDL-R is
structurally the most closely related to the LDL receptor (LDL-R). Its
amino acid sequence is highly uniform in a number of species. Compared
with the LDL-R, the ligand-binding domain of the VLDL-R contains an
extra cysteine-rich repeat that confers its specificity to the VLDL-R.
In vitro, the VLDL-R binds only apoE-containing lipoproteins. In
addition, the VLDL-R is essentially expressed in muscle and adipose
tissue, but not in the liver, suggesting that VLDL-R may be primarily
involved in triglyceride-rich lipoproteins (TRL)
metabolism in peripheral tissues. The human VLDL-R gene was cloned7 and located on the short arm of chromosome 9. Its gene structure is similar to that of the LDL-R gene. It differs by an additional exon that encodes for a cysteine-rich repeat. The 5'-untranslated region contains a polymorphic triplet (CGG) repeat sequence.7 To date, four to nine triplet repeat units have been described in random samples of population,8–10,19 resulting in as many alleles. Studies using the (CGG) triplet repeat as a genetic marker did not show any significant association between the VLDL-R polymorphism and dysbetalipoproteinemia, familial hypertriglyceridemia, familial combined lipemia, and type V hyperlipidemia.7 However, these studies were carried out in small samples without sufficient statistical power to definitely exclude any association. Furthermore, these disease-gene studies lacked the evidence of association with intermediate phenotypes, such as plasma lipoprotein levels.
In the last decade, evidence has accumulated that apolipoproteins play a critical role in lipoprotein metabolism. Apolipoprotein B is the structural component of VLDL, IDL, and LDL. ApoC-III and apoE at the surface of the B-containing lipoproteins modulate plasma enzyme activities and receptor interactions. These particles are triglyceride-rich lipoproteins (TRL) and bind to the LDL receptor of cultured cells.11 Their affinity for the LDL receptor depends on their relative contents in apoE and apoC-III.12
Although the VLDL-R functional properties are characterized in vitro, its physiological role in humans is not yet established. VLDL-R is expressed in adipose tissue, whereas lipoprotein clearance essentially takes place in the liver. Therefore, the question of the implication of the VLDL-R in TRL metabolism is important for the understanding of lipoprotein metabolism in the peripheral tissues. The goal of our study was to assess the association between VLDL-R gene polymorphism and plasma lipoprotein levels. Particular attention was paid to analyzing the particles that are enriched in apoE. These effects were further analyzed to account for the interactions with apoE phenotype, associated with functional differences in lipoprotein clearance.
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Methods |
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Subjects
The populations were selected in the course of a multicenter retrospective study of myocardial infarction, named ECTIM. This study was carried out between 1988 and 1991 in four European regions: Belfast (Northern Ireland), Toulouse (south of France), Strasbourg (Northeast of France), and Lille (north of France). Men between the ages of 25 and 64 were eligible: their family had to have been resident in the region for at least two generations and their four grandparents had to have been born in Europe. During the study period, all patients surviving myocardial infarction (MI) were eligible (n=609: 462 without lipid lowering drugs, 147 with lipid lowering drugs). Blood samples were obtained between 3 and 9 months after MI.
Controls were obtained from electoral rolls in France and from lists of general practitioners maintained by the Central Service Agency in Belfast (775 men: 723 without lipid lowering drugs, 52 with lipid lowering drugs). Stratification by age was used in the four study populations to match the age distribution of the control samples to that of cases in MI. Participants were examined at home or in clinics. A questionnaire was completed (personal history, presence of disease, drug intake); body weight and height were measured; 20 mL of blood was drawn on disodium EDTA.
Statistical analysis was conducted on controls without lipid lowering drugs. Two patients—having plasma triglyceride levels >12.5g/l—were excluded. This strategy equalized the variances of the lipid and lipoprotein variables tested in this work.
Lipid Analysis
A blood sample of 20 mL was drawn on EDTA
Na2 after the subjects had fasted for at least 10
hours, kept at 4°C, and centrifuged within 4 hours. After the
addition of conservative agents, the plasma was transported at 4°C to
the central laboratory in Lille, France, where all measurements were
performed within 5 days of venipuncture, as described
elsewhere.13 Human lipoprotein particles were
measured by a noncompetitive enzyme-linked immunosorbent assay
(sandwich ELISA) as described previously.14 The
bi-site ELISA assay allows for the determination of pairs of
apolipoproteins: C-III/B or E/B. The values are expressed as an amount
of apoB associated to either apoC-III or apoE. The intra- or
inter-assay coefficient variations (CV) were 8.7 and 11.1% for
LpC-III:B and 9.5 and 13.4% for LpE:B, respectively. Human
apoE15 and apoC-III16
levels were also measured by a noncompetitive enzyme-linked
immunosorbent assay (sandwich ELISA) as described previously. ApoA-I
and apoB were determined by immunonephelometry using the BNA system
(Behringwerke). Human LpA-I particles were measured by a differential
electroimmunoassay17 using commercially
available, ready-to-use plates from LpA-I hydragel kits (Sebia).
Genetic Analysis
Genomic DNA was prepared from white blood cells by
phenol-chloroform extraction. The (CGG)n repeat
genotype was detected for the 1384 subjects with PCR
amplification technique using previously described primer
pairs.7 PCR
amplifications18 were carried out using a Perkin
Elmer DNA Thermal Cycler model 4800 and Thermus aquaticus
polymerase (Gibco). After a first denaturing step at 95°C for 5
minutes, 30 cycles of amplification were performed as follows: in a
final volume of 50 µL, 200 ng of genomic DNA were mixed with 50
pmoles of each primer and 20 nmoles of each deoxynucleotide
triphosphate (Pharmacia) in the buffer recommended by the supplier
added with MgCl2 (1.5 mmol/L final).
Each cycle of the PCR reaction consisted of: 1 minute denaturation at
94°C, 1 minute annealing at 62°C followed by 1 minute elongation at
72°C. The amplified products were analyzed by
electrophoresis at 450V for 4 hours on 8%
acrylamide-bisacrylamide (19:1) gels stained by
ethidium bromide; pBR322/HaeIII DNA fragments (Appligene)
were used as molecular weight markers.
Statistical Analysis
The data were analyzed using the SAS statistical
software (release 6.10, SAS Institute Inc). Lipid and lipoprotein
levels were compared between genotypes by an analysis
of covariance. The tests were adjusted for age, sample origin,
BMI, tobacco, alcohol consumption and apoE phenotype.
Statistics were conducted on logarithmic variables for
triglycerides, VLDL-cholesterol, LpE:B and
LpC-III:B; results concerning these variables are expressed as
means at 95% confidence intervals.
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Results |
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VLDL-R Allele Frequencies
The study was carried out on European men between the ages of 25 and 64, recruited in the framework of the ECTIM Study, a case-control study intended to identify candidate genes predisposing to MI. The allelic distribution of the VLDL-R gene was assessed in the control group of this study (775 subjects; mean age 53.3±8.4 years). The VLDL-R gene locus was polymorphic with six different alleles corresponding to 5, 6, 7, 8, 9, and 11 (CGG) repeats, represented by 15 genotypes. The distribution of the genotypes was in Hardy-Weinberg equilibrium (Table 1
). The VLDL-R 5/5, 5/8, and
5/9 genotypes were the most frequent with each having an
average frequency of 20%. The 8/9 genotype was the second most
frequent at 17.4%. Each 8/8 and 9/9 genotype
represented an average frequency of 8%. The 5/6, 5/7,
5/11, 6/8, 6/9, 7/8, 7/9, 8/11, and 9/11 genotypes shared the
remaining 4% of the genotypic frequencies.
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Table 2 shows the corresponding
allele frequencies. Allele 5 was the most frequent allele
accounting for 41.3% of the whole sample. Alleles 8 and 9
represented each 27.5% and 29.2% of the sample
respectively. The frequency of the other
alleles6,7,11 was below 2% for each. The
allelic distribution of the VLDL-R polymorphism in the
ECTIM controls was significantly different from that of control
subjects of Japanese8,19 and
American10 (recruited in North Carolina) origin
(Table 2). The VLDL-R 5 allele frequency was low in the
North Carolina10 and the
Japanese8,19 samples. In addition, the 9 repeat
allele (VLDL-R 9) was rare in the Japanese populations.
In contrast, there was no difference of allelic distribution between
the ECTIM sample and another American sample9
(recruited in Iowa and Massachusetts).
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VLDL-R Interaction With ApoE
The association between VLDL-R genotypes and
the plasma lipid, lipoprotein and lipoparticle levels was assessed in
the control group of the ECTIM Study (n=723), excluding individuals
that were treated with lipid lowering drugs to avoid treatment bias.
Similarly, MI cases were excluded from this analysis to avoid
the possible confounding effects of cardiovascular
treatments (diet and drugs) on plasma lipid, lipoprotein, and
lipoparticle levels. To ensure enough subjects per group for the
statistical analyses, the calculations were restricted to
VLDL-R genotypes with a frequency greater than 5%.
The values were adjusted for age, body mass index (BMI), alcohol
consumption, cigarette smoking, and blood sample origin. Results are
presented in Table 3.
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A number of studies have demonstrated that the apoE phenotype
is an important source of plasma lipid, lipoprotein, and lipoparticle
level variability. Therefore, the data were analyzed including
apoE phenotypes (2/2, 2/3, 2/4, 3/3, 3/4, and 4/4) in the
model. Results are summarized in Table 3
. At the 0.05 level of
significance, an interaction between VLDL-R genotype
and apoE phenotype was found only for plasma
triglycerides (P<.04), suggesting that
there may be gene-gene interactions. In order to ensure a sufficient
number of subjects per group, the post hoc analysis of this
interaction was performed for the most frequent VLDL-R
genotypes (5/5, 5/8, 5/9) and apoE phenotypes (apoE
3/2, 3/3 and 3/4). Results are presented in Fig 1. This analysis showed no
difference in the mean value of triglycerides between
VLDL-R genotypes in the apoE 3/3 phenotype
subset. In contrast, in the apoE 3/2 group, the mean level of
triglycerides was lower in subjects bearing the
VLDL-R 8 or 9 allele than in those with the
VLDL-R 5 allele. Finally, in the apoE 3/4
phenotype group the mean level of triglycerides was
higher in individuals carrying the VLDL-R 8 or 9 allele
than in individuals homozygotes for the VLDL-R 5 allele.
A similar pattern was observed for LpC-III:B, although not reaching the
level of statistical significance.
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VLDL-R Principal Effect
There was evidence in this sample for a significant
VLDL-R genotype effect on LpE:B (P<.04)
and LpA-I (P<.04) levels. Individuals bearing at least one
VLDL-R 9 allele had lower levels of LpE:B than those
without this allele (XX: 0.38 [0.11 to 1.29]; 9X or 99: 0.35
[0.11 to 1.14] g/l; P<.05). Similarly, LpA-I
levels were significantly higher in subjects carrying at least one
VLDL-R 9 allele (XX: 0.47±0.13; 9X or 99: 0.49±0.14
g/l; P<.01).
VLDL-R and Hyperlipidemia, Myocardial Infarction,
and Body Fat
The relationship between the VLDL-R polymorphism
and plasma lipid and lipoprotein levels suggests that VLDL-R
may be a candidate gene for dyslipidemia and/or for
cardiovascular disease risk. Table 4 compares the allelic distribution of
VLDL-R (CGG) repeat in the control group between subjects
that were treated with lipid lowering drugs and those without such
treatment. The frequencies of VLDL-R 9 and VLDL-R
5 alleles were lower and higher, respectively, in treated patients
than in nontreated subjects, but the difference in VLDL-R
polymorphism distribution between treated and untreated controls
was not statistically significant (P=.054). Table 5 shows the allele distribution in
the ECTIM Study of cases with MI versus controls. There is no
statistically significant difference in the allele distribution
between cases and controls. Finally, since the VLDL-R gene
is expressed in adipose tissue, we tested the hypothesis that its
genetic variability was associated with differences in fat deposition
in peripheral tissues. Body fat was assessed using body
mass index. There was no statistically significant difference in body
mass index among VLDL-R genotypes either in the
control group or in the whole ECTIM sample (data not shown).
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Discussion |
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The VLDL-R belongs to the family of lipoprotein receptors and shares many structural domains with the LDL receptor.20 The physiological properties of the VLDL-R in humans are unknown. Since VLDL-R binds the apoE-containing lipoproteins, VLDL, IDL, and ß-VLDL,21,22 it is a candidate gene for study of its influence on the variability of these lipoprotein levels in humans. Indeed, VLDL-R gene polymorphism explains partially plasma triglycerides and variability in LpE:B and LpA-I levels, suggesting that VLDL-R may be implicated in triglyceride metabolism in humans. In the present sample, this association is less marked than that of the apoE phenotype. In addition, no statistically significant association was found between the VLDL-R polymorphism and myocardial infarction.
The results of the present study demonstrated the predominance of the 5, 8 and 9 (CGG) repeats at the genetic locus of VLDL-R in a sample of Europeans. The least frequent alleles have 4, 6, 7, 10 and 11 (CGG) repeats and are found in less than 3% of the study participants. The relative frequencies of VLDL-R alleles are remarkably different in Japanese and Europeans. These differences may be the consequence of the random genetic drift of a mutant allele that is neutral with respect to reproduction or may also result from a balance between selective forces. The probability that VLDL-R allele frequencies differences between Japanese and Caucasian Europeans result from a genetic drift or a new mutation is plausible.23 Such differences have been found in the blood group or in genes implicated in lipoprotein metabolism.24
On the basis of tissue expression,25 it has been proposed that the VLDL-R may have a central role in fat deposition in peripheral tissues.26 However, recent studies in mice deficient in LDL-R have shown that adenovirus-mediated gene transfer of the VLDL-R gene was accompanied by a lowering in the plasma apoE and apoB concentrations.27,28 The plasma lipoprotein fraction most affected by VLDL-R gene transfer was IDL, the clearance of which was clearly accelerated after treatment. These experiments demonstrated that apoE-enriched lipoproteins are important ligands for VLDL-R in vivo in rodents. One limitation, however, to the extension of these observations to the human metabolism is that adenovirus-mediated VLDL-R gene transfer leads to the preferential expression of VLDL-R in the liver, an organ where the processing of apoE-rich lipoproteins may differ substantially from that of peripheral tissues. We addressed the question of a potential implication of the VLDL-R in lipoprotein metabolism in humans by assessing the possible association between VLDL-R genetic polymorphism and circulating lipoprotein levels. Considering that apoE is the main ligand of VLDL-R and that apoE phenotype modulates lipoprotein binding to lipoprotein receptors, it was necessary to account for potential interactions between VLDL-R genotypes and apoE phenotypes. Accordingly, VLDL-R gene polymorphism was associated with plasma triglycerides level variability, suggesting that VLDL-R and apoE may interact to regulate TRL metabolism in humans.
In the present sample, the VLDL-R genotype was associated with plasma LpA-I level variability. Lipoparticles containing ApoA-I belong to plasma high density lipoproteins (HDL), which are heterogeneous in terms of size, lipid composition, and apolipoprotein content. LpA-I (particles containing apoA-I as the sole apolipoprotein) and LpA-I:A-II (particles containing apoA-II in addition to apoA-I) are two major HDL subclasses. The relationship between VLDL-R polymorphism and LpA-I plasma levels may appear unexpected since HDLs do not bind in vitro to the VLDL-R.29 This discrepancy is only apparent, since the plasma metabolism of VLDL and HDL are closely linked.30,31 Changes in circulating VLDL are usually associated with reciprocal changes in HDL, LpA-I, and LpA-I:A-II levels.32 Therefore, the association between VLDL-R gene polymorphism and LpA-I levels is very likely indirect, depending on an alteration of VLDL or apoE-rich lipoprotein metabolism.
The decrease in VLDL-R 9 allele frequency in subjects receiving lipid lowering drugs is consistent with the variation of plasma triglycerides and of LpE:B and LpA-I levels observed in controls. This suggests that the occurrence of dyslipidemia is related in part to the influence of the VLDL-R on lipid metabolism. However, this hypothesis needs to be confirmed in appropriate studies. Moreover, no association between VLDL-R genotype and MI was detected in the present study, suggesting that the VLDL-R polymorphism is not a genetic marker for MI.
The VLDL-R gene polymorphism used in this study concerns a (CGG) triplet repeat, a sequence that is highly polymorphic in the normal population and is thought to be unstable. Expansions of triplet repeat sequences have been linked to heritable genetic diseases in humans.33 Different conformations have been proposed for the (CGG) repeat: antiparallel double helix of base triads;34 aggregates of G-quadruplexes;35 tetrahelical structures,36 or intramolecular hairpins.37,38 These nonB structures can interfere with the VLDL-R gene transcription. Although this hypothesis seems very attractive in explaining the association between VLDL-R polymorphism and TRL level, it cannot be excluded that VLDL-R polymorphism acts only as a genetic marker in linkage disequilibrium with a mutation on the coding sequence of the protein.
In conclusion, the results of the present study demonstrated a modest association between VLDL-R gene polymorphism and plasma triglycerides, LpE:B and LpA-I levels. This finding suggests that VLDL-R may be physiologically implicated in TRL metabolism in humans, although the impact of this polymorphism appears less potent than that of apoE phenotype.
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Acknowledgments |
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The ECTIM Study was supported by grants from the Squibb and Parke-Davis Laboratories, the Mutuelle Générale de l'Education Nationale, the British Heart Foundation, the Institut National de la Santé et de la Recherche Médicale, and the Institut Pasteur de Lille.
Received February 14, 1997; accepted July 22, 1997.
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