Loci controlling plasma non-HDL and HDL cholesterol levels in a C57BL /6J × CASA /Rk intercross

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol.In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse. Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse. Plasma levels of non-HDL and HDL cholesterol have been shown to modulate the risk for cardiovascular diseases. Increased plasma levels of non-HDL cholesterol, especially in the form of LDL cholesterol, and decreased levels of HDL cholesterol are associated with increased risk for these diseases. Multiple studies have shown large individual-to-individual variation in plasma non-HDL and HDL cholesterol levels (1Havel R.J. Kane J.P. Introduction: structure and metabolism of plasma lipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. Vol. 2. McGraw-Hill, New York2001: 2705-2716Google Scholar). The causes of this variation and the regulation of non-HDL and HDL cholesterol levels are only partially understood. Current understanding supports a complex interaction between environmental and genetic determinants. Yet, whereas much is known about the nature and effect of environmental factors including cigarette smoking, total dietary fat intake, types of dietary fatty acids, physical activity, and alcohol consumption, relatively little is known about the genetic basis of this variation. Data from twin and family studies have shown that ∼50% of the interindividual variability in LDL cholesterol and HDL cholesterol can be ascribed to genetic determinants; however, only some of the genes involved have so far been identified (2Perusse L. Despres J.P. Tremblay A. Leblanc C. Talbot J. Allard C. Bouchard C. Genetic and environmental determinants of serum lipids and lipoproteins in French Canadian families.Arteriosclerosis. 1989; 9: 308-318Crossref PubMed Google Scholar, 3Rice T. Vogler G.P. Perry T.S. Laskarzewski P.M. Rao D.C. Familial aggregation of lipids and lipoproteins in families ascertained through random and nonrandom probands in the Iowa Lipid Research Clinics family study.Hum. Hered. 1991; 41: 107-121Crossref PubMed Scopus (41) Google Scholar, 4Austin M.A. King M.C. Bawol R.D. Hulley S.B. Friedman G.D. Risk factors for coronary heart disease in adult female twins. Genetic heritability and shared environmental influences.Am. J. Epidemiol. 1987; 125: 308-318Crossref PubMed Scopus (184) Google Scholar, 5Bucher K.D. Friedlander Y. Kaplan E.B. Namboodiri K.K. Kark J.D. Eisenberg S. Stein Y. Rifkind B.M. Biological and cultural sources of familial resemblance in plasma lipids: a comparison between North America and Israel–the Lipid Research Clinics Program.Genet. Epidemiol. 1988; 5: 17-33Crossref PubMed Scopus (35) Google Scholar, 6Tall A.R. Breslow J.L. Rubin E.M. Genetic disorders affecting plasma high-density lipoproteins.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. Vol. 2. McGraw-Hill, New York2001: 2915-2936Google Scholar). Studies in pedigrees that segregate mutations in critical genes largely expand the understanding of the physiological and metabolic aspects of lipoprotein carriers of non-HDL and HDL cholesterol. Yet, although in some populations variants of these genes are sufficiently common to have an impact on non-HDL and HDL cholesterol levels, it is likely that other genes are involved. Previous mapping studies in the mouse have identified loci in linkage with plasma non-HDL and HDL cholesterol levels. For example, Ko et al. used human apolipoprotein B (apoB) transgenics in a C57BL/6 × 129 cross to identify loci on chromosome 6 and chromosome 4 in linkage with plasma apoB levels, a signature apolipoprotein for plasma non-HDL cholesterol lipoproteins (7Ko C. Lee T.L. Lau P.W. Li J. Davis B.T. Voyiaziakis E. Allison D.B. Chua Jr., S.C. Huang L.S. Two novel quantitative trait loci on mouse chromosomes 6 and 4 independently and synergistically regulate plasma apoB levels.J. Lipid Res. 2001; 42: 844-855Abstract Full Text Full Text PDF PubMed Google Scholar). Mehrabian et al. used a C57BL/6J × CAST/Ei cross to map loci on chromosomes 2, 3, 5, 8, 9, 14, 16, 17, and 18 in linkage with plasma HDL cholesterol (8Mehrabian M. Castellani L.W. Wen P.Z. Wong J. Rithaporn T. Hama S.Y. Hough G.P. Johnson D. Albers J.J. Mottino G.A. Frank J.S. Navab M. Fogelman A.M. Lusis A.J. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J).J. Lipid Res. 2000; 41: 1936-1946Abstract Full Text Full Text PDF PubMed Google Scholar). Here we describe two mouse strains, C57BL/6J and CASA/Rk, that, when fed a chow diet, display different plasma levels of non-HDL cholesterol. To examine the genetic basis of this difference, an intercross was performed and linkage for non-HDL cholesterol as well as HDL cholesterol assessed in the F2 progeny. We identified three loci, on chromosomes 6, 4, and 8, in linkage with plasma non-HDL cholesterol, and two loci on chromosomes 9 and 8 in linkage with HDL cholesterol. Moreover, analysis of loci interactions identified a significant epistatic interaction that affects plasma non-HDL cholesterol levels. The inbred mouse strains C57BL/6J and CASA/Rk were purchased from The Jackson Laboratories (Bar Harbor, ME). CASA/Rk males were mated with C57BL/6J females to generate F1 animals. F1 males were intercrossed with F1 females to generate 369 F2 animals (185 males and 184 females). All animals were bred and housed in a single humidity- and temperature-controlled room with a 12 h dark-light cycle (6 AM–6 PM light-dark cycle) at the Laboratory Animal Research Center at Rockefeller University and fed with a single lot of Picolab Rodent Chow 20 (catalog #5053) pellet containing 0.02% w/w cholesterol. At the age of 11 weeks, food was removed from the cages at 10 AM and the animals were allowed access to water. At 3 PM, the mice were anaesthetized with ketamine/xylazine, tail tipped for DNA extraction, and blood samples collected by heart puncture into EDTA-containing tubes. Plasma was immediately separated, plasma density was adjusted to d = 1.063 gm/ml, and non-HDL and HDL fractions (d ≤ 1.063 g/ml and d > 1.063 g/ml, respectively) were isolated after overnight spinning at 40,000 rpm (Beckman L8-55M ultracentrifuge, rotor type 42.2 Ti) at 4°C. The non-HDL and HDL fractions were separated and kept at −80°C for analysis of cholesterol concentration as described below. All experiments were approved by the Institutional Animal Care and Research Advisory Committee. Total plasma cholesterol, non-HDL, and HDL cholesterol levels were determined enzymatically using a Sigma kit. It is of note that plasma total cholesterol, non-HDL, and HDL cholesterol levels in C57BL/6J, CASA/Rk, and F1 males and females were measured in one assay, whereas the measurements in F2 males and F2 females were determined in a another assay. The profile of plasma lipoprotein cholesterol was determined by on-line postcolumn analysis of Superose 6 gel-filtration as described previously (9Aalto-Setala K. Bisgaier C.L. Ho A. Kieft K.A. Traber M.G. Kayden H.J. Ramakrishnan R. Walsh A. Essenburg A.D. Breslow J.L. Intestinal expression of human apolipoprotein A-IV in transgenic mice fails to influence dietary lipid absorption or feeding behavior.J. Clin. Invest. 1994; 93: 1776-1786Crossref PubMed Google Scholar). Tail tips from parentals, F1, and F2 mice were digested with proteinase K, and DNA was precipitated with ethanol. Fluorescently labeled primers corresponding to 350 markers shown to be polymorphic between C57BL/6Jand CAST/Ei by Iakoubova et al. (10Iakoubova O.A. Olsson C.L. Dains K.M. Choi J. Kalcheva I. Bentley L.G. Cunanan M. Hillman D. Louie J. Machrus M. West D.B. Microsatellite marker panels for use in high-throughput genotyping of mouse crosses.Physiol. Genomics. 2000; 3: 145-148Crossref PubMed Scopus (15) Google Scholar) were tested to see if they were also polymorphic between C57BL/6J and CASA/Rk. This resulted in the identification of 255 markers that were used for genotyping in the current cross. The average spacing between these markers was 5.9 cM. Markers were subjected to PCR amplification using fluorescently labeled primers, and PCR products were analyzed by capillary electrophoresis using the ABI 3700 DNA sequencer. All PCR reactions and electrophoresis were automated using the Tecan, Genesis RST 100, and Robbins Scientific Hydra 384 robots and carried out by the Starr Center Genotyping Core Facility at the Rockefeller University. Allele scores were analyzed using the ABI Genotyper 3.6 NT software. The marker positions in cM correspond to mapping data found in the Mouse Genome Informatics Database at http://www.informatics.jax.org. Differences in plasma non-HDL, HDL, and total cholesterol levels between parentals and F1s and comparisons of plasma non-HDL and HDL cholesterol levels for F2s with the various combinations of genotypes at D6Mit63, D4Mit46, D8Mit171, D9Mit325, and D8Mit91 were analyzed using one-way ANOVA with Tukey's posttest. Differences in plasma non-HDL and HDL cholesterol between F2 males and females were analyzed using the unpaired Student t-test. Linkage, interval mapping (using the maximum likelihood algorithm), and loci interactions (using the Haley-Knott regression) were analyzed with the R/qtl software package, Version 0.94-17, with sex as a covariate. R/qtl was also used to permute the actual data sets for F2s to determine the significant LODs at the 95% genome-wide threshold level. In addition, R/qtl was used to identify locus-locus interactions by computing a joint LOD score. This joint LOD score represents a composite of the proportion of the trait variance explained by each locus, which together correspond to the additive component of the interaction and the proportion explained through epistasis. Finally, R/qtl was used to permute the data sets for F2s to determine the threshold LOD score for the joint and epistasis LODs at which 95% significance is achieved. This software package, developed by Karl Broman and Gary Churchill, is publicly available at The C57BL/6J and CASA/Rk at chromosome 6, 4, and 8 for non-HDL cholesterol and chromosomes 9 and 8 for were assessed in F2 males and F2 females for and using the software. To determine the plasma levels of non-HDL and HDL cholesterol, we subjected the plasma of chow-fed male and female C57BL/6J, CASA/Rk, and F1s to density and measured the of non-HDL cholesterol (d ≤ 1.063 and HDL cholesterol (d > 1.063 in each shown in CASA/Rk males and females plasma non-HDL cholesterol levels that were 87% and 25% higher than the corresponding values in C57BL/6J males and the levels of non-HDL cholesterol in F1 males and females were similar to in C57BL/6J out a for HDL cholesterol, although females to display levels that were lower than the corresponding values in no significant were found between C57BL/6J, CASA/Rk, and For of cholesterol different of we subjected the plasma of C57BL/6J, CASA/Rk, and F1 males and females to shown in when with C57BL/6J and F1, CASA/Rk males and females increased cholesterol, with no in LDL or HDL cholesterol non-HDL cholesterol, HDL cholesterol, and total plasma cholesterol levels in and F1 = C57BL/6J CASA/Rk, CASA/Rk F1, = C57BL/6J CASA/Rk, CASA/Rk F1, males and CASA/Rk F1, C57BL/6J = 5.9 CASA/Rk = F1 = C57BL/6J = CASA/Rk = F1 = = C57BL/6J CASA/Rk, CASA/Rk F1, = C57BL/6J CASA/Rk, CASA/Rk F1, males and CASA/Rk F1, in a To examine the of plasma non-HDL and HDL cholesterol levels, F1 animals were and the of non-HDL and HDL cholesterol were determined in 369 F2 mice (185 males and 184 females). To control for we the of non-HDL and HDL cholesterol in F2 males and females and found that, with F2 F2 males display significantly higher non-HDL and levels cholesterol of and HDL cholesterol of in males and a of plasma non-HDL and HDL cholesterol in F2 males and with lower levels in that in plasma non-HDL and HDL cholesterol levels are by than one Moreover, the data a effect on plasma non-HDL and HDL cholesterol levels, and linkage analysis with sex as a covariate. To identify loci that control plasma levels of non-HDL and HDL cholesterol, a was on the F2 mice. For non-HDL cholesterol, quantitative trait mapping using the R/qtl with sex as a significant linkage on chromosomes 6, 4, and The interval for these chromosomes are shown in With regard to chromosome 6, the analysis a of linkage at 49 with a maximum LOD score of that between markers at cM and at cM. With regard to chromosome 4, the a of linkage at 55 cM with a maximum LOD score of that between markers at cM and at cM. for the on chromosome 8, the analysis found a of linkage at 7 cM with a maximum LOD score of that between markers at cM and at 8 cM. analysis of HDL cholesterol with sex as significant linkage on chromosomes 9 and The interval for these chromosomes are shown in With regard to chromosome 9, the analysis a of linkage at cM with a maximum LOD score of that between markers at 14 cM and at cM. Finally, for chromosome 8, the analysis disclosed a of linkage at cM with a maximum LOD score of that between markers D8Mit91 at cM and at 9 and 8 linkage of plasma HDL cholesterol in The marker positions correspond to mapping data found in the Mouse Genome Informatics At the of linkage for plasma non-HDL cholesterol, the in by the were for F2 males and F2 females and are shown in 2. For the loci on chromosome 6 and chromosome 4, for the C57BL/6J had higher plasma non-HDL cholesterol levels than did and for the CASA/Rk At the chromosome 6 locus, the effect of the CASA/Rk a of whereas at the chromosome 4 locus, the CASA/Rk a and of in males and At the chromosome 8 locus, for the CASA/Rk had higher plasma non-HDL cholesterol levels than did and for the C57BL/6J At this locus, the effect of the CASA/Rk a of In the loci on chromosomes 6, 4, and 8 to and of the variance in non-HDL cholesterol levels, in females the loci to and of the effect on non-HDL cholesterol in F2 males and in in = = = = = = in a At the of linkage for HDL cholesterol, the in by the were for F2 males and F2 females and shown in For the on chromosome 9, for the CASA/Rk had higher plasma HDL cholesterol levels than did and for the C57BL/6J At this locus, the effect of the CASA/Rk a of For the chromosome 8 locus, for the C57BL/6J had higher plasma HDL cholesterol levels than did and for the CASA/Rk At this locus, the effect of the CASA/Rk a and of in males and Finally, in F2 it that the on chromosomes 9 and the on chromosome 8 each of the variance in HDL cholesterol levels, in F2 females the loci to and of the effect on HDL cholesterol in F2 males and HDL in HDL in = = = = = = = = in a interactions in plasma non-HDL and HDL cholesterol levels were The R/qtl software the positions of the loci, the joint LOD score of the the LOD score of in the of 2, the LOD score of 2 in the of and an LOD score of epistasis. For non-HDL cholesterol, significance for a joint LOD score was for the interactions of of were of an additive nature In as shown in 4, a interaction was observed between loci on chromosomes 2 and 5, for which the joint LOD was significant the epistasis LOD of was For HDL cholesterol, significance for a joint LOD was for the interactions of of loci, which were of an additive nature interaction in plasma non-HDL cholesterol 2 95% significance for and of and 2 95% significance for and of and 95% significance for and of and in a In the we describe two mouse inbred strains, C57BL/6J and CASA/Rk, which on a chow display different plasma levels of non-HDL cholesterol. these strains in a genetic we have loci in linkage with plasma non-HDL and HDL cholesterol levels. This analysis three loci on chromosomes 6, 4, and 8 in linkage with plasma levels of non-HDL cholesterol, and two loci on chromosomes 9 and 8 in linkage with plasma levels of HDL cholesterol. In addition, for plasma non-HDL cholesterol levels, a significant epistatic interaction was Previous studies in the mouse have identified loci in linkage with non-HDL cholesterol levels. Ko et al. performed an intercross between C57BL/6J × 129 mouse strains with the F2s for a human apoB (7Ko C. Lee T.L. Lau P.W. Li J. Davis B.T. Voyiaziakis E. Allison D.B. Chua Jr., S.C. Huang L.S. Two novel quantitative trait loci on mouse chromosomes 6 and 4 independently and synergistically regulate plasma apoB levels.J. Lipid Res. 2001; 42: 844-855Abstract Full Text Full Text PDF PubMed Google Scholar). human apoB as a marker for plasma non-HDL they found linkage on chromosome 6 that at and on chromosome 4 that at on chromosome 6 with non-HDL cholesterol on chromosome 6 at 49 on chromosome 4 is about cM to on chromosome 4 at 55 cM. It is of note that in both the 129 and CASA/Rk the plasma levels of apoB and non-HDL cholesterol, In the Ko the of both the chromosome 6 and chromosome 4 129 to in a In in the chromosome 6 and chromosome 4 CASA/Rk to in a of this difference the 129 and CASA/Rk segregate different at the loci on chromosome 6 and chromosome 4, the 129 and CASA/Rk segregate the that the two genetic and the linkage in the two is to different genes in each The is that 129 and CASA/Rk different of separated by one of Mouse and New Scholar). The non-HDL cholesterol we to chromosome 8 been in Previous studies in the mouse have identified loci in linkage with HDL cholesterol levels. Mehrabian et al. fed C57BL/6J × CAST/Ei F2s with a chow for and measured HDL cholesterol, the animals to an for an 5 and HDL cholesterol (8Mehrabian M. Castellani L.W. Wen P.Z. Wong J. Rithaporn T. Hama S.Y. Hough G.P. Johnson D. Albers J.J. Mottino G.A. Frank J.S. Navab M. Fogelman A.M. Lusis A.J. Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J).J. Lipid Res. 2000; 41: 1936-1946Abstract Full Text Full Text PDF PubMed Google Scholar). chow they found linkage on chromosome 9 that at and on chromosome 8 that at on chromosome 9 and chromosome 8 with HDL cholesterol on chromosome 9 at cM and on chromosome 8 at cM. In the chromosome 9 CAST/Ei increased and the chromosome 8 CAST/Ei decreased HDL cholesterol levels. loci to in a This is similar to we observed for the CASA/Rk in cross. This is the inbred strains CAST/Ei and CASA/Rk were both from the of mice at the Jackson and have the at the chromosome 9 and chromosome 8 It is in that have higher non-HDL cholesterol levels than analysis in the F2s CASA/Rk at both chromosome 6 and chromosome 4 that non-HDL cholesterol levels This type of been observed in other M. J. J.S. S. J. J.D. Breslow J.L. A genetic novel loci in the PubMed Google and that the CASA/Rk at these loci are in the of the CASA/Rk non-HDL cholesterol levels in the of one or C57BL/6J This the of significant The mouse was for known genes in the of loci for non-HDL cholesterol on chromosome 6 chromosome 4 and chromosome 8 The chromosome 6 interval the at which an that and fatty This interval also the apoB at which an of the complex that into The is to the chromosome 6 at 49 whereas the is 10 cM The chromosome 4 interval the at which the for a by that and the at which a fatty the 2 at which a that fatty into the for and the at which an that the of into The and genes are to the chromosome 4 at 55 whereas the and genes are 8 cM and 12 cM to the The chromosome 8 interval the at 6 which an in the This is to the chromosome 8 at 7 cM. The was also for genes in the of loci for HDL cholesterol on chromosome 9 and chromosome 8 The chromosome 9 interval the and at cM. The genes at this are known to physiological in plasma HDL cholesterol is the of HDL, and and have been shown in both mouse and human studies to regulate levels, which are with HDL cholesterol levels. This is 8 cM to the chromosome 9 at cM. The chromosome 8 interval did genes known to a in lipoprotein or lipid In summary, a cross between two strains that in plasma non-HDL cholesterol loci on chromosomes 6, 4, and 8 that control the plasma levels of non-HDL cholesterol, and loci on chromosomes 9 and 8 that control the plasma levels of HDL cholesterol. Moreover, we found a significant epistatic interaction that the levels of plasma non-HDL cholesterol. It is that the of genes at these loci at of the variability in non-HDL and HDL cholesterol levels in and to the of novel for of

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