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Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:923-928
Published online before print February 1, 2007, doi: 10.1161/01.ATV.0000258945.76141.8a
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:923.)
© 2007 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

High Frequency of a Retinoid X Receptor {gamma} Gene Variant in Familial Combined Hyperlipidemia That Associates With Atherogenic Dyslipidemia

Atsushi Nohara; Masa-aki Kawashiri; Thierry Claudel; Mihoko Mizuno; Masayuki Tsuchida; Mutsuko Takata; Shoji Katsuda; Kenji Miwa; Akihiro Inazu; Folkert Kuipers; Junji Kobayashi; Junji Koizumi; Masakazu Yamagishi; Hiroshi Mabuchi

From the Departments of Lipidology (A.N., M.M., J. Kobayashi, H.M.) and Cardiovascular Medicine (M.K., M. Tsuchida, M. Takata, S.K., K.M., M.Y.), Graduate School of Medical Science, Kanazawa University, Japan; Center for Liver, Digestive, and Metabolic Diseases, Laboratory of Pediatrics (T.C., F.K.), University Medical Center Groningen, Groningen, the Netherlands; School of Health Sciences, Faculty of Medicine (A.I.), and Department of General Medicine (J. Koizumi), Kanazawa University Hospital, Japan.

Correspondence to Atsushi Nohara, Department of Lipidology, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8641, Japan. E-mail a-nohara{at}med.kanazawa-u.ac.jp


*    Abstract
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 *Abstract
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Objective— The genetic background of familial combined hyperlipidemia (FCHL) has not been fully clarified. Because several nuclear receptors play pivotal roles in lipid metabolism, we tested the hypothesis that genetic variants of nuclear receptors contribute to FCHL.

Methods and Results— We screened all the coding regions of the PPAR{alpha}, PPAR{gamma}2, PPAR{delta}, FXR, LXR{alpha}, and RXR{gamma} genes in 180 hyperlipidemic patients including 60 FCHL probands. Clinical characteristics of the identified variants were evaluated in other 175 patients suspected of coronary disease. We identified PPAR{alpha} Asp140Asn and Gly395Glu, PPAR{gamma}2 Pro12Ala, RXR{gamma} Gly14Ser, and FXR –1g–>t variants. Only RXR{gamma} Ser14 was more frequent in FCHL (15%, P<0.05) than in other primary hyperlipidemia (4%) and in controls (5%). Among patients suspected of coronary disease, we identified 9 RXR{gamma} Ser14 carriers, who showed increased triglycerides (1.62±0.82 versus 1.91±0.42 [mean±SD] mmol/L, P<0.05), decreased HDL-cholesterol (1.32±0.41 versus 1.04±0.26, P<0.05), and decreased post-heparin plasma lipoprotein lipase protein levels (222±85 versus 149±38 ng/mL, P<0.01). In vitro, RXR{gamma} Ser14 showed significantly stronger repression of the lipoprotein lipase promoter than RXR{gamma} Gly14.

Conclusion— These findings suggest that RXR{gamma} contributes to the genetic background of FCHL.

We investigated prevalence of genetic variants of several nuclear receptors in FCHL. We found a specific RXR{gamma} variant to be significantly more frequent in FCHL and to be associated with atherogenic dyslipidemia and reduced lipoprotein lipase expression. We suggest a contribution of this RXR{gamma} variant to FCHL.


Key Words: apolipoproteins • gene mutations • lipoprotein lipase • familial combined hyperlipidemia • nuclear receptors


*    Introduction
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 *Introduction
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Familial combined hyperlipidemia (FCHL) is the most common form of inherited hyperlipidemia. FCHL shows strong genetic susceptibility resembling an autosomal dominant disease,1–3 but most of the underlying causal mechanisms remain to be elucidated. Lipoprotein lipase (LPL) has been implicated as one of the genes that modify the lipid phenotype in FCHL.4,5 "Intra-individual variability" of the lipoprotein phenotype is often included as a criterion in diagnosis.6 However, a recent prospective study of FCHL families suggests that this variability may even include normolipidemic periods in affected subjects.7 This feature indicates that FCHL could be a "disease of regulation" rather than a genetic defect in certain peripheral components of lipid metabolism.

Nuclear receptors are transcription factors that can be activated by specific ligands. Recent studies have shown that nuclear receptors, especially retinoid X receptor (RXR) and its heterodimerization partners,8 play important roles in maintenance of lipid homeostasis on their activation by a variety of ligands derived from dietary cholesterol and fatty acids.9 The peroxisome proliferator-activated receptors (PPARs) family, the oxysterol sensor liver X receptor (LXR), and the bile acid sensor farnesoid X receptor (FXR) are all involved in control of plasma lipid concentrations.10 Thus, we tested the hypothesis that variants of these nuclear receptors, ie, PPAR{alpha}, PPAR{gamma}2, PPAR{delta}, LXR{alpha}, FXR, and RXR{gamma}, could constitute part of the genetic background of atherogenic dyslipidemia, particularly of FCHL.


*    Methods
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 *Methods
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Subjects
The study design consists of 2 parts. First, we screened for frequent variants in the nuclear receptor candidate genes among 180 patients with primary hyperlipidemia, including 60 unrelated patients with FCHL (clinical characteristics are presented in supplemental Table I, available online at http://atvb.ahajounals.org). Patients with familial hypercholesterolemia and secondary hyperlipidemia were excluded. Diagnosis of FCHL was based on the fulfillment of all of the following three criteria: (1) Phenotype IIb, IIa, or IV hyperlipidemia according to the Fredrickson classification; (2) Presence of phenotype IIb, IIa, or IV hyperlipidemia in a first-degree relative and at least one family member with phenotype IIb; (3) Exclusion of familial hypercholesterolemia. Two hundred ninety-eight anonymous samples from healthy males were used as controls for frequency analysis of identified mutations. All blood samples in this study were obtained after an overnight fast.

Second, we evaluated the clinical impact of potentially relevant variants in another 175 patients who were suspected of having coronary artery disease based on any of the following reasons: ECG abnormalities, cumulative coronary risk factors, and/or chest symptoms. The group included 105 patients who had undergone coronary angiography. Patients with familial hypercholesterolemia were excluded because of their clear genetic background for hyperlipidemia. The extent and severity of atherosclerotic changes in coronary angiography were assessed by assigning scores to each of the 15 segments, according to the classification of the American Heart Association Grading Committee. The coronary stenosis index (CSI) was defined as the sum of the following scores11: A normal coronary angiogram was graded 0, stenosis of less than 25% was graded 1, 25% to 50% stenosis was graded 2, 50% to 75% stenosis was graded 3, and more than 75% stenosis was graded 4. CSI is a useful index for evaluating mild-moderate coronary atherosclerotic changes.

All the subjects and controls enrolled were inhabitants of the Hokuriku district of Japan. Written informed consent was obtained from each of the subjects. The study protocol was approved by the ethics committee of the Graduate School of Medical Science, Kanazawa University.

Laboratory Analyses
Total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL)-cholesterol, apolipoproteins, glucose, and thyroid hormones were measured according to standard clinical laboratory techniques. HDL-cholesterol fractions were obtained by dextran sulfate-magnesium chloride precipitation and assayed using a commercial kit (Daiichi, Tokyo, Japan).12 Separation of lipoproteins by ultracentrifugation was performed as described by Havel et al.13 Plasma remnant-like particle (RLP)-cholesterol was determined by immuno-absorption using the commercial RLP-C JIMRO kit.14 Plasma cholesteryl ester transfer protein (CETP) concentrations were determined by enzyme-linked immunosorbent assay using the monoclonal antibody TP2 and a rabbit polyclonal antibody raised against recombinant human CETP.15 For LPL assessment, blood samples were obtained 10 minutes after an intravenous injection of 30 IU heparin/kg body weight. LPL activity was measured using radio-labeled triolein emulsion after hepatic lipase (HL) inhibition by SDS as previously described.16 LPL mass was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibody against LPL (Daiichi Pure Chemicals Co Ltd, Tokyo, Japan).17

Genetic Analyses of Candidate Genes
Genomic DNA was isolated from peripheral white blood cells using standard phenol-chloroform extraction techniques. We screened all the coding regions of PPAR{alpha} (NM_032644), PPAR{delta} (NM_006238), PPAR{gamma}2 (NM_015869), LXR{alpha} (NM_005693), FXR (NM_005123), and RXR{gamma} (NM_006917) genes with flanking exon-intron boundaries by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) using the DCode system (Bio-Rad), which is highly accurate in detecting changes in nucleic acids.18 The structural organization and nucleotide sequences of these genes were retrieved from the gene database of NCBI. Lists of all GC-clamped primers used in DGGE analysis are available online (supplemental Table II). Samples with a variant detected by DGGE analysis were directly sequenced on an ABI310 analyzer (Applied Biosystems). PCR-restriction-fragment-length polymorphisms analysis on the RXR{gamma} Ser14 variant was performed with the primers 5'- AGCCGAGAGAGGCGGTAATA-3' (forward) and 5'-TACAGGTCCACGCAGTGAAG-3' (reverse) in patients suspected of coronary artery disease. Digestion with AluI resulted in a 76-bp fragment for Ser allele and a 120-bp fragment for Gly allele.

Cell Culture and Transfection Assays
Cos7 cells were grown in DMEM supplemented with 10% FCS, penicillin/streptomycin, sodium pyruvate, glutamine, and nonessential amino acids (Gibco BRL, Invitrogen). The medium was changed every 48 hours. Cos7 cells were transfected using FuGENE 6 reagent (Roche): 150 ng of the indicated LPL firefly luciferase reporter plasmid (a generous gift of Dr B. Staels, Institut Pasteur de Lille, France), that contains the proximal 466-bp of the human LPL promoter in front of the ATG cloned into the HindIII site of the pGL3 plasmid, was cotransfected with or without 100 ng of the human RXR{gamma} expressing vector (a generous gift of Dr W. Lamph, Ligand Pharmaceuticals Inc, San Diego, Calif). After an overnight incubation, cells were incubated with medium containing 10% FCS with or without the retinoid LGD1069, (1 µmol/L, Sigma) and luciferase activity was assayed 48 hours later using an Orion luminometer (Berthold). Transfection studies were performed at least 3 times in triplicate. Transfection efficiency was monitored by cotransfection of 150 ng of a SV40-driven ß-galactosidase expression plasmid. A positive RXRE TKpGL3 construct was made by cloning 3 copies of the direct repeat AGGTCA spaced by 5 nucleotides in the TKpGL3 plasmid.

Plasmid Site-Directed Mutagenesis
Nucleotide substitution was introduced in the plasmid expressing human RXR{gamma} using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, The Netherlands) and the primer 5'-CATGAAGTTTCCCGCAAGCTATGGAGGCTCCCCTGG C-3' in which the nucleotide in bold indicates the mutation.

Statistical Analysis
The frequency distribution of genotypes was compared using standard {chi}2 tests. Student t test was used for normally distributed parameters and the Kruskal–Wallis test was used for non-normally distributed parameters: triglycerides levels, LPL levels, and CSI. JMP 5.1.2 software (SAS Institute Inc) was used for statistical calculation.


*    Results
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 *Results
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Identified Variants in Nuclear Receptor Genes
With PCR-DGGE analysis, we identified 4 variants with amino acid changes, ie, Asp140Asn and Gly395Glu in the PPAR{alpha} gene, Pro12Ala in the PPAR{gamma}2 gene, Gly14Ser in the RXR{gamma} gene, and 1 nucleotide substitution in a flanking coding region, ie, FXR –1g–>t variant. The PPAR{gamma}2 Pro12Ala polymorphism has already been well-described,19 whereas the others represent novel variants identified in this study. In humans, variants in the RXR{gamma} gene have been associated with elevated triglyceride levels in familial type 2 diabetes, but none of these variants showed an altered coding sequence.20 Therefore, this is the first description of a RXR{gamma} variant with an amino acid substitution. In the PPAR{alpha} gene, the Leu162Val variant has been reported in Western countries,21 but this variant was not identified in this study. We also identified some silent nucleotide substitutions, ie, 891C–>G (rs13306747) and 1431C–>T (rs1724155) in the PPAR{gamma}2 gene, 1233C–>T (rs9658166) in the PPAR{delta} gene, and 1134A–>G (rs1131379) in the LXR{alpha} gene. We did not identify variants with amino acid changes in the PPAR{delta} and LXR{alpha} genes. We further investigated the variants with amino acid substitutions and the –1g–>t FXR variant, because of the likelihood that these induced altered physiological function.

Higher Frequency of RXR{gamma} Variant in FCHL
We evaluated the frequencies of the 5 identified polymorphisms in subjects with FCHL, subjects with other forms of primary hyperlipidemia and in the general population (Table 1). Only the RXR{gamma} Ser14 variant was found to be significantly more frequent in FCHL patients (15%) compared with that in other forms of primary hyperlipidemia (4%) or the general population (5%).


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  		TABLE 1. Frequencies of Nuclear Receptor Genes Variants Identified in This Study

Atherogenic Plasma Lipids Profiles and Coronary Atherosclerosis Associated With the RXR{gamma} Ser14 Variant
To establish the impact of the identified RXR{gamma} variant on metabolic parameters and on coronary atherosclerosis, we evaluated anthropometric parameters and laboratory data from 175 patients suspected of coronary disease. The RXR{gamma} Ser14 variant was identified in 9 patients, all of whom were heterozygotes. Eight of the RXR{gamma} Ser14 carriers had hyperlipidemia, while the remaining 1 demonstrated an isolated low HDL cholesterol level. Clinical characteristics of patients with or without the RXR{gamma} Ser14 allele are shown in Table 2. There was no difference in age or body mass index between the two groups. In their lipid profiles, RXR{gamma} Ser14 carriers had higher TG, lower HDL cholesterol especially in the HDL2 subfraction, and lower apolipoprotein A-II levels. There was no difference in CETP protein levels between the groups. Furthermore, we found that the RLP cholesterol level was significantly higher in the RXR{gamma} Ser14 carriers than in the wild-type. Subjects with this variant also showed significantly lower LPL activities and protein levels in post-heparin plasma. Separation of lipoproteins demonstrated that the Ser14 carriers had higher TG levels in very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions, higher cholesterol levels in VLDL, and lower cholesterol levels in HDL (supplemental Table III).


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  		TABLE 2. Clinical Characteristics of Patients With RXR{gamma} Variant

Two RXR{gamma} Ser14 carriers were diagnosed as FCHL (22%), and 2 additional carriers were suspected of FCHL with hyperlipidemic siblings without information on first-degree relatives. Among non-carriers, 22 of 166 patients were diagnosed as FCHL (13%). One hundred twenty-five patients suspected of coronary disease showed hyperlipidemia and the intraindividual variability of lipoprotein phenotype was significantly more frequent in RXR{gamma} Ser14 carriers (7 of 8 hyperlipidemic patients; 88%) than in wild-type (32 of 117 hyperlipidemic patients; 27%, Table 2).

There was no significant difference in the thyroid hormone levels between the two groups.

Four males and 1 female were identified as RXR{gamma} variant carriers among 105 patients who underwent coronary angiography. The carriers of RXR{gamma} Ser14 demonstrated significantly higher CSI than those with the wild-type (Table 2).

RXR{gamma} Variant Represses More Efficiently the LPL Promoter Activity
Because RXR{gamma} Ser14 carriers showed significantly lower LPL activities and protein levels in post-heparin plasma, we hypothesized that activated-RXR{gamma} downregulates LPL gene expression by a transcriptional mechanism and that RXR{gamma} variant is more effective in repressing the LPL promoter activity. Therefore, transfection assays were performed using the LPL promoter cotransfected with either wild-type RXR{gamma} or the variant (Figure). Interestingly, RXR{gamma} Gly14 significantly repressed (–40%) the LPL promoter activity, whereas the RXR{gamma} Ser14 repressed even more strongly (–60%, P<0.001, Figure A). Moreover, the RXR{gamma}Ser14 was a more potent activator of a positive RXRE cloned in front of a TKpGL3 plasmid (note the different scales in Figure B). Taken together, our results indicate that RXR{gamma} downregulates human LPL gene expression, at least partially by a transcriptional mechanism, and that the newly identified RXR{gamma} variant is a more potent repressor than the wild-type in this respect, as well as a more potent transactivator of a positive RXR response element.


Figure 1
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  		A, Cos7 cells were cotransfected with RXR{gamma} wild-type or the Ser14 variant and activated with retinoid in presence of the LPL promoter. B, Cos7 cells were cotransfected with RXR{gamma} wild-type or the Ser14 variant and activated with retinoid in presence of a positive RXRE cloned in the TKpGL3 plasmid.

Gain of Function Variant of PPAR{alpha} and Increased LDL-C Levels
The carriers of the PPAR{alpha} variant Gly395Glu tended to have higher frequency in the FCHL population, although not statistically significant. Four subjects were identified as PPAR{alpha} Glu395 carriers in the coronary artery disease-suspected group and showed significantly higher LDL-cholesterol levels (supplemental Table IV). On in vitro functional analysis, Glu395 showed a moderately but significantly increased transcriptional activity compared with wild-type PPAR{alpha} (supplemental Figure I, available online at http://atvb.ahajournals.org). The previously described Leu162Val variant of the PPAR{alpha} gene has been shown to give gain of function in in vitro,24 has been associated with raised LDL-cholesterol levels.21,22 Our results appear to be in accordance with these previous reports.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The main findings of the present study are the following: (1) identification of novel polymorphisms in plasma lipid levels-associated nuclear receptor genes, (2) a higher frequency of the RXR{gamma} gene variant Gly14Ser in subjects with FCHL, (3) RXR{gamma} Ser14 variant carriers showed more atherogenic dyslipidemia associated with coronary atherosclerosis, (4) the RXR{gamma} variant showed a stronger response to its ligand in repression of the LPL promoter than the wild-type RXR{gamma}.

RXRs are major heterodimerization partners of nuclear receptors such as PPARs, LXRs, and FXR. Three RXR isotypes have been identified: RXR{alpha}, RXRß, and RXR{gamma}. Synthetic RXR ligands induce hypertriglyceridemia through decreased clearance of VLDL by LPL-dependent pathways,23,24 except in 1 study.25 In contrast to the embryonic lethality observed in RXR{alpha}- and RXRß-deficient mice, RXR{gamma}-deficient mice develop apparently normal.26 Yet, RXR{gamma}-deficient mice showed reduced fasting plasma TG levels and increased skeletal muscle LPL activity when fed a high fat diet.27 The human RXR{gamma} gene is located on chromosome 1q21-q23, ie, the so-called "FCHL locus",28 and both linkage analysis and a twin study have indicated that the RXR{gamma} gene is linked with dyslipidemia in Chinese and German families.29,30

To our knowledge, there are only few data concerning the physiological roles and targets of RXR{gamma} in humans. The RXR{gamma} gene is mainly expressed in skeletal muscles, central nervous system, skin, intestine, and lung. In the present study, LPL protein mass and activity were significantly decreased in RXR{gamma} variant carriers. Because LPL is mainly expressed in adipose tissues and in skeletal muscles, we assume that this is attributable to the fact that the presence of the RXR{gamma} variant affects LPL expression in skeletal muscles. RXR{gamma} mRNA is detectable in adipose tissue only at a low level,31 but it has been reported that RXR{gamma} could replace RXR{alpha} in heterodimerization with PPAR{gamma} in adipose tissue.32 Therefore, there is a possibility that RXR{gamma} variant expression in adipose tissue contributes to the changes in LPL.

It has been reported that RXR{gamma}-deficient mice show a 17% increase in serum thyroid hormone (T4) and a 20% increase in thyroid-stimulating hormone (TSH) levels.33 In the present study, thyroid hormone levels did not appear to differ sufficiently between variant carriers and non-carriers to explain the differences observed in lipid levels.

It has been shown that low LPL levels contribute to disorders associated with TG-rich lipoprotein catabolism with low HDL, especially in HDL2,34,35 and are associated with increased risk for future coronary disease.36 Thus, the low LPL could well contribute to the increase in TG and the decrease in HDL-cholesterol levels in subjects with the RXR{gamma} variant.

We assessed the functional consequence of the RXR{gamma} Ser14 variant in vitro. The activation function-1 (AF-1) domain of RXR{gamma} is located between amino acids 1 and 103, and is required for optimal ligand-dependent transactivation of RXR response element.37 Fourteen amino acids are located within the AF-1 domain and are conserved among humans, mice, and chickens. In a transfection assay, RXR{gamma} Ser14 repressed LPL promoter activity more strongly than the wild-type RXR{gamma}. In addition, the Ser14 variant was a more potent inducer of a positive RXR response element. Therefore, we speculate that the Ser14 variant induces a better recruitment and/or stabilization of RXR cofactors. Further studies will be required to understand the precise molecular mechanism(s) involved in the LPL regulation by RXR{gamma} Ser14.

Within the so-called FCHL locus, on chromosome 1q21-q23, several genes have been reported to be associated with the FCHL phenotype28,30,38 and with type 2 diabetes.39 First, the thioredoxin interacting protein gene was shown to be associated with combined hyperlipidemia in mice, but no disease-causing mutation has been found in humans so far.40,41 Currently upstream stimulatory factor 1 (USF1) is considered the most promising candidate gene of FCHL.42 In the USF1 gene, no amino acid substitution has been identified in the coding regions, but single nucleotide polymorphisms in the 3'untranslated region and in intron 7 have been reported to be associated with FCHL, metabolic syndrome, or type 2 diabetes mellitus quite reproducibly.43–45 However, populations did not show any such association have also been reported.46–48 These reports emphasize the complexity of phenotypic expression in multi-factorial diseases such as FCHL. RXR{gamma} had been reported to show an association with TG and cholesterol levels on linkage analysis,29,30 and we identified novel RXR{gamma} variant that associated with atherogenic dyslipidemia. However, the changes in lipid levels attributable to the RXR{gamma} variant alone were not sufficient to cause FCHL. Thus, we suggest the RXR{gamma} gene variant to be a strong modifier rather than a causative gene in development of the FCHL phenotype.

In conclusion, the present study suggests that a variant of RXR{gamma} gene contributes to genetic dyslipidemia, including FCHL, based on the increased frequency of this variant in FCHL, its association with an atherogenic lipid profile, and initial functional studies.


*    Acknowledgments
 
The authors thank Sachio Yamamoto for technical assistance. Drs William W. Lamph and Bart Staels are kindly acknowledged for the generous gift of plasmids.

Sources of Funding

This work has been supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (No.17790603) and ONO Medical Research Foundation. Thierry Claudel was supported by Grant 2002B017 from the Netherlands Heart Foundation.

Disclosures

None.


*    Footnotes
 
Original received August 28 2006; final version accepted January 6, 2007.


*    References
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 *References
 

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