Rat model of familial combined hyperlipidemia as a result of comparative mapping

Takahiro Ueno1, Johanne Tremblay1, Jaroslav Kunes2, Josef Zicha2, Zdenka Dobesova2, Zdenka Pausova1, Alan Y. Deng1, Yu-Lin Sun1, Howard J. Jacob3 and Pavel Hamet1

1 Centre de recherche du Centre hospitalier de l’Université de Montréal, Montreal, Quebec, Canada
2 Institute of Physiology, Czech Academy of Sciences, Czech Republic
3 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Total genome scan was carried out in 266 F2 intercrosses from the Prague hypertriglyceridemic (HTG) rat that shares several clinical characteristics with human metabolic syndrome. Two loci for plasma triglycerides (TG) were localized on chromosome 2 (Chr 2) (LOD 4.4, 3.2). The first locus overlapped with the rat syntenic region of the human locus for the metabolic syndrome and for small, dense LDL, while the second overlapped with the syntenic region of another locus for small, dense LDL in humans by the comparative mapping approach. Loci for TG on rat Chr 13 (LOD 3.3) and Chr 1 (LOD 2.7) overlapped with the syntenic region of loci for human familial combined hyperlipidemia (FCHL) in Finnish and Dutch populations, respectively. The concordances of loci for TG localized in this study with previously reported loci for FCHL and its related phenotypes are underlying the generalized importance of these loci in dyslipidemia. These data suggest the close relationship between dyslipidemia in HTG rats and human FCHL, establishing a novel animal model for exploration of pathophysiology and therapy based on genomic determinants.

Prague hypertriglyceridemic rat; quantitative trait locus; triglycerides; cholesterol; metabolic syndrome


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE METABOLIC SYNDROME is one of the potential targets of therapy in the primary prevention of coronary heart disease characterized by abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, and insulin resistance (17, 50) present in more than 20% of the US adult population (40). Although the metabolic syndrome is a broad category, it may include several distinct genetic diseases that share similar clinical features.

Familial combined hyperlipidemia (FCHL) was originally described in the early 1970s (19, 52). The affected members of FCHL families present different lipid phenotypes: hypercholesterolemia, hypertriglyceridemia, or combined hyperlipidemia and high serum apolipoprotein B. FCHL is also known to share features of the metabolic syndrome in higher prevalence compared with the general population, which include insulin resistance, obesity, and hypertension (2, 25, 48). Therefore, individuals with FCHL seem to form, at least partially, a subset of the metabolic syndrome. The metabolic defect in lipoprotein metabolism is associated with a predominance of small, dense LDL particles (6, 23, 24) and appears to be a consequence of hepatic apolipoprotein B-100 overproduction (60). FCHL is the most common of the dyslipidemias, with 1% to 2% prevalence in the general population and in up to 20% of patients with premature coronary heart disease (4).

Genetic studies of FCHL have been complicated by uncertain phenotype definition, genetic heterogeneity, and unknown modes of inheritance. In Finnish FCHL families, genome-wide scan revealed a significant locus for the FCHL trait on chromosome 1q21–23 (Chr 1q21–23), between but not including the flanking apolipoprotein A-II gene and the P, L, and E selectin genes (38, 39). In Dutch FCHL families, a significant locus for FCHL on Chr 11p was determined by a two-step genome scan approach (5). Candidate gene studies have provided evidence that common variations of many genes, including lipoprotein lipase (7, 66), protein AI-CIII-AIV cluster (6365), fatty acid binding protein 2 (45), hormone-sensitive lipase (47), hepatic lipase (43), ß3-adrenergic receptor and uncoupling protein 1 (46), as well as peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) (44), can influence lipid levels in affected individuals, but none of these genes has been found to be a primary determinant. Numerous factors, such as a lack of unequivocal diagnostic criteria, impact of the environment, and age dependence of the lipid phenotype, are increasing the heterogeneity of affected subjects (21, 22, 41).

Physiological genomics approaches to cardiovascular disease include single and complex genetic manipulations (18). One of the most productive approaches to the study of FCHL is the development of animal models that closely resemble both the clinical and genetic features of this disease. Currently, there are three animal models of FCHL: the Hyplip1 mutant mouse (8, 37), the LDLR1/APOC3/CETP combined transgenic mouse (35), and the St. Thomas Hospital rabbit (14). However, these models have not been reported to share genetic abnormalities with human FCHL. The Prague hypertriglyceridemic (HTG) rat is a novel strain that develops features of human FCHL, such as hypertriglyceridemia, age-dependent changes of plasma total cholesterol, and overproduction of triglyceride-rich lipoproteins (31, 55, 56, 61, 62), as well as of human metabolic syndrome and hypertension. The use of rodent models for complex disorders has considerably advanced our understanding of polygenic diseases (32, 58). Until recently, the utility of the comparative genomics approach was limited by the lack of rodent and human dense, gene-based maps. However, with progress in human, mouse, and rat genome projects, dense maps now exist that allow the construction of comparative maps for these models and the human genome. The purpose of this study was to investigate the relationship of the genetic backgrounds of the HTG strain and human FCHL using comparative genomics.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal procedure.
HTG were originally derived from a colony of Wistar rats (61), and their characteristics have been described previously (55). This rat strain develops several major features of the metabolic syndrome, such as hypertension, hypertriglyceridemia, hyperinsulinemia, and impaired glucose tolerance (56, 62).

Lewis and HTG rats were reciprocally mated to produce F1 hybrids. Females and males of the F1 generation were randomly mated to provide F2 cohorts (31).

All the animals used in this study were housed under standard laboratory conditions (23 ± 1°C, 12-h light-dark cycle) and fed standard laboratory chow as well as tap water ad libitum. Under light ether anesthesia, polyethylene catheters were inserted into the left carotid artery and jugular vein and exteriorized in the interscapular region. Blood pressure was recorded in conscious animals after 24-h recovery. The procedures and experimental protocols were approved by the local animal Ethics Committee of the Institute of Physiology, Academy of Sciences of the Czech Republic. Body weight, plasma triglycerides, plasma cholesterol, and systolic and diastolic blood pressure traits of the progenitors and 266 (137 male, 129 female) F2 hybrids were measured at the age of 5–6 mo.

Genotyping and mapping.
A two-step genome-wide scan was performed using markers based on simple sequence length polymorphisms (SSLP). The aim of the first step was to create a genetic map in the HTG x Lewis cross and to identify chromosomal regions of interest, while the second step comprised detailed mapping of these regions as used previously for blood pressure determinants (58). The first step was carried out as follows: 135 SSLP markers covering 21 chromosomes were selected to provide a genomic map with an intermarker average distance of ~20 cM. This distance was expected to limit the number of false-negative results, as previous theoretical computations have suggested (3, 13). To increase the efficacy of the first map, an approach was adopted to select animals with extreme phenotypes; 46 F2 rats, representing extreme values for plasma triglycerides, total cholesterol, and mean arterial pressure, were chosen. This approach maximized genetic contrast and potential linkage data. Complete genome-wide study allowed us to build a genetic linkage map for our cross and provided preliminary mapping information with greater efficiency than scanning in all F2 rats (11, 12).

The second step was performed as follows: chromosomal regions of interest were identified on the basis of a logarithm of likelihood (LOD) >1.0. For these regions, the density of the markers was augmented to reach an intermarker distance of less than 10 cM. The number of genotyped animals was also increased to comprise all 266 F2 hybrids for 137 of 191 markers, including all markers on Chr 1, 2, 5, 8, 12, 13, and 16. Finally, 191 markers covered the genetic map length of 1,634.9 cM, and the averaged genetic distance between adjacent markers was 10.5 cM. The average distance in the region of interest was 6.1 cM.

SSLP marker information and mapping data were taken from the Whitehead Institute/Massachusetts Institute of Technology Rat Database (Cambridge, MA; http://www.ratmap.gen.gu.se), Mouse Genome Informatics/Jackson Laboratory (Bar Harbor, ME; www.informatics.jax.org), the Wellcome Trust Centre for Human Genetics (Oxford, UK; http://www.well.ox.ac.uk), the Rat Genome Database (RGD)/Medical College of Wisconsin (Milwaukee, WI; http://www.rgd.mcw.edu), the National Center for Biotechnology Information (NCBI)/National Library of Medicine (Bethesda, MD; http://www.ncbi.nlm.nih.gov), and the Journal of Clinical Investigation (16).

Statistical analysis.
The normality of all phenotypes was examined by application of the Kolmogorov-Smirnov test. Phenotypes that did not pass the normality test were then corrected by log transformation. The significance of differences within and between groups was determined by one-way analysis of variance (ANOVA), followed by Tukey multiple comparison tests. Construction of linkage maps and quantitative trait locus (QTL) mapping were achieved with the Map Manager QT program (Version 3.0b; Ref. 34). The significance of each potential association was measured by likelihood ratio statistics (LRS). Then, LRS were converted to conventional base-10 LOD scores by division with 4.61. Each of the traits reported here was evaluated as "model free." A permutation test, randomly assigning phenotypes relative to genotypes in 10,000 replicated tests, was used to determine the threshold of significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Clinical phenotypes in parental and F2 populations.
As summarized in Table 1, plasma triglyceride levels were significantly higher in HTG rats than in Lewis rats for both sexes. Plasma total cholesterol levels were lower in HTG than in Lewis rats but significantly lower only in females. Plasma triglycerides and total cholesterol levels were higher in HTG males than in HTG females, but these differences did not reach statistical significance. Plasma triglycerides and total cholesterol levels were significantly lower in Lewis males than in Lewis females. HTG rats showed significantly higher systolic blood pressure compared with Lewis rats for both sexes and diastolic blood pressure in males. However, in the F2 population, plasma triglycerides and blood pressure levels were not significantly different between males and females.


View this table:
[in this window]
[in a new window]
 
Table 1. Plasma lipids, blood pressures, and body weight in HTG and Lewis progenitors and F2 hybrids

 
QTL on rat Chr 2.
In the whole rat F2 population, the most significant QTL for contributing to plasma triglycerides with significant linkage by the permutation test was located on Chr 2 between D2Rat182 and D2Mit8 (LOD 4.4) (Fig. 1A). At the nearest marker from this peak (D2Rat210), the homozygous HTG genotype (HH) was associated with significantly higher plasma triglycerides than the homozygous Lewis genotype (LL) and the heterozygous genotype (HL) (ANOVA P = 0.0001, Tukey/Kramer P < 0.05) (Table 2). Another QTL for plasma triglycerides was located on Chr 2, between D2Rat55 and D2Rat111 (LOD 3.1) (Fig. 1A). At this locus, rats with the HH genotype also showed significantly higher plasma triglyceride levels than rats with LL and HL genotypes (ANOVA P = 0.0035, Tukey/Kramer P < 0.05) (Table 2). The region between D2Rat136 and D2Mgh9 presented suggestive linkage for body weight in the whole F2 population (LOD 3.2) (Fig. 1A). At D2Rat40, rats with HH and HL genotypes had significantly greater body weight than rats with the LL genotype (ANOVA P = 0.0013, Tukey/Kramer P < 0.05) (Table 2). All three loci on Chr 2 were sex-specific, and we could localize them in the whole F2 and female populations.



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1. A: quantitative trait locus (QTL) plots for plasma triglycerides (solid line) and body weight (gray, dotted line) on rat chromosome 2 (Chr 2). The significance of each potential association is measured by logarithm of likelihood (LOD). *Significant linkage and {dagger}suggestive linkage of the QTL for each phenotype after the permutation test. B: comparative map between rat Chr 2, mouse Chr 13 and 3, and human Chr 5, 8, 3, 1, and 4. The bar for rat QTL in the HTG x Lewis map shows a region with the above suggestive level of LOD by the permutation test for each phenotype. Scale bar = 10 cM in the HTG x Lew map. Sources of the established maps are as follows: the Rat Genome Database (RGD)/Medical College of Wisconsin (Milwaukee, WI; http://www.rgd.mcw.edu); the Mouse Genome Database (MGD)/Jackson Laboratory (Bar Harbor, ME; http://www.informatics.jax.org); and National Center for Biotechnology Information (NCBI)/National Library of Medicine (Bethesda, MD; http://www.ncbi.nlm.nih.gov). Ccnb1, cyclin B1; Pik3r1, phosphoinositide-3-kinase, regulatory unit, polypeptide 1; Gzmk, granzyme; Cyp7b1, oxysterol 7{alpha}-hydroxylase; Slc2a2, solute carrier family 2A2 (facilitated glucose transporter); Fgf2, fibroblast growth factor 2; Mme, membrane metalloendopeptidase; Il17, interleukin 17; Gca (NPR1), natriuretic peptide receptor A/guanylate cyclase A; Hsd3b1, hydroxysteroid dehydrogenase-6, delta5-3-ß; Tshb, thyroid stimulating hormone, ß-subunit; Ngfb, nerve growth factor ß; Adora3, adenosine A3 receptor; Vcam1, vascular cell adhesion molecule 1; F3, coagulation factor 3; Glclr(Gclm), glutamate-cysteine ligase, modifier subunit; Nfkb1, nuclear factor of {kappa}-light polypeptide gene enhancer; Egf, epidermal growth factor; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Cenph, kinetochore protein CENP-H; Hmgcs, 3-hydroxy-3-methylglutaryl-CoA synthase; Hey1, hairy/enhancer-of-split related to the YRPW motif; Pmp2, peripheral myelin protein; Car1, carbonic anhydrase I; Car2, carbonic anhydrase II; Pld1, phospholipase D1; Pik3ca, phosphoinositide-3-kinase, catalytic, {alpha}-polypeptide; Cpa3, carboxypeptidase A3; FABP4, fatty acid binding protein 4; Anxa5, annexin 5; Ccna2, cyclin A2; Il2, interleukin 2; Il12a, interleukin 12a; Mtx, metaxin; Notch2, notch gene homolog 2; Atp1a1, ATPase, Na+-K+ transporting, {alpha}-subunit; GJA8, gap junction membrane channel protein {alpha}8; Fabp2, intestinal fatty acid binding protein; Abcd3, ATP-binding cassette, subfamily D, member 3; Abca4, ATP-binding cassette, subfamily A, member 4; Mtp, microsomal triglyceride transfer protein; ELF2, E74-like factor 2; CDS1, CDP-diacylglycerol synthase.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Mean values of phenotypes in each genotype at the nearest marker from peak LOD. All F2 rats (n = 266)

 
These two loci for plasma triglycerides on rat Chr 2 are novel loci and did not overlap with any previously reported rat loci for lipid phenotypes. No significant or suggestive loci were localized for plasma total cholesterol on this Chr. To examine the relationship between the located QTL of this novel strain of experimental rats and previously located loci for human metabolic syndrome, FCHL, and their related phenotypes, we constructed an interspecies map between rat, mouse, and human chromosomes based on our QTL. Figure 1B shows this interspecies map of identified loci for plasma triglycerides on rat Chr 2. In these regions, many genes were mapped on the RGD radiation hybrid (RH) map. They were also mapped on the Mouse Genome Database (MGD) RH map on mouse Chr 13 or 3 and on the NCBI human gene sequence map on Chr 5, 8, 3, 1, or 4. The region between D2Rat19 and D2Rat136 showed above significant or suggestive linkage with plasma triglycerides and body weight in the whole F2 and female populations. The syntenic region of this locus on the human Chr overlapped with previously reported loci for human metabolic syndrome (24) and for cholesterol levels of small, dense LDL on human Chr 3 (49). The human syntenic region of another locus for plasma triglycerides on rat Chr 2 located between D2Rat55 and D2Rat111 overlapped with a previously reported locus for cholesterol level of small, dense LDL (49).

QTL on rat Chr 13.
QTL with significant linkage after the 10,000 permutation test were found for plasma triglycerides on Chr 13 between D13Wox8 and D13Mit4 (LOD 3.3) in our whole F2 population (Fig. 2A). At this locus, rats with HH and HL genotypes showed significantly higher plasma triglyceride levels than rats with the LL genotype (ANOVA P = 0.0011, Tukey/Kramer P < 0.05) (Table 2). Suggestive linkage for plasma triglycerides was localized in this region for the female population but not for males by the interspecies map (Fig. 2A). Genes in this region of the RGD RH map were mapped on Chr 1 of the mouse MGD RH map and Chr 1 of the NCBI human gene sequence map (Fig. 2B). In the human syntenic region of this locus, significant evidence of linkage with FCHL phenotypes was localized in Finnish FCHL patients (38). This QTL region did not show linkage with any other phenotypes examined.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2. A: QTL plots for plasma triglycerides on rat Chr 13. The significance of each potential association is measured by LOD. *Significant linkage and {dagger}suggestive linkage of the QTL for each phenotype after the permutation test. B: comparative map between rat Chr 13, mouse Chr 1, and human Chr 1. The bar for rat QTL in the HTG x Lewis map shows a region with above suggestive level of LOD by the permutation test for each phenotype. Scale bar = 10 cM in the HTG x Lew map. See legend to Fig. 1 for the sources of the established maps. Rxrg, retinoid X receptor, {gamma}; Selp, P selectin; Cd3z, CD3 antigen, {zeta}-polypeptide; Rgs4, regulator of G protein signaling 4; Drd2, discoidin domain receptor family, member 2; Pigm, phosphatidylinositol glycan, class M; Fcer1a, Fc receptor, IgE, high-affinity I, {alpha}-polypeptide; Sele, E selectin; Sell, L selectin; Pou2f1, POU domain, class 2, transcription factor 1; Pbx1, pre-ß cell leukemia transcription factor 1; Apoa2, apolipoprotein AII; Dfy (fy), Duffy blood group; Crp, C reactive protein; NR1I3, nuclear receptor subfamily 1, group 1, member 3; Kcnj9, potassium inwardly-rectifying channel, subfamily J, member 9.

 
QTL on rat Chr 1.
Suggestive linkage for plasma triglycerides was localized only in the male population on Chr 1 (between D1Rat64 and D1Rat71: LOD 2.7). This locus also showed suggestive linkage with plasma total cholesterol only in the male population (LOD 2.5). Another locus for plasma total cholesterol was determined to have significant linkage in the male population between D1Rat137 and D1Rat35 (LOD 4.6). This locus showed suggestive linkage with plasma total cholesterol in the whole F2 population (Fig. 3A). Genes mapped in this region of the rat RGD RH map were mapped on Chr 7 or 19 of the mouse MGD RH map and Chr 16, 10, or 11 of the human NCBI gene sequence map (Fig. 3B). Part of the human syntenic region of this locus overlapped with a previously reported human FCHL locus in the Dutch population (5).



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 3. A: QTL plots for plasma triglycerides (solid line) and total cholesterol (gray, dotted line) on rat Chr 1. The significance of each potential association is measured by LOD. *Significant linkage and {dagger}suggestive linkage of the QTL for each phenotype after the permutation test. B: comparative map between rat Chr 1, mouse Chr 7 and 19, and human Chr 16, 10, and 11. The bar for rat QTL in the HTG x Lewis map shows a region with the above suggestive level of LOD by the permutation test for each phenotype. Scale bar = 10 cM in the HTG x Lew map. See legend to Fig. 1 for the sources of the established maps. PST-1 (Sult1a1), minoxidil sulfotransferase; Spn, sialophorin (gpL115, leukosianin, CD43); Lat, linker for activation of T-cells; Cox6a2, cytochrome c oxidase subunit VIa polypeptide 2; Itgax, integrin-{alpha} X (Cd11c); Aldoa, aldolase A; Sglt2 (SLC5A2), low-affinity Na-dependent glucose transporter; Mgmt, O6-methylguanine-DNA methyltranferase; Oat, ornithine aminotransferase; Cox8h, cytochrome c oxidase subunit VIII-H; Drd4, dopamine receptor D4; Igf2, insulin-like growth factor II; Ins2, insulin 2; Cd81, CD81 antigen (target of antiproliferative antibody 1); Cpt1a, carnitine palmitoyltransferase 1{alpha}; Cttnb (EMS1), cortactin isoform B; Adrbk1, adrenergic receptor kinase, ß 1; Gstp, glutathione-S-transferase; Pc, pyruvate carboxylase; Mdu1 (Slc3a2), antigen identified by monoclonal antibodies 4F2; Hrev107 (HRASLS3), hras-revertant gene 107; Plcb3, phospholipase C, ß3; Cntf, ciliary neurotropic factor; Aqp8, aquaporin 8; Coro1a, coronin, actin-binding protein 1A; Fgfr2, fibroblast growth factor receptor 2; Cyp2e1, cytochrome P450, 2e1, ethanol inducible; Rrm, ribonucleotide reductase M1 polypeptide; Fadd, fas-associating protein with death domain; Hras1, Harvey rat sarcoma virus oncogene; Map3k11, mitogen-activated protein kinase kinase kinase 11; Capn1, calpain 1; Ddb1, damage-specific DNA-binding protein 1; Osbp, oxysterol-binding protein; Ms4a1, membrane-spanning 4-domains, subfamily A, member 1; Rom1, retinal outer segment membrane protein 1.

 
QTL on rat Chr 5 and 12.
For plasma total cholesterol, two significant QTL were localized on Chr 5 (between D5Rat147 and D5Rat49: LOD 5.6) and on Chr 12 (between D12Rat12 and D12Rat40: LOD 3.8) in the whole F2 population. At these loci, plasma total cholesterol levels in rats with the HH genotype were significantly lower than in those with HL or LL genotypes. These negatively contributing loci were also sex- specific, as the locus on Chr 5 was significant in the whole F2 and female populations while the locus on Chr 12 was relevant on the whole F2 and male populations (Table 2).

Another locus on Chr 5 suggestively linked with plasma triglyceride levels, and rats with the HH genotype at this locus showed significantly higher plasma triglyceride levels than rats with LL or HL genotypes (ANOVA P = 0.0035, Tukey/Kramer P < 0.05). This locus presented suggestive linkage in the whole F2 population and in females (Table 2).

All the cosegregation results tested by one-way ANOVA were confirmatory to the effects of genotypes on each phenotypic trait as obtained by Map Manager (Table 2).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Dense gene maps were established recently for the rat by the RGD and for the mouse by MGD. The outcome of the human genome project has enabled construction of the human genome map, currently available from the NCBI. These maps are applicable as translations to compare the results of quantitative genetics on rat chromosomes with previously established loci on human chromosomes. Recently, Stoll et al. (57) reported a strategy to apply data from rat quantitative trait genetics to the human genome, using a genomic-systems biology map for cardiovascular functions. Identification of the overlapping regions of loci for similar traits between humans and other species by comparative mapping should empower us to find animal models for human complex trait diseases as well as to narrow down target regions during steps of the positional cloning of candidate genes. Such is the case in the present study.

The metabolic syndrome, characterized by abdominal obesity, atherogenic dyslipidemia, heightened blood pressure, insulin resistance, and prothrombotic and proinflammatory states, has been recognized as an important target of risk-reduction therapy of coronary artery disease (CAD) (20). Human FCHL is also known to increase the risk for CAD, expression of diverse lipid abnormalities in the same family (19, 36, 52), and sometimes, to associate insulin resistance and hypertension (51). Because of these similarities with clinical features, at least a part of FCHL patients are diagnosed as having metabolic syndrome. The search for causal genes of the metabolic syndrome in human studies has been very difficult because of its complex, heterogeneous, and multifactorial nature resulting from the interplay of genetic and environmental factors (15, 54). Several strains of animals have been reported as models for human metabolic syndrome or FCHL, and several loci for metabolic phenotypes have been proposed (1, 2, 8, 14, 28).

The HTG rat is a novel strain expressing hereditary hypertriglyceridemia and overproduction of lipoprotein as in human FCHL. It also associates hyperinsulinemia and hypertension, which are known to be highly prevalent in individuals with FCHL (31, 51, 61). In a previous study using a pharmacogenetic approach, we have determined genetic components of major blood pressure controlling systems in this strain (59). In this study, we successfully identified loci for plasma triglycerides, and the majority of them did not overlap with previously reported rat loci for plasma lipid with the exception of the suggestive locus on Chr 1, which overlapped with the previously reported region for plasma triglycerides in a diabetic rat strain (28). QTL for plasma triglycerides located on rat Chr 2 are novel and syntenic to previously reported human loci for the increased cholesterol concentration of small, dense LDL on Chr 3 and 4, one of the clinical characteristics of human FCHL (49). The human syntenic region for the other locus for plasma triglycerides on rat Chr 2 also overlapped with the previously reported locus for the metabolic syndrome phenotypes, such as body mass index, waist, hip, and plasma insulin (26) and the locus for small, dense LDL on human Chr 3 (49). In this region, Kissebah et al. (26) predicted solute carrier family member 2 (GLUT2) and phosphoinositide-3-kinase as candidate genes involved in glucose metabolism. Moreover, the phospholipase D1 gene can also be an attractive candidate gene involved in lipid metabolism. The second locus for plasma triglycerides on rat Chr 2 overlaps with the locus for small, dense LDL, including microsomal triglyceride transfer protein (MTP) and intestinal fatty acid binding protein (FABP2) genes as positional candidates. MTP is known to have an important role in lipoprotein production by the liver, and FABP2 is thought to have a significant function in lipoprotein production by the small intestine. The locus for plasma triglycerides localized on rat Chr 13 overlaps with the human locus for FCHL on Chr 1 reported in Finnish, German, Chinese, and American populations (10, 38, 39, 42). Pei et al. (42) predicted the retinoid X receptor-{gamma} gene as a possible candidate gene at this region. The suggestive locus for plasma triglycerides on rat Chr 1 overlaps with the human FCHL region in the Dutch population (5) containing the oxysterol-binding protein (OSBP) gene. OSBP is believed to transport sterols from lysosomes to the nucleus where LDL receptor and 3-hydroxy-3-methylglutaryl-CoA synthase downregulation occur. Our data indicate that the plasma triglyceride level of this rat strain was controlled for up to 30% of its variance in females by the same gene loci as lipids in human FCHL patients, and these loci include relevant candidate genes involved in lipid and insulin-glucose metabolism. Overlapping of loci for plasma triglycerides in hypertensive, hyperlipidemic, hyperinsulinemic animals with loci for plasma triglycerides or metabolic phenotypes in human subjects with hyperlipidemia or metabolic syndrome clearly suggests the importance of these loci for plasma lipid determination in metabolic abnormalities. Although several strains of animals share clinical characteristics with human metabolic syndrome or FCHL, and many loci have been localized as QTL for traits related to these diseases, concordance of the results of genetic studies in animals with those in humans has not been reported. We used here a novel strain of HTG rats as a first animal model providing evidence of sharing its clinical and genetic basis with human complex trait diseases.

As the HTG rat strain is hypertensive as well as hyperlipidemic, like human subjects with metabolic syndrome, loci affecting both blood pressure and plasma triglycerides were our targets of interest. We have determined loci linked with blood pressure phenotypes during pharmacological interventions in this F2 population, and significant linkage with blood pressure phenotypes has been observed at loci on Chr 1, 3, 5, and 8 (59). However, the located region for plasma triglycerides in this study did not overlap with these loci for blood pressure, except for one suggestive, male sex-specific locus for triglycerides on rat Chr 1. This observation was consistent with the results of Kovacs et al. (30) on the dissection of loci for plasma lipids and blood pressure in hypertensive hypertriglyceridemic Wistar-Ottawa-Karlsburg rats with the RT1u haplotype. Although the association of hypertriglyceridemia and hypertension in the HTG rat cannot be explained by one or more causative genes affecting both phenotypes, it may be subject to gene-gene interaction or epigenetic factors that affect both hypertensive and dyslipidemic phenotypes.

QTL mapping showed obvious sex differences for all of the loci determined in this study while both sexes of rats had the same genomic DNA sequences for each allele at every QTL. Sex differences of QTL for factors of the metabolic syndrome have been reported by Kloting et al. (27), but these loci did not include the locus for plasma triglycerides. Although sexual specificity with the QTL effects can be explained by a sex chromosome action, hormonal interaction at the transcriptional and posttranscriptional levels represents an alternative possibility that needs to be addressed in future studies. Moreover, changing of the hyperlipidemic phenotype is frequently observed in affected patients from FCHL families, and it is one of the clinical features of this disease. Kovacs et al. (29) reported age-dependent changes of the QTL effect for lipid loci on rat chromosomes. Their locus for triglycerides had maximal impact at 20 wk of age but disappeared at age 32 wk. As we phenotyped at almost the same rat age, we were evaluating phenotypes only at one point in life for each rat. The different sets of QTL in both sexes may be reflecting the effect of QTL at different time points of their life.

On Chr 5 and 12, we located significant negatively linked loci for plasma total cholesterol, and both loci also displayed sex differences. A significantly lower plasma total cholesterol level in the HTG progenitor strain can be explained by these loci in both sexes. In contrast to humans, the major cholesterol-containing lipoprotein in rodents is HDL rather than LDL and VLDL. The low plasma total cholesterol level induced by the HTG allele may reflect the decline of the amount of HDL particles as well as of ß-lipoprotein particles, and low HDL cholesterol is known to be one of the common features of human FCHL. However, this observation is limited by the fact that in the rat, LDL and HDL particles are of similar size, and therefore separation is incomplete as we have verified by additional experiments (T. Ueno, unpublished observations). Sequential ultracentrifugation has been used to isolate lipoprotein fractions in "pooled" rat serum (33), which makes it currently inaccessible in F2 hybrid studies where individual samples have to be analyzed in the entire set of rats.

This novel strain of rat is a relevant model of FCHL and metabolic syndrome, available for the assessment of the effect of agents affecting lipoprotein metabolism and insulin resistance. Chvojkova et al. (9) reported the plasma triglyceride- lowering effect of PPAR{gamma} activators in a strain of the same origin as the one used in this investigation, yet bred for several generations under different dietary and environmental conditions. Moreover, it could be interesting to perform a pharmacogenetical study by administration of PPAR{alpha}, PPAR{gamma}, or other agents affecting lipid or glucose metabolism using F2 intercross from this strain, as demonstrated to be useful in other models of metabolic syndrome (O. Seda, L. Kazdova, D. Krenova, and V. Kren, unpublished observations).

In this study, we successfully showed by comparative mapping that the HTG rat is a model of FCHL. This is the first animal model that both clinically and genetically confirmed the sharing of characteristics with human complex trait disorders. Comparative genomics, using the interspecies map of rat, mouse, and human chromosomes, is a powerful tool to compare the results of genetic analysis of animal disease models and of human complex trait diseases. It also narrows down the target region derived from quantitative genetic study during the positional cloning of disease-causing genes. Concordance of loci for lipids in animal models and in human subjects by comparative mapping unraveled the importance of these loci for the determination of plasma lipid levels in metabolic syndrome.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Canadian Institutes of Health Research Grants MOP-11463, MT-12574, and MOP36378; the Academy of Sciences of the Czech Republic Grant A7011711; Grant Agency of the Czech Republic Grant 305/03/0769; and American Heart Association National Center Grant AD-12-FMCQ 0140149N. T. Ueno is a recipient of a Servier Canada fellowship.


    ACKNOWLEDGMENTS
 
The technical assistance of Gilles Corbeil and Carole Long and the editorial help of Ovid Da Silva are appreciated.

J. Pelletier served as the review editor for this manuscript submitted by Editor H. J. Jacob.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: P. Hamet, Laboratory of Molecular Medicine, Centre de recherche du CHUM, 3850, rue Saint-Urbain, Montreal, Quebec H2W 1T7, Canada (E-mail: pavel.hamet{at}umontreal.ca).

10.1152/physiolgenomics.00043.2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aitman TJ, Glazier AM, Wallace CA, Cooper LD, Norsworthy PJ, Wahid FN, al Majali KM, Trembling PM, Mann CJ, Shoulders CC, Graf D, St Lezin E, Kurtz TW, Kren V, Pravenec M, Ibrahimi A, Abumrad NA, Stanton LW, and Scott J. Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nat Genet 21: 76–83, 1999.[CrossRef][ISI][Medline]
  2. Aitman TJ, Godsland IF, Farren B, Crook D, Wong HJ, and Scott J. Defects of insulin action on fatty acid and carbohydrate metabolism in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 17: 748–754, 1997.[Abstract/Free Full Text]
  3. Almasy L and Blangero J. Multipoint quantitative-trait linkage analysis in general pedigrees. Am J Hum Genet 62: 1198–1211, 1998.[CrossRef][ISI][Medline]
  4. Aouizerat BE, Allayee H, Bodnar J, Krass KL, Peltonen L, de Bruin TW, Rotter JI, and Lusis AJ. Novel genes for familial combined hyperlipidemia. Curr Opin Lipidol 10: 113–122, 1999.[CrossRef][ISI][Medline]
  5. Aouizerat BE, Allayee H, Cantor RM, Davis RC, Lanning CD, Wen PZ, Dallinga-Thie GM, de Bruin TW, Rotter JI, and Lusis AJ. A genome scan for familial combined hyperlipidemia reveals evidence of linkage with a locus on chromosome 11. Am J Hum Genet 65: 397–412, 1999.[CrossRef][ISI][Medline]
  6. Austin MA, Brunzell JD, Fitch WL, and Krauss RM. Inheritance of low density lipoprotein subclass patterns in familial combined hyperlipidemia. Arteriosclerosis 10: 520–530, 1990.[Abstract]
  7. Babirak SP, Brown BG, and Brunzell JD. Familial combined hyperlipidemia and abnormal lipoprotein lipase. Arterioscler Thromb 12: 1176–1183, 1992.[Abstract]
  8. Castellani LW, Weinreb A, Bodnar J, Goto AM, Doolittle M, Mehrabian M, Demant P, and Lusis AJ. Mapping a gene for combined hyperlipidaemia in a mutant mouse strain. Nat Genet 18: 374–377, 1998.[CrossRef][ISI][Medline]
  9. Chvojkova S, Kazdova L, and Divisova JA. A comparison of the effects of troglitazone and vitamin E on the fatty acid composition of serum phospholipids in an experimental model of insulin resistance. Physiol Res 50: 261–266, 2001.[ISI][Medline]
  10. Coon H, Myers RH, Borecki IB, Arnett DK, Hunt SC, Province MA, Djousse L, and Leppert MF. Replication of linkage of familial combined hyperlipidemia to chromosome 1q with additional heterogeneous effect of apolipoprotein A-I/C-III/A-IV locus. The NHLBI Family Heart Study. Arterioscler Thromb Vasc Biol 20: 2275–2280, 2000.[Abstract/Free Full Text]
  11. Darvasi A. The effect of selective genotyping on QTL mapping accuracy. Mamm Genome 8: 67–68, 1997.[CrossRef][ISI][Medline]
  12. Darvasi A and Soller M. Selective genotyping for determination of linkage between a marker locus and a quantitative trait locus. Theor Appl Genet 85: 353–359, 1992.[ISI]
  13. Darvasi A and Soller M. Advanced intercross lines, an experimental population for fine genetic mapping. Genetics 141: 1199–1207, 1995.[Abstract/Free Full Text]
  14. de Roos B, Caslake MJ, Ardern HA, Martin BG, Suckling KE, and Packard CJ. Insulin resistance in the St. Thomas’ mixed hyperlipidaemic (SMHL) rabbit, a model for familial combined hyperlipidaemia. Atherosclerosis 156: 249–254, 2001.[CrossRef][ISI][Medline]
  15. Delawi D, Meijssen S, and Castro CM. Intra-individual variations of fasting plasma lipids, apolipoproteins and postprandial lipemia in familial combined hyperlipidemia compared to controls. Clin Chim Acta 328: 139–145, 2003.[CrossRef][ISI][Medline]
  16. Deng AY, Dene H, Pravenec M, and Rapp JP. Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes. J Clin Invest 93: 2701–2709, 1994.[ISI][Medline]
  17. Ferrannini E. Syndrome X. Horm Res 39, Suppl 3: 107–111, 1993.
  18. Glueck SB and Dzau VJ. Physiological genomics: implications in hypertension research. Hypertension 39: 310–315, 2002.[Abstract/Free Full Text]
  19. Goldstein JL, Schrott HG, Hazzard WR, Bierman EL, and Motulsky AG. Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder, combined hyperlipidemia. J Clin Invest 52: 1544–1568, 1973.[ISI][Medline]
  20. Grundy SM. United States Cholesterol Guidelines 2001: expanded scope of intensive low-density lipoprotein-lowering therapy. Am J Cardiol 88: 23J–27J, 2001.[ISI][Medline]
  21. Hamet P. Apolipoprotein E alleles and hypertension: controversy or lack of understanding? J Hypertens 20: 1941–1942, 2002.[CrossRef][ISI][Medline]
  22. Hamet P, Pausova Z, Adarichev S, Adaricheva K, and Tremblay J. Hypertension: genes and environment. J Hypertens 16: 397–418, 1998.[CrossRef][ISI][Medline]
  23. Hokanson JE, Austin MA, Zambon A, and Brunzell JD. Plasma triglyceride and LDL heterogeneity in familial combined hyperlipidemia. Arterioscler Thromb 13: 427–434, 1993.[Abstract]
  24. Hokanson JE, Krauss RM, Albers JJ, Austin MA, and Brunzell JD. LDL physical and chemical properties in familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 15: 452–459, 1995.[Abstract/Free Full Text]
  25. Keulen ET, Voors-Pette C, and de Bruin TW. Familial dyslipidemic hypertension syndrome: familial combined hyperlipidemia, and the role of abdominal fat mass. Am J Hypertens 14: 357–363, 2001.[CrossRef][ISI][Medline]
  26. Kissebah AH, Sonnenberg GE, Myklebust J, Goldstein M, Broman K, James RG, Marks JA, Krakower GR, Jacob HJ, Weber J, Martin L, Blangero J, and Comuzzie AG. Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome. Proc Natl Acad Sci USA 97: 14478–14483, 2000.[Abstract/Free Full Text]
  27. Kloting I, Kovacs P, and van den Brandt J. Sex-specific and sex-independent quantitative trait loci for facets of the metabolic syndrome in WOKW rats. Biochem Biophys Res Commun 284: 150–156, 2001.[CrossRef][ISI][Medline]
  28. Kovacs P and Kloting I. Quantitative trait loci on chromosomes 1 and 4 affect lipid phenotypes in the rat. Arch Biochem Biophys 354: 139–143, 1998.[CrossRef][ISI][Medline]
  29. Kovacs P, van den Brandt J, and Kloting I. Effects of quantitative trait loci for lipid phenotypes in the rat are influenced by age. Clin Exp Pharmacol Physiol 25: 1004–1007, 1998.[ISI][Medline]
  30. Kovacs P, van den Brandt J, and Kloting I. Genetic dissection of the syndrome X in the rat. Biochem Biophys Res Commun 269: 660–665, 2000.[CrossRef][ISI][Medline]
  31. Kunes J, Dobesova Z, and Zicha J. Altered balance of main vasopressor and vasodepressor systems in rats with genetic hypertension and hypertriglyceridaemia. Clin Sci (Lond) 102: 269–277, 2002.[CrossRef][Medline]
  32. Kwitek-Black AE and Jacob HJ. The use of designer rats in the genetic dissection of hypertension. Curr Hypertens Rep 3: 12–18, 2001.[Medline]
  33. Lefebvre AM, Peinado-Onsurbe J, Leitersdorf I, Briggs MR, Paterniti JR, Fruchart JC, Fievet C, Auwerx J, and Staels B. Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct but complementary mechanism relative to fibrates. Arterioscler Thromb Vasc Biol 17: 1756–1764, 1997.[Abstract/Free Full Text]
  34. Manly KF and Olson JM. Overview of QTL mapping software and introduction to Map Manager QT. Mamm Genome 10: 327–334, 1999.[CrossRef][ISI][Medline]
  35. Masucci-Magoulas L, Goldberg IJ, Bisgaier CL, Serajuddin H, Francone OL, Breslow JL, and Tall AR. A mouse model with features of familial combined hyperlipidemia. Science 275: 391–394, 1997.[Abstract/Free Full Text]
  36. Nikkila EA and Aro A. Family study of serum lipids and lipoproteins in coronary heart-disease. Lancet 1: 954–959, 1973.[Medline]
  37. Pajukanta P, Bodnar JS, Sallinen R, Chu M, Airaksinen T, Xiao Q, Castellani LW, Sheth SS, Wessman M, Palotie A, Sinsheimer JS, Demant P, Lusis AJ, and Peltonen L. Fine mapping of Hyplip1 and the human homolog, a potential locus for FCHL. Mamm Genome 12: 238–245, 2001.[CrossRef][ISI][Medline]
  38. Pajukanta P, Nuotio I, Terwilliger JD, Porkka KV, Ylitalo K, Pihlajamaki J, Suomalainen AJ, Syvanen AC, Lehtimaki T, Viikari JS, Laakso M, Taskinen MR, Ehnholm C, and Peltonen L. Linkage of familial combined hyperlipidaemia to chromosome 1q21-q23. Nat Genet 18: 369–373, 1998.[ISI][Medline]
  39. Pajukanta P, Terwilliger JD, Perola M, Hiekkalinna T, Nuotio I, Ellonen P, Parkkonen M, Hartiala J, Ylitalo K, Pihlajamaki J, Porkka K, Laakso M, Viikari J, Ehnholm C, Taskinen MR, and Peltonen L. Genomewide scan for familial combined hyperlipidemia genes in Finnish families, suggesting multiple susceptibility loci influencing triglyceride, cholesterol, and apolipoprotein B levels. Am J Hum Genet 64: 1453–1463, 1999.[CrossRef][ISI][Medline]
  40. Park YW, Zhu S, Palaniappan L, Heshka S, Carnethon MR, and Heymsfield SB. The metabolic syndrome: prevalence and associated risk factor findings in the US population from the Third National Health and Nutrition Examination Survey, 1988–1994. Arch Intern Med 163: 427–436, 2003.[Abstract/Free Full Text]
  41. Pausova Z, Tremblay J, and Hamet P. Gene-environment interactions in hypertension. Curr Hypertens Rep 1: 42–50, 1999.[Medline]
  42. Pei W, Baron H, Muller-Myhsok B, Knoblauch H, Al Yahyaee SA, Hui R, Wu X, Liu L, Busjahn A, Luft FC, and Schuster H. Support for linkage of familial combined hyperlipidemia to chromosome 1q21-q23 in Chinese and German families. Clin Genet 57: 29–34, 2000.[CrossRef][ISI][Medline]
  43. Pihlajamaki J, Karjalainen L, Karhapaa P, Vauhkonen I, Taskinen MR, Deeb SS, and Laakso M. G-250A substitution in promoter of hepatic lipase gene is associated with dyslipidemia and insulin resistance in healthy control subjects and in members of families with familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 20: 1789–1795, 2000.[Abstract/Free Full Text]
  44. Pihlajamaki J, Miettinen R, Valve R, Karjalainen L, Mykkanen L, Kuusisto J, Deeb S, Auwerx J, and Laakso M. The Pro12A1a substitution in the peroxisome proliferator activated receptor gamma 2 is associated with an insulin-sensitive phenotype in families with familial combined hyperlipidemia and in nondiabetic elderly subjects with dyslipidemia. Atherosclerosis 151: 567–574, 2000.[CrossRef][ISI][Medline]
  45. Pihlajamaki J, Rissanen J, Heikkinen S, Karjalainen L, and Laakso M. Codon 54 polymorphism of the human intestinal fatty acid binding protein 2 gene is associated with dyslipidemias but not with insulin resistance in patients with familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 17: 1039–1044, 1997.[Abstract/Free Full Text]
  46. Pihlajamaki J, Rissanen J, Valve R, Heikkinen S, Karjalainen L, and Laakso M. Different regulation of free fatty acid levels and glucose oxidation by the Trp64Arg polymorphism of the beta3-adrenergic receptor gene and the promoter variant (A-3826G) of the uncoupling protein 1 gene in familial combined hyperlipidemia. Metabolism 47: 1397–1402, 1998.[ISI][Medline]
  47. Pihlajamaki J, Valve R, Karjalainen L, Karhapaa P, Vauhkonen I, and Laakso M. The hormone sensitive lipase gene in familial combined hyperlipidemia and insulin resistance. Eur J Clin Invest 31: 302–308, 2001.[CrossRef][ISI][Medline]
  48. Purnell JQ, Kahn SE, Schwartz RS, and Brunzell JD. Relationship of insulin sensitivity and ApoB levels to intra-abdominal fat in subjects with familial combined hyperlipidemia. Arterioscler Thromb Vasc Biol 21: 567–572, 2001.[Abstract/Free Full Text]
  49. Rainwater DL, Almasy L, Blangero J, Cole SA, VandeBerg JL, MacCluer JW, and Hixson JE. A genome search identifies major quantitative trait loci on human chromosomes 3 and 4 that influence cholesterol concentrations in small LDL particles. Arterioscler Thromb Vasc Biol 19: 777–783, 1999.[Abstract/Free Full Text]
  50. Reaven GM. Syndrome X. Blood Press Suppl 4: 13–16, 1992.[Medline]
  51. Reaven GM. Role of insulin resistance in human disease (syndrome X): an expanded definition. Annu Rev Med 44: 121–131, 1993.[CrossRef][ISI][Medline]
  52. Rose HG, Kranz P, Weinstock M, Juliano J, and Haft JI. Inheritance of combined hyperlipoproteinemia: evidence for a new lipoprotein phenotype. Am J Med 54: 148–160, 1973.[ISI][Medline]
  53. Shamir R, Tershakovec AM, Gallagher PR, Liacouras CA, Hayman LL, and Cortner JA. The influence of age and relative weight on the presentation of familial combined hyperlipidemia in childhood. Atherosclerosis 121: 85–91, 1996.[CrossRef][ISI][Medline]
  54. Stolba P, Dobesova Z, Husek P, Opltova H, Zicha J, Vrana A, and Kunes J. The hypertriglyceridemic rat as a genetic model of hypertension and diabetes. Life Sci 51: 733–740, 1992.[CrossRef][ISI][Medline]
  55. Stolba P, Opltova H, Husek P, Nedvidkova J, Kunes J, Dobesova Z, Nedvidek J, and Vrana A. Adrenergic overactivity and insulin resistance in nonobese hereditary hypertriglyceridemic rats. Ann NY Acad Sci 683: 281–288, 1993.[Abstract]
  56. Stoll M, Cowley AW Jr, Tonellato PJ, Greene AS, Kaldunski ML, Roman RJ, Dumas P, Schork NJ, Wang Z, and Jacob HJ. A genomic-systems biology map for cardiovascular function. Science 294: 1723–1726, 2001.[Abstract/Free Full Text]
  57. Stoll M and Jacob HJ. Genetic rat models of hypertension: relationship to human hypertension. Curr Hypertens Rep 3: 157–164, 2001.[Medline]
  58. Ueno T, Tremblay J, Kunes J, Zicha J, Dobesova Z, Pausova Z, Deng AY, Sun YL, Jacob HJ, and Hamet P. Resolving the composite trait of hypertension into its pharmacogenetic determinants by acute pharmacological modulation of blood pressure regulatory systems. J Mol Med 81: 51–60, 2003.[ISI][Medline]
  59. Venkatesan S, Cullen P, Pacy P, Halliday D, and Scott J. Stable isotopes show a direct relation between VLDL apoB overproduction and serum triglyceride levels and indicate a metabolically and biochemically coherent basis for familial combined hyperlipidemia. Arterioscler Thromb 13: 1110–1118, 1993.[Abstract]
  60. Vrana A and Kazdova L. The hereditary hypertriglyceridemic nonobese rat: an experimental model of human hypertriglyceridemia. Transplant Proc 22: 2579, 1990.[ISI][Medline]
  61. Vrana A, Kazdova L, Dobesova Z, Kunes J, Kren V, Bila V, Stolba P, and Klimes I. Triglyceridemia, glucoregulation, and blood pressure in various rat strains. Effects of dietary carbohydrates. Ann NY Acad Sci 683: 57–68, 1993.[ISI][Medline]
  62. Wijsman EM, Brunzell JD, Jarvik GP, Austin MA, Motulsky AG, and Deeb SS. Evidence against linkage of familial combined hyperlipidemia to the apolipoprotein AI-CIII-AIV gene complex. Arterioscler Thromb Vasc Biol 18: 215–226, 1998.[Abstract/Free Full Text]
  63. Wojciechowski AP, Farrall M, Cullen P, Wilson TM, Bayliss JD, Farren B, Griffin BA, Caslake MJ, Packard CJ, and Shepherd J. Familial combined hyperlipidaemia linked to the apolipoprotein AI-CII-AIV gene cluster on chromosome 11q23-q24. Nature 349: 161–164, 1991.[CrossRef][ISI][Medline]
  64. Xu CF, Talmud P, Schuster H, Houlston R, Miller G, and Humphries S. Association between genetic variation at the APO AI-CIII-AIV gene cluster and familial combined hyperlipidaemia. Clin Genet 46: 385–397, 1994.[ISI][Medline]
  65. Yang WS, Nevin DN, Iwasaki L, Peng R, Brown BG, Brunzell JD, and Deeb SS. Regulatory mutations in the human lipoprotein lipase gene in patients with familial combined hyperlipidemia and coronary artery disease. J Lipid Res 37: 2627–2637, 1996.[Abstract]