Quantitative Trait Loci Influencing Blood and Liver Cholesterol Concentration in Rats
Objective— The LEW/OlaHsd and BC/CpbU rat inbred strains differ markedly in blood and hepatic cholesterol levels before and after a cholesterol-rich diet. To define loci controlling these traits and related phenotypes, an F2 population derived from these strains was genetically analyzed.
Methods and Results— For each of the 192 F2 animals, phenotypes were determined, and genomic DNA was screened for polymorphic microsatellite markers. Significant quantitative trait loci (QTLs) were detected for basal serum cholesterol level on chromosome 1 (D1Rat335-D1Rat27: total population, lod score 9.6; females, lod score 10.3) and chromosome 7 (D7Rat69: males, lod score 4.1), for postdietary serum cholesterol level on chromosome 2 (D2Rat69: total population, lod score 4.4) and chromosome 16 (D16Rat6-D16Rat44: total population, lod score 3.3), for postdietary serum phospholipid level on chromosome 11 (D11Rat10: total population, lod score 4.1; females, lod score 3.6), and for postdietary serum aldosterone level on chromosome 1 (D1Rat14: females, lod score 3.7) and chromosome 18 (D18Rat55-D18Rat8: females, lod score 2.9). In addition, QTLs with borderline significance were found on chromosomes 3, 5 to 11, 15, and 18.
Conclusions— QTLs involved in blood and/or hepatic cholesterol concentrations (or related phenotypes) in the rat were identified. This contributes to the value of the rat as an animal model in studies researching the role of cholesterol in the pathogenesis of atherosclerosis and other cholesterol-related diseases.
Atherosclerosis is a complex disorder in which genetic and environmental factors play a role. A high serum cholesterol concentration is one of the risk factors in the development of this disease. Circulating cholesterol levels do not exclusively reflect dietary habits; epidemiological studies have revealed consistently higher than average serum cholesterol levels only in particular individuals after a high dietary cholesterol intake.1 Individual differences in serum cholesterol concentration also exist after a diet with low-fat and/or low-cholesterol content. Similar variability in serum cholesterol levels can also be observed in laboratory animals such as mice,2 rabbits,3 and rats4 in response to control diets and to high-fat and high-cholesterol diets.5–8⇓⇓⇓ Differences observed between inbred strains of these species indicate that the basal serum cholesterol concentration and the rate of increase in serum cholesterol levels after a cholesterol-rich diet are under genetic control.
To genetically analyze these phenotypes in laboratory rats, we performed a total genome scan of an F2 population derived from the BC/CpbU and LEW/OlaHsd rat inbred strains. Cholesterol (basal and postdietary serum level and postdietary liver concentration) and cholesterol-related traits (postdietary serum phospholipid, aldosterone, and corticosterone levels) were measured in the F2 animals. The cholesterol-related traits were included into the present study because in the F2 population these parameters were significantly associated with postdietary serum cholesterol levels. The results of this quantitative trait loci (QTL) analysis and possible candidate genes located in the vicinity of some QTLs will be discussed.
The Methods section can be accessed online (http://www.atvb.ahajournals.org).
For parental strains, please see Table 1. At the beginning of the test period, all rats were of the same age, but the body weight of the LEW rats, compared with the BC rats, was higher. Males and females of the BC strain had equal body weights. In contrast, initial body weight of the LEW males was higher than that of the LEW females. As would be expected, body weight increased, in an identical fashion, in the 2 inbred strains during the course of the experiment. At the end of the test period, the LEW rats had a higher body weight than the BC rats, and the males were significantly heavier than the females.
Pre-experimental serum cholesterol levels (baseline values) were significantly higher in the BC rats than in the LEW rats. Baseline serum cholesterol levels were similar in males and females of the BC strain. These levels were significantly higher in LEW females compared with LEW males. Because there were significant differences in initial body weight, group means of baseline serum cholesterol levels were also compared by ANOVA, with initial body weight as a covariate. The effect of the rat’s sex on baseline serum cholesterol concentration disappeared, but there tended to be a strain effect.
The high-fat high-cholesterol diet produced an increase in serum total cholesterol levels in LEW rats but not in BC rats (in fact, in BC males there was a significant decrease). This increase was more pronounced in female compared with male LEW rats. Sex and strain significantly affected the final serum cholesterol concentration. However, after correction for body weight, the sex effect was of borderline significance, but the strain effect was still highly significant. Group mean postdietary serum phospholipid levels were higher in the LEW strain than in the BC strain; this strain effect reached the level of statistical significance only in the females. After correction for body weight, there was a significant strain effect.
Although serum cholesterol levels in males of the BC strain responded with a decrease to a high-fat high-cholesterol diet, the postdietary liver cholesterol concentration was significantly higher in male BC rats compared with male LEW rats. Female rats compared with male rats of the LEW strain, but not of the BC strain, had significantly elevated hepatic cholesterol levels. In the 2-way ANOVA with final body weight as a covariate, these effects did not reach the level of statistical significance. Group mean circulating adrenal steroids levels were higher in BC rats than in LEW rats. However, after correction for body weight, this strain effect was significant only for serum aldosterone concentration.
Genetic Mapping of Quantitative Traits
For genetic mapping of quantitative traits, please see Tables 2 and 3⇓, Figure 1, and online Figures I to IV (available at http://www.atvb.ahajournals.org). The results of the QTL analysis using the MapQTL software are summarized in Table 2. For initial body weight, male- or female-specific QTLs were mapped on chromosomes 5 and 7 (please see online Figure IA), respectively. QTLs for males plus females were found for initial body weight on chromosomes 15 (please see online Figure IB) and 18 and for final body weight (females, males plus females) on chromosome 17. Kovács et al9 and Klöting et al10 also found QTLs influencing body weight on rat chromosomes 5 and 18.
The genome-wide scan revealed regions with significant linkage of the cholesterol and related phenotypes to loci on chromosomes 1, 2, 7, 11, 16, and 18 (Figures 1, online Figures II to IV, and Table 2). QTLs with borderline significance were found on rat chromosomes 3, 5 to 11, 15, and 18 (Table 2). Table 2 also illustrates the existence of sex-specific and sex-independent QTLs.
Table 3 shows the phenotypes of each of the 3 genotypes (LL, LB, and BB) segregating at selected loci on chromosomes that were significantly associated with a trait. The allele frequencies were not statistically different (P>0.05) from the expected ratio, 1:2:1 (LL:LB:BB), with 1 exception, D1Rat335 in females. However, the closely linked marker D1Rat27 showed no segregation deviation in females, and the ANOVA results of D1Rat27 and D1Rat335 (P<0.0005) point to a QTL also in females; the lod score for baseline serum cholesterol level was somewhat higher with D1Rat335 as a marker. Therefore, taken together, the segregation distortion at D1Rat335 is not a serious problem for quantitative trait linkage analysis.
A 2-way ANOVA was used for evaluating genetic interactions (Table 4 and Figure 2) among selected loci, ie, among the loci that were flanking a segment that contains a candidate gene (see Discussion). Table 4 summarizes the significant interactions. Figure 2 illustrates the interaction between the D1Rat335 and D11Rat10 loci for baseline serum cholesterol levels. When rats were homozygous for the BC allele at the D1Rat335 locus and homozygous for the LEW allele at the D11Rat10 locus, the baseline serum cholesterol levels were highest.
In the present study, genome-wide scanning of 192 F2 animals derived from the rat inbred strains BC and LEW for associations between marker genotypes and quantitative traits related to dietary cholesterol susceptibility resulted in the localization of quite a few QTLs with borderline significance (on rat chromosomes 3, 5 to 11, 15, and 18; Table 2) and 7 significant QTLs (on rat chromosomes 1, 2, 7, 11, 16, and 18; Tables 2 and 3⇑, Figure 1, and online Figures I to IV). Between body weight and cholesterol or related phenotypes, there were in the F2 rats weak, but significant, associations (please see online Table I, available at http://www.atvb.ahajournals.org). This prompted us to perform a 1-way (Table 3) or 2-way (Table 4 and I) ANOVA with a covariate (body weight). Despite the use of a covariate, the genotypes at selected loci were still significantly associated with the traits (Table 3). Furthermore, the body weight QTLs did not localize to regions where the QTLs for cholesterol and related phenotypes were found (Table 2; please see online Figure IA versus Figure 1B). Thus, it could be excluded that the QTLs for the cholesterol and related phenotypes are QTLs controlling interstrain differences in body weight. It is a common practice to study the genetic background of phenotypes with multiple environmental and genetic components by crossing 2 inbred strains that differ for the parameter under study and subsequently intercrossing or backcrossing the F1 progeny. In most studies, the progenitor strains also differ regarding body weight. We believe that in these studies, body weight should be considered, at least when there is evidence of an association between the parameters under study and body weight.
Kovács et al11 reported significant linkage of the marker D1Mit14 with basal serum cholesterol levels in a study of a backcross derived from SHR/Mol and BB/OK rats. Genetic analyses of OLETF×(OLETF×Fischer 344) rats revealed statistically significant linkage between D1Rat306 and basal serum cholesterol levels.12 These QTL regions are located at the telomeric part of the q arm of rat chromosome 1 and do not colocalize with the QTL region D1Rat335-D1Rat27 as found in the present study (Figure 1A). The markers D1Rat306 and D1Mit14 are located at ≈133 cM in the rat genome database map, whereas D1Rat27 is located at 45 cM.
Interestingly, the region around D1Rat27 (Figure 1A) contains the Apoe gene.13 ApoE plays a pivotal role in the catabolism of triglyceride-rich lipoproteins by serving as a ligand for lipoprotein receptors. The Apoe gene might be a positional candidate for this QTL. In humans, allelic variation of the Apoe gene has been associated with differences in serum total cholesterol14 and LDL cholesterol15 levels. Transfer of a segment of chromosome 1 containing the Apoe gene from the BN/Cr rat inbred strain into the genetic background of the SHR/Ola rat16 caused a significant increase in LDL/HDL1 cholesterol level. The SHR.BN-D1Wox6-D1Mgh11/Ipcv (n=6) compared with the SHR/Ola (n=6) had increased LDL/HDL1 cholesterol (on average, 65% higher level; P=0.001 by unpaired Student t test; unpublished data).
For mice, several genetic analyses have been published that revealed QTLs influencing hepatic or plasma cholesterol concentration after a high-cholesterol diet.2,18,19⇓⇓ However, to the best of our knowledge, this is the first report dealing with rats in which a genome-wide search successfully identified multiple chromosomal regions linked to circulating cholesterol levels after a high-fat high-cholesterol diet (Tables 2 and 3⇑ and online Figure II). The present study also supports our previous findings of a QTL on rat chromosome 2 influencing postdietary IDL cholesterol levels.20
Bottger et al21 localized the gene (Lpl) coding for lipoprotein lipase (LPL) to rat chromosome 16. In the radiation hybrid map, Lpl is located near the marker D16Rat6.13 In the present study, the latter marker is located in the vicinity of the QTL controlling serum cholesterol levels on rat chromosome 16. LPL plays a major role in lipoprotein metabolism,22 and in humans, LPL mutations have been associated with atherosclerosis and dyslipidemia.23 The SHR.BN-D16Mit5/Cub congenic strain is a rat strain with the genetic background of the spontaneously hypertensive rat (SHR) onto which a segment from the BN strain, containing the Lpl gene, has been transferred.16 This SHR-BN congenic strain (n=4) has a significantly increased serum cholesterol level compared with the SHR/Ola (n=6) progenitor strain after the feeding of a high-fat high-cholesterol diet (on average, 81% higher level; P=0.014 by unpaired Student t test).17 Thus, Lpl could be the gene on rat chromosome 16 that is controlling postdietary circulating cholesterol levels.
Up until now, 2 QTLs controlling serum phospholipid levels have been described in the rat. Bottger et al21 and Kovács et al24 found a QTL for basal serum phospholipid levels on rat chromosome 4. Postdietary HDL2 phospholipids were associated with the same region of rat chromosome 424 or with rat chromosome 20.21 In the present study, we could not confirm the aforementioned associations. However, we now found a QTL for postdietary serum phospholipid levels on rat chromosome 11 (Tables 2 and 3⇑ and online Figure III). As to the gene involved, we can only speculate. This segment of rat chromosome 11 is homologous with mouse chromosome 16 and human chromosome 3q, where the gene for CTP:phosphocholine cytidylyltransferase (Pcyt1a) is located. This gene is involved in hepatic phospholipid metabolism.25 On the basis of homology, one might speculate that this gene is responsible for the strain difference in serum phospholipid level in rats fed high-fat high-cholesterol diets.
For humans, a significant interaction between Apoe and Lpl variants for circulating HDL cholesterol levels has been described.26 In the present study, there was no interaction between the segment of rat chromosome 1 that contains the Apoe gene and the Lpl containing chromosomal segment (rat chromosome 16). However, we detected a significant association between chromosome 11 and 1 or 16 (Table 4). This association points to a gene-gene interaction of Pcyt1a with Apoe and Lpl. To the best of our knowledge, such an interaction has not been described previously.
The reciprocal relationship between cholesterol metabolism and adrenal steroid hormone activity is well established. Therefore, we anticipated that QTLs affecting serum cholesterol concentration might also be associated with variations in serum corticosterone. However, no significant QTLs for corticosterone were identified, even though there were significant interactions between the markers flanking a cholesterol-QTL (D1Rat335, D16Rat6, and D16Rat44) and the D11Rat10 marker (Table 4), thus suggesting a relationship between circulating cholesterol and corticosterone levels. Interestingly, QTLs for aldosterone were revealed on regions of chromosomes 1 and 18, where QTLs for high blood pressure have previously been identified.27,28⇓ The genes responsible for these aldosterone variations are not known but may, indirectly, relate to the regulation of electrolyte metabolism.27,29⇓
In the mouse, several QTLs also involved in cholesterol metabolism have been localized.18,19,30–35⇓⇓⇓⇓⇓⇓⇓ The chromosomal positions of the QTLs as found in the present study have been compared with the chromosomal locations of QTLs found in the mouse. A homologous location of the QTL for basal serum cholesterol levels on rat chromosome 1 (Figure 1) has been confirmed by 2 studies in the mouse. Gu et al18 and Purcell-Huynh et al30 found a QTL on mouse chromosome 7 in the vicinity the Apoe gene. For serum total cholesterol levels, a QTL on rat chromosome 2 was found (please see online Figure II). On the homologous region of mouse chromosome 3, a QTL for serum cholesterol levels was also identified.35 Furthermore, on mouse chromosome 18, a QTL for phospholipids levels has been found.36 Mouse chromosome 18 is homologous to rat chromosome 18, where we have found a suggestive QTL for this trait (Table 2). These findings seem to indicate that these traits have been conserved in the evolutionary differentiation of these species and thus may play a major role in cholesterol metabolism.
In summary, the present study indicates that rat chromosomes 1 to 3, 5 to 11, 15, 16, and 18 each contain at least 1 QTL that is involved in blood and/or hepatic lipid concentrations (or related phenotypes). Because the QTL mapping data were obtained with a relatively small number of animals, further experiments, including the development of (double) congenic strains or knockout strains after gene cloning, are necessary to precisely map the QTLs and to confirm the role of the suggested candidate genes.
Received May 24, 2002; revision accepted September 19, 2002.
- ↵Pitman WA, Hunt MH, McFarland C, Paigen B. Genetic analysis of the difference in diet-induced atherosclerosis between the inbred mouse strains SM/J and NZB/BINJ. Arterioscler Thromb Vasc Biol. 1998; 18: 615–620.
- ↵Kato N, Tamada T, Nabika T, Ueno K, Gotoda T, Matsumoto C, Mashimo T, Sawamura M, Ikeda K, Nara Y, Yamori Y. Identification of quantitative trait loci for serum cholesterol levels in stroke-prone spontaneously hypertensive rats. Arterioscler Thromb Vasc Biol. 2000; 20: 223–229.
- ↵Bottger A, Den Bieman M, Lankhorst Æ, Van Lith HA, Van Zutphen LFM. Strain-specific response to hypercholesterolaemic diets in the rat. Lab Anim. 1996; 30: 149–157.
- ↵Meijer GW, Van Der Palen JGP, Geelen MJH, Versluis A, Van Zutphen LFM, Beynen AC. Secretion of lipoprotein cholesterol by perfused livers from rabbits hypo- or hyperresponsive to dietary cholesterol: greater dietary cholesterol-induced secretion in hyperresponsive rabbits. J Nutr. 1992; 122: 1164–1173.
- ↵Yamasaki Y, Watanabe TK, Okuno S, Ono T, Oga K, Mizoguchi-Miyakita A, Goto Y, Shinomiya H, Momota H, Miyao H, Hayashi I, Asai T, Suzuki M, Harada Y, Hishigaki H, Wakitani S, Takagi T, Nakamura Y, Tanigami A. Quantitative trait loci for lipid metabolism in the study of OLETF x (OLETF x Fischer 344) backcross rats. Clin Exp Pharmacol Physiol. 2000; 27: 881–886.
- ↵Watanabe TK, Bihoreau MT, McCarthy LC, Kiguwa SL, Hishigaki H, Tsuji A, Browne J, Yamasaki Y, Mizoguchi-Miyakita A, Oga K, Ono T, Okuno S, Kanemoto N, Takahashi E, Tomita K, Hayashi H, Adachi M, Webber C, Davis M, Kiel S, Knights C, Smith A, Critcher R, Miller J, Thangarajah T, Day PJR, Hudson JR Jr, Irie Y, Takagi T, Nakamura Y, Goodfellow PN, Lathrop GM, Tanigami A, James MR. A radiation hybrid map of the rat genome containing 5,255 markers. Nat Genet. 1999; 22: 27–36.
- ↵Hagberg JM, Wilund KR, Ferrell RE. APOE gene and gene-environment effects on plasma lipoprotein-lipid levels. Physiol Genomics. 2000; 4: 101–108.
- ↵Gylling H, Kontula K, Koivisto UM, Miettinen HE, Miettinen TA. Polymorphisms of the genes encoding apoproteins A-I, B, C-III, and E and LDL receptor, and cholesterol and LDL metabolism during increased cholesterol intake: common alleles of the apoprotein E gene show the greatest regulatory impact. Arterioscler Thromb Vasc Biol. 1997; 17: 38–44.
- ↵Kren V, Simakova M, Musilova A, Zidek V, Pravenec M. SHR.BN-congenic strains for genetic analysis of multifactorially determined traits. Folia Biol (Praha). 2000; 46: 25–29.
- ↵Deleted in press.
- ↵Gu L, Johnson MW, Lusis AJ. Quantitative trait locus analysis of plasma lipoprotein levels in an autoimmune mouse model: interactions between lipoprotein metabolism, autoimmune disease, and atherogenesis. Arterioscler Thromb Vasc Biol. 1999; 19: 442–453.
- ↵Schwarz M, Davis DL, Vick BR, Russell DW. Genetic analysis of cholesterol accumulation in inbred mice. J Lipid Res. 2001; 42: 1812–1819.
- ↵Bottger A, Van Lith HA, Van Zutphen LFM, Kren V, Pravenec M. Genetic analysis of the relative intermediate density lipoprotein cholesterol levels using recombinant inbred strains. In: Harmonization of Laboratory Animal Husbandry. Proceedings of the Sixth Symposium of the Federation of European Laboratory Animal Science Associations; June 19–21, 1996; Basel, Switzerland.
- ↵Bottger A, Van Lith HA, Kren V, Krenová D, Bílá V, Vorlícek J, Zídek V, Musilová A, Zdobinská M, Wang J-M, Van Zutphen BFM, Kurtz TW, Pravenec M. Quantitative trait loc influencing cholesterol and phospholipid phenotypes map to chromosomes that contain genes regulating blood pressure in the spontaneously hypertensive rat. J Clin Invest. 1996; 98: 856–862.
- ↵Kovács P, Van Den Brandt J, Bonné ACM, Van Zutphen LFM, Van Lith HA, Klöting I, Congenic BB. SHR rat provides evidence for effects of a chromosome 4 segment (D4 Mit6-Npy∼1 cM) on total serum and lipoprotein lipid concentration and composition after feeding a high-fat, high-cholesterol diet. Metabolism. 2001; 50: 458–462.
- ↵Kast HR, Nguyen CM, Anisfeld AM, Ericsson J, Edwards PA. CTP: phosphocholine cytidylyltransferase, a new sterol- and SREBP responsive gene. J Lipid Res. 2001; 42: 1266–1272.
- ↵Corella D, Guillen M, Saiz C, Portoles O, Sabater A, Folch J, Ordovas JM. Associations of LPL and APOC3 gene polymorphisms on plasma lipids in a Mediterranean population: interaction with tobacco smoking and the APOE locus. J Lipid Res. 2002; 43: 416–427.
- ↵Zagato L, Modica R, Florio M, Torielli L, Bihoreau M-T, Bianchi G, Tripodi G. Genetic mapping of blood pressure quantitative trait loci in Milan hypertensive rats. Hypertension. 2000; 36: 734–739.
- ↵Welch CL, Xia YR, Shechter I, Farese R, Mehrabian M, Mehdizadeh S, Warden CH, Lusis AJ. Genetic regulation of cholesterol homeostasis: chromosomal organization of candidate genes. J Lipid Res. 1996; 37: 1406–1421.
- ↵Mehrabian M, Castellani LW, Wen PZ, Wong J, Rithaporn T, Hama SY, Hough GP, Johnson D, Albers JJ, Mottino GA, Frank JS, Navab M, Fogelman AM, Lusis AJ. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J). J Lipid Res. 2000; 41: 1936–1946.
- ↵Paigen B, Schork NJ, Svenson KL, Cheah YC, Mu JL, Lammert F, Wang DQ, Bouchard G, Carey MC. Quantitative trait loci mapping for cholesterol gallstones in AKR/J and C57L/J strains of mice. Physiol Genomics. 2000; 4: 59–65.
- ↵Mu JL, Naggert JK, Svenson KL, Collin GB, Kim JH, McFarland C, Nishina PM, Levine DM, Williams KJ, Paigen B. Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J. J Lipid Res. 1999; 40: 1328–1335.