Novel QTLs for HDL levels identified in mice by controlling for Apoa2 allelic effects: confirmation of a chromosome 6 locus in a congenic strain

Carrie L. Welch1, Sara Bretschger1,2, Ping-Zi Wen3, Margarete Mehrabian3, Nashat Latib1, Jamila Fruchart-Najib6, Jean Charles Fruchart6, Christy Myrick7 and Aldons J. Lusis3,4,5

1 Division of Molecular Medicine, Department of Medicine
2 Institute of Human Nutrition, Columbia University, New York, New York 10032
3 Departments of Medicine
4 Microbiology and Molecular Genetics
5 Molecular Biology Institute, University of California, Los Angeles, California 90095
6 Department of Atherosclerosis, Pasteur Institute, Lille 59019, France
7 University of Florida, Gainesville, Florida 32610


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Atherosclerosis is a complex disease resulting from the interaction of multiple genes, including those causing dyslipidemia. Relatively few of the causative genes have been identified. Previously, we identified Apoa2 as a major determinant of high-density lipoprotein cholesterol (HDL-C) levels in the mouse model. To identify additional HDL-C level quantitative trait loci (QTLs), while controlling for the effect of the Apoa2 locus, we performed linkage analysis in 179 standard diet-fed F2 mice derived from strains BALB/cJ and B6.C-H25c (a congenic strain carrying the BALB/c Apoa2 allele). Three significant QTLs and one suggestive locus were identified. A female-specific locus mapping to chromosome 6 (Chr 6) also exhibited effects on plasma non-HDL-C, apolipoprotein AII (apoAII), apoB, and apoE levels. A Chr 6 QTL was independently isolated in a related congenic strain (C57BL/6J vs. B6.NODc6: P = 0.003 and P = 0.0001 for HDL-C and non-HDL-C levels, respectively). These data are consistent with polygenic inheritance of HDL-C levels in the mouse model and provide candidate loci for HDL-C and non-HDL-C level determination in humans.

atherosclerosis; lipoprotein; quantitative trait locus


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PLASMA CHOLESTEROL LEVELS are major risk factors for atherosclerosis, a disease that causes up to 50% of all mortality in industrialized countries (2). Specifically, plasma low-density lipoprotein cholesterol (LDL-C) levels are positively associated with atherosclerosis, and high-density lipoprotein cholesterol (HDL-C) levels are negatively associated with the disease (10, 24). Data from family and twin studies indicate that genetic factors account for 40–60% of the interindividual variation of plasma cholesterol levels (10). Although a number of single gene mutations have been identified that give rise to rare dyslipidemias and increased risk for atherosclerosis (12), few of the genes involved in common variations of plasma cholesterol levels are known. Association studies focusing on known candidate genes have revealed that polymorphisms of the apolipoprotein E (apoE) (28, 47, 58) and cholesterol 7{alpha}-hydroxylase (9, 64) genes contribute to variations of LDL-C levels in the general population. Similarly, polymorphisms of the hepatic lipase promoter have been associated with HDL-C levels in a sex-specific manner (21), and heterozygosity for mutations of the ATP-binding cassette, subfamily A, member 1 (ABCA1) gene have been associated with low HDL-C levels (7). Taken together, allelic variations of these genes explain less than 10% of the variation in plasma cholesterol levels; the genes underlying the vast majority of the genetic variation are unknown.

Because of the inherent difficulties of carrying out linkage analysis for complex traits in humans, many mammalian geneticists have turned to animal models. In the mouse model, an Apoa2-linked locus on distal chromosome 1 (Chr 1) has been associated with HDL-C levels in multiple crosses involving different strains of mice (11, 37, 42, 51, 62). In mice fed a standard diet, the locus accounts for 18–60% of the genetic variance of HDL-C levels depending on the genetic context. The results of both classic and molecular genetic studies suggest that structural variations in Apoa2 can account for the variations in HDL-C levels attributed to the Chr 1 locus (11, 15, 23, 42, 51, 62, 66). Although polymorphisms of human APOA2 have been associated with moderate effects on HDL-C level and/or composition (3, 5, 22, 55), clearly other factors are yet to be identified.

To identify genetic loci influencing HDL-C levels in the absence of segregating Apoa2 alleles, we performed quantitative trait locus (QTL) analysis in a cross between B6.C-H25c/(HW65)By (B6.C-H25c, a congenic strain carrying an Apoa2-containing donor interval from BALB/cBy) and BALB/cJ mice. BALB/cBy and BALB/cJ are substrains carrying identical forms of Apoa2 based on sequence analysis, isoelectric focusing, and plasma apolipoprotein concentrations (15, 31, 35, 44). In the current study, all of the F2 progeny were homozygous for the BALB-derived Apoa2 allele. The results suggest that controlling for major gene effects in an experimental cross can facilitate the identification of unique loci having significant effects on a phenotype. The polygenic model of plasma HDL-C levels described here provides candidate loci for HDL-C, as well as non-HDL-C, level determination in humans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice.
B6.C-H25c (stock no. 000114) and BALB/cJ (stock no. 000651) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). (The B6.C-H25c strain was originally constructed to study the H25c histocompatibility locus on mouse Chr 1; see Ref. 1.) B6.C-H25c females were mated with BALB/cJ males to produce F1 mice. Brother-sister matings were set up to produce F2 mice. Mice were weaned onto standard diet at 21 days and housed 3–6 animals/cage. At 5–6 mo of age, the mice were bled following an 8.5-h fast. The breeding colony was produced and maintained in a conventional facility at the University of California, Los Angeles. Age-matched (5–6 mo old) parental and F1 mice were purchased from the Jackson Laboratory for plasma cholesterol analyses.

B6.NODc6 mice, a congenic strain carrying an NOD-derived region of distal Chr 6 on the B6 background (69), were bred and housed at the University of Florida Department of Pathology Mouse Colony, a specific pathogen-free facility. The NOD-derived interval is ~27 cM in length, extending from D6Mit108 (at 48.1 cM) (69) to D6Mit304 (at 75 cM) (determined herein, data not shown). B6 controls were also maintained at the University of Florida facility. Mice were weaned onto standard diet at 21 days. At 3 mo or 6–10 mo, as indicated, the mice were bled without fasting.

For crosses with Ldlr knockout mice, B6.129S7-Ldlrtm1Her (B6.LdlrKO, a congenic strain carrying a targeted Ldlr gene on the B6 background) (stock no. 002207) mice were purchased from the Jackson Laboratory. B6.NODc6 mice were transferred from the University of Florida to the Columbia University Institute of Comparative Medicine, a specific pathogen-free facility. B6.NODc6 females were mated with B6.LdlrKO males to produce F1 progeny. Brother-sister matings were set up to produce F2 mice homozygous for the LdlrKO allele, with or without the NODc6 congenic interval. Mice were weaned onto standard diet at 21 days and housed 2–5 mice/cage. At 2–3 mo of age, the mice were bled following a 5-h fast. The mice were then switched to a high-cholesterol diet (described below) for 2 wk and bled again.

The mice were allowed free access to food and water throughout the course of the study. The relevant protocols at each institution were approved by the respective Institutional Animal Care and Research Advisory Committees.

Diets.
Mice were fed either P-5001 standard diet (TD 99479; Harlan Teklad, Madison, WI) or a 1.25% cholesterol/15% fat/0.5% cholic acid ("Paigen") diet (TD 88051, Harlan Teklad), as indicated.

Plasma lipoprotein measurements.
Retro-orbital bleeding was performed under Forane anesthesia (Baxter, Deerfield, IL). Blood was collected directly into heparinized capillary tubes (Becton-Dickinson). Plasma was separated from cells by centrifugation and stored at -70°C. Isolation of HDL-C by chemical precipitation (HDL reagent, Sigma), as well as enzymatic measurements of cholesterol and triglycerides (Wako Pure Chemical Industries), were carried out according to the manufacturers’ instructions. Non-HDL-C was calculated by subtracting HDL-C from total cholesterol (TC).

Apolipoprotein measurements.
Mouse plasma apoAI, apoAII, apoCIII, and apoE were quantified by a sandwich enzyme-linked immunosorbent assay (ELISA) using anti-synthetic peptide antibodies generated in rabbit as previously described (61). Mouse plasma apoB was measured by nephelometry using polyclonal antibodies generated in rabbit against purified mouse apoB.

Construction of linkage map.
Genomic DNA was isolated using a standard phenol/chloroform extraction method. Microsatellite markers (13, 34) were typed by PCR amplification using primers purchased from Research Genetics (Huntsville, AL) and following the manufacturer’s protocol. The markers were screened for polymorphic bands using parental and F1 DNA. The segregation patterns of parental alleles among the F2 mice were entered into a Map Manager QT database (38, 39). Marker orders were estimated from published data (Mouse Genome Database) and confirmed in this cross by minimizing the number of double and multiple recombination events between markers within a linkage group. A small number of anomalous typings (double recombinations which could not be accounted for by scoring errors) were not included in the calculation of genetic length or QTL mapping. A total of 110 markers distributed across the 19 autosomes and X chromosome were used for statistical analyses. A list of markers and the resulting linkage map is available from the authors.

Genetic screening of (B6.NODc6 x B6.LdlrKO)F2 mice.
DNA was extracted from tail tips by a quick alkaline lysis protocol (63). Briefly, the tail tips were incubated in 50 mM NaOH for 1 h at 95°C, vortexed, and neutralized in 1 M Tris (pH 8). Cellular debris was pelleted by centrifugation, and the supernatant was used for PCR amplification of Ldlr alleles and microsatellite markers defining the ends of the Chr 6 congenic region. Primer sequences and a protocol for Ldlr genotyping were obtained from http://www.jax.org/resources/documents/imr/protocols/Ldlr_KO.html (August 5, 1998). Ldlr typings were confirmed by measuring plasma TC levels. Primers for the microsatellite markers D6Mit108 (proximal) and D6Mit14 (distal) were purchased from Research Genetics.

Statistical analysis.
ANOVA, regression analyses, and correlation analyses were performed using Statview 5.0 (Abacus Concepts) for Macintosh computers. Linkage analyses were performed using MAPMAKER/QTL 3.0 and Map Manager QTb28ppc as described for F2 intercrosses (39, 48); both analyses yielded comparable results. Results are expressed as logarithm of the odds ratio (LOD) scores. Significance thresholds were determined by permutation analysis with 10,000 permutations in 2-cM steps. Support intervals are defined by a 1-unit decrease in LOD score on either side of the peak marker. Recessivity and dominance are relative to the B6-derived alleles. Because of the strong effect of sex on lipoprotein phenotypes, all analyses were performed separately for males and females.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Plasma lipoprotein cholesterol profiles of B6.C-H25c, BALB/cJ, F1, and F2 intercross mice.
B6.C-H25c mice exhibited 20–30% higher levels of plasma HDL-C and TC, as well as 2.5-fold higher levels of non-HDL-C, relative to BALB/cJ mice (P < 0.01 for all traits, Table 1); these data are in agreement with a previous report (42). (B6.C-H25c x BALB/cJ)F1 mice exhibited similar levels of HDL-C and TC relative to BALB/cJ mice, suggesting the action of a dominant HDL-C-lowering factor(s) contributed by the BALB/cJ genome. Non-HDL-C levels in F1 mice were lower than the levels observed in either parental strain, suggesting additive or interacting effects of more than one gene. Male mice had ~20% higher levels of HDL-C and TC than female mice of the same strain (P < 0.03); the only exception was no difference in TC levels between males and females of the B6.C-H25c strain. No effect of sex was observed for levels of non-HDL-C.


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Table 1. Plasma lipoprotein levels in standard diet-fed B6.C-H25c, BALB/cJ, and (B6.C-H25c x BALB/cJ)F1 mice

 
To further characterize the lipoprotein differences between the parental strains, plasma apolipoprotein levels were measured in B6.C-H25c, BALB/cJ, and F1 mice (Table 2). ApoAII and apoAI represent the predominant protein components of HDL particles. B6.C-H25c mice exhibited higher mean plasma apoAII levels (P < 0.05) but lower apoAI levels (P < 0.01) relative to BALB/cJ mice. Since the parental strains carry the same apoAII structural gene, the observed differences in plasma levels of apoAII must be due to allelic variations at another locus acting in trans. The lower levels of apoAI observed in B6.C-H25c mice suggest complex regulation of HDL-C levels; apoAI-independent regulation of HDL-C levels has been observed in segregation analysis of families as well (27). Levels of apoAII and apoAI in F1 mice overlapped the levels observed in both parental strains. Somewhat surprisingly, the most striking difference in apolipoprotein levels observed between the strains was for apoB (the sole apoprotein component of LDL particles). B6.C-H25c mice exhibited nearly twofold higher levels of apoB compared with BALB/cJ or F1 mice (P < 0.0001). In addition, B6.C-H25c mice had lower apoCIII (P < 0.01 vs. BALB/cJ; P < 0.05 vs. F1) and higher apoE levels (P < 0.01 vs. BALB or F1) than BALB/cJ or F1 mice.


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Table 2. Plasma apolipoprotein levels in standard diet-fed, female B6.C-H25c, BALB/cJ, and (B6.C-H25c x BALB/cJ)F1 mice

 
The distributions of HDL-C and TC levels among standard diet-fed male and female (B6.C-H25c x BALB/cJ)F2 mice are shown in Fig. 1. As expected for mice fed standard diet, there was a highly significant correlation between HDL-C and TC levels in the animals (correlation coefficient = 0.96, P < 0.0001). The distributions of both traits were continuous and approached normality in both sexes. The range of F2 values exceeded mean ± 1SD parental intervals for each trait and sex. Male mice had ~20% higher mean levels of HDL-C and TC than female mice (P < 0.0001 for both traits). Taken together, these data suggested complex inheritance of plasma HDL-C levels in this cross.



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Fig. 1. Distributions of total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels among 179 (B6.C-H25c x BALB/cJ)F2 mice fed standard diet. Values are expressed in milligrams per deciliter. Horizontal bars represent the mean ± 1SD intervals for B6.C-H25c (heavy black line), BALB/cJ (thin black line), and F1 (dotted line) mice.

 
Construction of a complete linkage map.
A linkage map was constructed using a panel of 179 (B6.C-H25c x BALB/cJ)F2 mice and 110 microsatellite markers which spanned the entire mouse genome (excluding Chr Y). The resulting map has an average marker spacing of 12 cM and an overall genetic length of 1,300 cM, slightly less than previous estimates of 1,361–1,600 cM (8, 14, 16, 19). Although strain-specific differences in recombination rates have been documented (reviewed in Ref. 57), the relatively short genetic length observed in this study is likely due to the disproportionate number of males used (102 males vs. 65 females). On average, male mice exhibit rates of recombination 50–85% of that observed in females (57). A 34-cM region of nonpolymorphism was identified on proximal Chr 2, between markers D2Mit355 and D2Mit12, indicating a region of nondivergence between B6 and BALB/cJ (this is not unusual since inbred strains of mice have been observed to carry blocks of low diversity, sometimes extending across tens of megabases; Ref. 67). Three gaps in the linkage map, with marker spacing greater than 20 cM, occur on proximal Chr 3 (26 cM gap between D3Mit221 and D3Mit22), proximal Chr 6 (21 cM between D6Mit1 and D6Mit4), and distal Chr 18 (29 cM between D18Mit53 and D18Mit213).

Refined mapping of the Apoa2-containing donor interval on Chr 1.
The boundaries of the Apoa2-containing congenic region, on Chr 1, were previously defined by restriction fragment length polymorphism analysis (42). The proximal site of recombination was reported to reside between At3 (currently listed at 84.6 cM distal to the centromere; 46) and Fcgr2b (92.3 cM), whereas the distal site of recombination was between Spna1 (95.2 cM) and Crry (erroneously listed at 106.6 cM, see below). We have confirmed these results and refined the distal site using microsatellite markers (Fig. 2). Thus D1Mit33 and D1Mit424 (both at 81.6 cM) were mapped proximal to the congenic interval; D1Mit209 (106.1 cM), D1Mit17 (106.3 cM), and D1Mit223 (106.3 cM) mapped within the interval; and D1Mit155 (112 cM) mapped distal to the interval. A discrepant marker, D1Mit293 (109.6 cM), was nonpolymorphic between B6.C-H25c and BALB/cJ but polymorphic between B6.C-H25c and B6, indicating that it mapped within the congenic interval. These data suggest an apparent error in the relative localization of Crry and D1Mit293 in the Mouse Genome Database (MGD) but are consistent with data obtained from the Ensembl mouse genome browser (ver. 14.30.1; http://www.ensembl.org/Mus_musculus/) (May 6, 2003) which lists Crry as residing distal to both D1Mit293 and D1Mit155.



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Fig. 2. Refined mapping of the Apoa2-containing congenic interval on chromosome 1 (Chr 1). Chr 1 is drawn with the centromeric end positioned at the top, and distal end is at the bottom. The donor segment exhibiting nonpolymorphism between B6.C-H25c and BALB/cJ but polymorphism between B6.C-H25c and B6 is represented by a solid black rectangle. Sites of recombination occurred proximally between At3 and Fcgr2b (42) and distally between D1Mit293 and D1Mit155; the lengths of the donor segments in-between these markers are unknown and are indicated by hatched rectangles. D1Mit33 and D1Mit424 are polymorphic between B6.C-H25c and BALB/cJ but nonpolymorphic between B6.C-H25c and B6, confirming that this region of Chr 1 lies outside of the congenic interval. Relative locations of markers/genes, taken from the Mouse Genome Database (http://www.informatics.jax.org/), are given in centimorgans (cM) and are indicated to the left of the chromosome. An exception is for Crry, the location of which is apparently in error in MGD and is shown in the relative position suggested by data from the current study as well as the Ensembl mouse genome browser(ver. 14.30.1). The angled brackets ("<...>") indicate genes not mapped in this study.

 
Localization of plasma HDL-C QTLs to Chrs 6, 13, 15, and 19.
Three statistically significant HDL-C QTLs were identified on Chr 6, 13, and 15 (Table 3). In addition, one suggestive QTL was localized to Chr 19 (Table 3). As expected for mice fed standard diet, the HDL-C loci were completely coincident with loci underlying TC levels (Table 3). Two of the loci, on Chrs 6 and 13, showed a high degree of sex specificity; the other loci, on Chrs 15 and 19, exerted similar effects in males and females (Tables 3 and 4). Together, the loci explain 37% and 52% of the total variation of plasma HDL-C levels among F2 males and females, respectively. The significant QTLs on Chr 6, 13, and 15 have been deposited into MGD as Lipq1–3 (lipoprotein QTL 1–3).


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Table 3. QTLs contributing to plasma HDL-C and TC levels in a panel of standard diet-fed (B6.C-H25c x BALB/cJ)F2 mice

 

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Table 4. Genotypic effects of significant QTLs contributing to variations in plasma cholesterol levels in a panel of standard diet-fed (B6.C-H25c x BALB/cJ)F2 mice

 
Lipq1 (Chr 6) exerted the strongest effect on HDL-C levels in F2 females, explaining 28% and 34% of the variance of HDL-C and TC levels, respectively (Table 3). The locus was supported by peak LOD scores of 5.0 (HDL-C) and 6.3 (TC) at D6Mit15. Linkage was detected in females but not males. Similarly, ANOVA analysis indicated a strong sex-specific effect of the locus (Table 4). Inheritance of two copies of the B6-derived allele resulted in 25% higher HDL-C and TC levels relative to inheritance of two copies of the BALB/cJ allele in females (P < 0.0001 for both traits); no effect of genotype was observed in males. Mean plasma cholesterol levels in female heterozygotes were intermediate relative to the levels in homozygotes for either allele, but the ranges of values overlapped the ranges among BALB/cJ homozygotes. When we tested different Mendelian models of inheritance in the female population, an additive model yielded a similar result as a free (unconstrained) model and was highly favored over a dominant model [likelihood ratio (LR), 1,778:1]. The 1-LOD support interval extended ~10 cM (64 cM to the telomere) (Fig. 3).



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Fig. 3. Likelihood plots for HDL-C quantitative trait loci (QTLs) in (B6.C-H25c x BALB/cJ)F2 mice. Plots were obtained using the interval mapping function of Map Manager QTb28ppc under constrained models of inheritance. Black plots reflect the likelihood ratio statistics calculated at 1-cM intervals; gray plots reflect the dominant, additive, or recessive effects, as indicated. Genome-wide significance thresholds of P = 0.001, P = 0.05, and P = 0.63 are indicated by the straight vertical lines. A: female data for Chr 6, additive model. B: male data for Chr 13, additive model. C: female data for Chr 15, dominant model. D: male data for Chr 15, dominant model. E: female data for Chr 19, recessive model. F: male data for Chr 19, recessive model.

 
Lipq2 (Chr 13) exerted the strongest effect on HDL-C levels in F2 males, explaining 18% and 20% of the variance of HDL-C and TC, respectively (Table 3). The Chr 13 QTL was supported by peak LOD scores of 4.8 (HDL-C) and 5.5 (TC) at D13Mit11. Males carrying two copies of the B6-derived allele had 15% lower HDL-C and TC levels relative to mice carrying two copies of the BALB allele (P < 0.0001); no differences in plasma cholesterol levels were observed among females (Table 4). Male heterozygotes had intermediate levels of cholesterol relative to homozygotes for either parental allele, with the range of values overlapping the range among B6 homozygotes. Additive and dominant models were favored over a recessive model of inheritance by LR values greater than 400:1. The 1-LOD support interval for Lipq2 was ~13 cM (Fig. 3).

Lipq3 (Chr 15) and the suggestive locus on Chr 19 affected males and females similarly, explaining 8–14% of the variance of plasma cholesterol levels (Table 3). Lipq3 and the Chr 19 locus were supported by sex-combined LOD scores of 5.2 (HDL-C) and 5.4 (TC) at D15Mit159 and 4.2 (HDL-C) and 4.7 (TC) at D19Mit68, respectively. The B6-derived alleles at these loci conferred different effects on plasma cholesterol levels: increased cholesterol levels at Lipq3 and decreased levels at the Chr 19 locus (Table 4). A dominant model of inheritance of the Chr 15 allele was highly favored over a recessive model (LR, 5,000:1) but only modestly favored over an additive model (LR, 20:1). In contrast, a recessive model was favored for the Chr 19 allele (LR, 100:1).

Lipq1 (Chr 6) has pleiotropic effects on lipoprotein and apolipoprotein levels.
Although the LOD scores for TC tended to be slightly higher than analogous scores for HDL-C levels at each of the QTLs, the difference in TC and HDL-C LOD scores was greater than 1 LOD unit at Lipq1. The difference in LOD scores suggested an additional effect of the Chr 6 QTL on non-HDL-C levels. To test this hypothesis, we performed linkage analysis using non-HDL-C levels in male and female F2 mice. Linkage to Chr 6 was detected in females but not males; the peak was coincident with the peaks for HDL-C and TC levels (data not shown). The locus accounted for 21% of the variance of non-HDL-C levels among females and was supported by a LOD score of 3.7. Homozygosity for the B6-derived allele conferred a twofold increase in plasma non-HDL-C levels relative to homozygosity for the BALB/cJ allele in females (P < 0.0007) but not males (Fig. 4). Heterozygosity resulted in levels of non-HDL-C that were similar to levels in BALB/cJ homozygotes (P < 0.0004 vs. B6 homozygotes). These data suggested that the gene(s) underlying Lipq1 plays a role in determining both HDL-C and non-HDL-C levels.



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Fig. 4. The plasma cholesterol QTL on distal Chr 6 determines non-HDL-C levels in female but not male (B6.C-H25c x BALB/cJ)F2 mice. Values are means ± SD. BB, animals homozygous for the C57BL/6-derived allele at D6Mit15; CC, animals homozygous for the BALB/cJ allele at D6Mit15; Het, animals heterozygous at the marker. The number of mice per group is given in parentheses above each bar. *P < 0.0007 vs. BB.

 
To investigate the effects of Lipq1 on apolipoprotein levels, we compared plasma levels in a subset of female F2 mice grouped by genotype at D6Mit15 (Table 5). F2 mice homozygous for the B6-derived allele exhibited higher levels of plasma apoAII (P < 0.002) as well as apoB (P < 0.01) and apoE (P < 0.05) than mice homozygous for the BALB/cJ allele. No differences in levels of apoAI or apoCIII were observed between the two groups of F2 mice. The relative effects of Lipq1 alleles on HDL-C, non-HDL-C, apoAII, apoB, and apoE levels among the F2 mice reflected the differences observed between the parental strains (Table 2). Thus Lipq1 represents a major determinant of the lipoprotein profile variation between B6 and BALB/cJ strains.


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Table 5. Plasma apolipoprotein levels in a subset of female (B6.C-H25c x BALB/cJ)F2 mice selected by genotype at D6Mit15

 
Confirmation of a Chr 6-linked QTL in both normolipidemic and hypercholesterolemic congenic strains.
QTLs identified by linkage analysis require confirmation in mice having a uniform genetic background (a congenic strain). B6.NODc6 mice were generated previously to test an insulin-dependent (type 1) diabetes-related QTL localized to distal Chr 6; histological examination of the congenic mice revealed no signs of pancreatitis or insulitis (69). We tested the NOD donor interval for the presence of a genetic variant determining lipoprotein levels by comparing TC, HDL-C, and non-HDL-C levels of 3-mo-old control (B6) and congenic (B6.NODc6) mice fed standard diet (Fig. 5A). Female B6.NODc6 mice exhibited 14% higher HDL-C and 4.5-fold lower non-HDL-C levels compared with female wild-type mice (P < 0.003 and P < 0.0001, respectively). Similar results were obtained from a second set of mice bled at 6–10 mo of age (data not shown). Consistent with the original observation of a female-specific effect of the locus, no significant differences were observed between male control and congenic mice (data not shown). These data confirm the existence of a female-specific plasma lipoprotein-regulating gene within the genomic interval. However, the opposite effects of the BALB- and NOD-derived alleles on HDL-C levels in females suggest that either the strains carry different alleles of a single gene affecting lipoprotein levels or two lipoprotein determinants exist within the interval.



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Fig. 5. Plasma lipoprotein cholesterol levels in C57BL/6J and the Chr 6 congenic strain, B6.NODc6. A: females fed standard diet. B: 3-wk Paigen diet-fed females. Values are means ± SD. P values represent significant differences between B6 and B6.NODc6 mice for a given phenotype.

 
Mice fed standard diet exhibit plasma lipoprotein profiles (high HDL-C relative to apoB-containing lipoproteins) that are in stark contrast to the lipoprotein profiles of humans (high non-HDL-C relative to HDL-C). To test the effect of the Chr 6 locus in the presence of a more human-like lipoprotein profile, plasma lipoprotein levels were compared between B6 and B6.NODc6 mice following the feeding of a diet high in cholesterol and fat (Paigen diet) for 3 wk. Although both strains of mice exhibited dramatic shifts in lipoprotein profiles following feeding of the high-fat diet, female congenic mice exhibited 42% lower non-HDL-C levels compared with B6 controls (44 ± 11 vs. 76 ± 13 mg/dl, respectively, mean ± SD; P < 0.002) and higher HDL-C:non-HDL-C ratios (0.710 ± 0.226 vs. 0.319 ± 0.194, respectively, mean ± SD; P = 0.01) (Fig. 5B). No significant differences were observed between males (data not shown).

To further investigate the effect of the Chr 6 locus under the condition of hypercholesterolemia, B6.NODc6 mice were crossed with B6-Ldlr knockout (B6-LdlrKO) mice. Lipoprotein cholesterol levels of homozygous B6.LdlrKO mice, with and without the NODc6 congenic interval, were compared following the feeding of standard diet or Paigen diet (Table 6). Standard diet-fed B6-LdlrKO, NODc6 mice exhibited 24% lower non-HDL-C levels relative to mice without the congenic interval (58 ± 5 vs. 76 ± 7, mg/dl, respectively, mean ± SD; P < 0.008). A trend toward lower non-HDL-C levels in the B6-LdlrKO, NODc6 group was observed following the feeding of the Paigen diet; the difference did not reach statistical significance in the small group of animals studied. Surprisingly, the feeding of the Paigen diet resulted in a marked difference in plasma TG levels: B6-LdlrKO, NODc6 mice exhibited 64% lower mean TG levels relative to B6-LdlrKO mice without the congenic interval (P < 0.01). These studies of B6.NODc6 congenic mice confirm the existence of a Chr 6-linked lipoprotein QTL having pleiotropic effects on plasma lipoprotein levels.


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Table 6. Plasma lipoprotein cholesterol and TG levels in homozygous B6.LdlrKO mice, with and without the NODc6 congenic interval

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have identified novel QTLs for plasma HDL-C levels by controlling for the major gene effect of Apoa2 in an experimental cross. Together, the loci account for ~37–52% of the total variance of HDL-C levels in the population. The remaining ~48–63% of the genetic variation in HDL-C levels is likely due to small gene effects (requiring increased sample size) or gene-gene interactions (not tested in this study). One of the loci mapped in this study, Lipq1 on distal Chr 6, affected apoAII levels independent of the Apoa2 structural gene and explained 28% of HDL-C level variation among females. Somewhat surprisingly, the locus also affected levels of apoB and apoB-containing lipoproteins. In a related congenic strain, an interval donated by the NOD strain was also observed to affect both HDL-C and apoB-containing lipoprotein levels. Lastly, in the presence of hypercholesterolemia resulting from transfer of the Ldlr null allele onto the congenic background, presence of the donor interval significantly affected triglyceride levels. These studies reveal a novel locus on distal Chr 6 having pleiotropic effects on lipoprotein levels and underscore the utility of congenic strains in defining the phenotype attributed to individual QTLs.

Positional candidate genes have been identified for some but not all of the Lipq1–3 loci. The genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Hmgcr) (52) and HMG-CoA synthase (Hmgcs1) (41) both reside within the confidence interval for Lipq2 on Chr 13. Statin-mediated inhibition of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, increases plasma HDL-C levels in humans (49). Inbred strains of mice exhibit large genetic variations in HMG-CoA mRNA and activity levels; notably, B6 and BALB mice differ by about fivefold in hepatic reductase activity, with a similar difference in mRNA levels (26). We compared the complete coding sequences and partial promoter regions (328 nt upstream of the transcription start site) of Hmgcr derived from B6 and BALB/cJ and found only conservative amino acid substitutions in the coding region (Z. Zhang, C. Welch, and A. J. Lusis, unpublished observation). Previously, Garcia and colleagues (18) reported a high degree of sequence conservation in the coding region of the human gene, HMGCR, among both normal and dyslipidemic subjects. Thus, although it is unlikely that sequence variations in the coding region of Hmgcr underlie the Chr 13 QTL, we cannot rule out a possible effect of upstream cis-acting regulatory elements.

The gene encoding peroxisome proliferator-activated receptor-{alpha}, Ppara, resides on Chr 15 within the interval for Lipq3. Activation of the apoAI promoter by the protein product of Ppara, PPAR{alpha}, is the likely mechanism underlying statin-mediated increases in HDL-C levels (40). Two other genes involved in lipid metabolism reside within the Chr 15 interval: Srebf2, encoding a key regulator of cholesterol metabolism (4, 25), and Dgat1, encoding an enzyme required for triacylglycerol synthesis (6). The gene encoding the oxysterol-binding protein (Osbp1) maps to proximal Chr 19 under the peak of our suggestive locus. The oxysterol-binding protein has been implicated in the regulation of intracellular vesicular transport and cholesterol homeostasis (33). No obvious candidate genes reside within the confidence interval for Lipq1 on distal Chr 6.

More than 20 QTLs contributing to plasma HDL-C levels have been identified in the mouse (11, 20, 36, 37, 43, 50, 51, 62, 65). Table 7 lists loci supported by LOD scores greater than 3.3 for a backcross or 4.3 for an intercross (30). Several additional loci were included if the HDL-C QTL was coincident with a related trait meeting the above criteria. For example, a locus on proximal Chr 3 appears to have a primary effect on cholesterol-7-{alpha}-hydroxylase (C7AH) levels secondarily effecting HDL-C levels (37). Several of the loci were detected in more than one cross: the Apoa2-linked locus on distal Chr 1 as well as loci on Chrs 2 (proximal), 5, and 6 (distal). As discussed earlier, the Chr 1 locus is most likely due to genetic variation in Apoa2 in each of the crosses. The Chr 2 locus, detected in two crosses utilizing CAST and different Mus domesticus strains as parent strains, under similar conditions of high-fat, cholic acid-enriched diet feeding, may also be due to genetic variation at a single locus. However, the linkages to Chr 5, representing a rather large region of medial Chr 5 which has been associated with HDL-C levels (and C7AH) and/or non-HDL-C levels, depending on the cross, may be due to variation in different genes residing in the region. Similarly, the Chr 6 loci are likely to be due to different genes: the locus reported in this paper exhibited strong female specificity and additional phenotypic effects on non-HDL-C levels, whereas the two Chr 6 loci reported by Lyons and colleagues (36), at 48 cM and 74 cM, were detected using males only and showed no associations with non-HDL-C levels. Ultimately, fine genetic or physical mapping of each of the loci will provide more definitive proof as to whether the same genes or closely linked genes underlie the QTLs mapped to overlapping chromosomal regions.


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Table 7. HDL-C QTLs detected in various genetic crosses

 
The majority of the HDL-C QTLs listed in Table 7 have been detected only in a specific cross. This is not surprising for a complex trait, since the progeny derived from each set of parental strains represent unique mapping panels for the identification of subsets of trait-related QTLs. Both allelic variation (between parental strains) and genetic context (strain background, type of cross) are critical determinants of whether a given QTL can be detected in a particular cross (see Ref. 17). Thus the total number of genes contributing to variation of a particular trait within a species is best estimated by comparing the results of multiple crosses rather than a single cross. Second, the presence or absence of similar QTLs in different crosses can suggest 1) the degree of polymorphism of a gene, 2) the requirement of modifier genes to detect a locus (54), or 3) the degree of QTL clustering for a given trait (45, 56). Thus the screening of multiple genetic crosses for loci contributing to variation of a particular trait provides a powerful means of understanding the genetic complexity of that trait.

The Chr 6 locus mapped in this study, Lipq1, exhibited effects on apoB and non-HDL-C levels in addition to HDL-C levels. Ko and colleagues (29) have reported a Chr 6 QTL, Abrg1, regulating plasma levels of apoB derived from a human transgene. No effect on HDL-C or apoAII levels was reported. Although both of these QTLs have been mapped to Chr 6, the peak markers reported for each of the loci are separated by ~30 cM (D6Mit55, at 45.9 cM, for Abrg1 vs. D6Mit15, at 75 cM, for Lipq1) and the confidence intervals are not overlapping (40.4–57.9 cM for Abrg1 vs. 64.0–74.0 for Lipq1). These data suggest that different genes underlie the phenotypic effects of Abrg1 and Lipq1. However, variation in location estimates for QTLs contributing to complex traits is likely to occur in independent linkage analyses (53). Refined mapping of Abrg1 and Lipq1 to subcongenic intervals and ultimate identification of the underlying genes will provide direct evidence as to whether the loci represent the same or different QTLs.

Conserved homology between mouse and human chromosomes can be used to predict the human chromosomal locations corresponding to the QTLs reported herein. There is extensive homology between distal mouse Chr 6 and human Chr 12pter-12p13: 82 mouse/human orthologies have been reported without overlap of other regions of conserved homology. Thus the human homolog of Lipq1 most likely resides on human 12pter-12p13. In contrast, the mouse chromosomal regions containing Lipq2, Lipq3, and the suggestive locus on Chr 19 exhibit homologies with two or more human chromosomal regions. The Chr 13-linked QTL overlaps an evolutionary breakpoint in which the proximal half of the interval contains orthologs of human genes mapped to human Chr 5, 7p13–15, 7q35–36, and 9q21–22, but the peak and distal half of the interval overlap a region of exclusive homology with Chr 5. The Chr 15-linked QTL overlaps two breakpoints: 8q/22q12–13 and 22q12–13/12q11–13. The human homolog of the Chr 19-linked QTL most likely resides on 11q12–13 or 9q13–21. These predictions can be utilized, particularly in the case of Lipq1, to direct genetic searches for loci affecting HDL-C level variation in humans.

The set of genes contributing to common variation in HDL-C levels in humans is unlikely to be identical to the set of genes contributing to interstrain variation in the mouse. However, it seems likely that some of the HDL-C level QTLs will be common to both species. For example, QTLs contributing to body fat and plasma insulin levels were localized to distal Chr 2 in two independent mouse crosses (43, 51) as well as the homologous region of human Chr 20q (32). In addition, five regions predicted to harbor blood pressure-related QTLs in rat models were recently identified in genome-wide scans for human hypertension loci (59). Providing further evidence for conservation of QTLs across species, a subset of the blood pressure loci detected in rat and human have also been detected in homologous regions of the mouse genome (60, 68). Ultimately, identification of the gene(s) underlying a particular QTL will be necessary to determine which subset of genes contributes to genetic variation of a trait across species.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
C. L. Welch was supported by National Research Service Award Postdoctoral Fellowship F32-HL-09930-03.


    ACKNOWLEDGMENTS
 
We thank Mikhail Bezouevski, Nick Pleskac, and Anna Gorelik for assistance with animal husbandry.

The QTL mapping data for this paper have been uploaded to the Mouse Genome Informatics database (accession no. MGI J:87344).


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

Address for reprint requests and other correspondence: C. L. Welch, Dept. of Medicine/Division of Molecular Medicine, P&S 8-401, 630 W. 168th St., New York, NY 10032 (E-mail: cbw13{at}columbia.edu).

10.1152/physiolgenomics.00124.2003.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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