Abstract
Plasma plant sterol levels differ among humans due to genetic and dietary factors. A disease characterized by high plasma plant sterol levels, β-sitosterolemia, was recently found to be due to mutations at the ABCG5/ABCG8 locus. To detect variants at this and other loci, a genetic cross was carried out between two laboratory mouse strains. Parental C57BL/6J had almost twice the campesterol and sitosterol levels compared with parental CASA/Rk mice, and F1 mice had levels halfway between the parentals. An intercross between F1s was performed and plasma plant sterol levels measured in 102 male and 99 female F2 mice. Plasma plant sterols in F2s displayed a unimodal distribution, suggesting the effects of several rather a single major gene. In the F2 mice, a full genome scan revealed significant linkages on chromosomes 14 and 2. With regard to chromosome 14, analysis showed a single peak for linkage at 17 cM with a logarithm of odds (LOD) score of 9.9, designated plasma plant sterol 14 (Plast14). With regard to chromosome 2, analysis showed two significant peaks for linkage at 18 and 65 cMs with LOD scores of 4.1 and 3.65, respectively, designated Plast2a and Plast2b, respectively. Four interactions between loci, predominantly of an additive nature, were also demonstrated, the most significant between Plast14 and Plast2b (LOD 16.44). No significant linkage or gene interaction was detected for the ABCG5/ABCG8 locus on chromosome 17. Therefore, other genes besides ABCG5/ABCG8 influence plasma plant sterol levels and now become candidates to explain differences in plasma plant sterol levels between humans.
Plasma plant sterol levels may serve as a marker for aspects of cholesterol metabolism, such as intestinal absorption and biliary excretion (1–4). The most abundant plant sterols in the human diet and in plasma are sitosterol and campesterol. These sterols are structurally similar to cholesterol, except for an ethyl or a methyl group, respectively, attached to carbon 24. Diet is the only source of plant sterols, containing 100–200 mg/day compared with 300–500 mg/day cholesterol. Yet in normal individuals, plasma plant sterol concentrations are 0.3–1 mg/dl, whereas cholesterol concentrations are 150–200 mg/dl (1). In a rare disease, β-sitosterolemia, plasma plant sterol levels are increased to 15–60 mg/dl due to mutations in two hemitransporters, ABCG5 and ABCG8 (1, 5, 6). These genes map to human chromosome 2, which is syntenic to mouse chromosome 17 (7). In the general population, plasma plant sterol levels are highly heritable and, although some role has been ascribed to the human ABCG5/ABCG8 locus, most of the genetic variance is as yet unexplained (8).
In an attempt to identify genes besides ABCG5 and ABCG8 that regulate plasma plant sterol levels, a genetic cross was undertaken between inbred mouse strains with high and low plasma plant sterol levels, C57BL/6J and CASA/Rk, respectively. This cross revealed a locus on chromosome 14 and two loci on chromosome 2 that together explain 40% of the variance in plasma plant sterol levels in the F2 animals. There was no linkage to mouse chromosome 17, implying that the chromosome 14 and chromosome 2 loci contain novel genes involved in regulating plasma plant sterol levels and perhaps cholesterol metabolism as well.
Materials and Methods
Animals and Diets.
The inbred mouse strains C57BL/6J (stock no. 00664) and CASA/Rk (stock no. 000735) were purchased from The Jackson Laboratory. CASA/Rk males were mated with C57BL/6J females to generate F1 animals. F1 males were intercrossed with F1 females to generate 201 F2 animals (102 males and 99 females). All animals were bred and housed in a single humidity- and temperature-controlled room with a 12-h dark/light cycle (6:00 a.m.–6:00 p.m. light/dark cycle) at the Laboratory of Animal Research Center at The Rockefeller University and fed with a single lot of Picolab (Bouncbrook, NJ) Rodent Chow 20 (catalog no. 5053) pellet containing 0.02% wt/wt cholesterol. At the age of 11 wk, food was removed from the cages at 10:00 a.m. and the animals allowed access to water. At 3:00 p.m., the mice were tail tipped for DNA extraction and blood samples collected by heart puncture into EDTA-containing tubes. Plasma was immediately separated and stored at −80°C. In a separate experiment, C57BL/6Jand CASA/Rk males were placed in metabolic cages for 2 d and then food consumption and animal weight measured for 2 consecutive days. All experiments were approved by the Institutional Animal Care and Research Advisory Committee.
Plasma Plant Sterols and Cholesterol Measurements.
Plasma sterols were extracted, subjected to alkaline hydrolysis, and derivatized with trimethylsilane as described (9). Plasma total cholesterol levels were measured by using gas–liquid chromatography (GC), and plant sterol levels determined by GC–mass spectrometry by using 5α-cholestane and epicoprostanol as internal standards, respectively. The ratio of plasma plant sterol to total cholesterol levels (μg/mg) was calculated.
Genotyping.
Tail tips from parentals and F1 and F2 mice were digested with proteinase K and DNA precipitated with ethanol. Fluorescently labeled primers corresponding to 350 markers shown to be polymorphic between C57BL/6Jand CAST/Ei by Iakoubova et al. (10) were tested to see whether they were also polymorphic between C57BL/6J and CASA/Rk, resulting 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 by using fluorescently labeled primers, and PCR products were analyzed by capillary electrophoresis by using the Applied Biosystems 3700 DNA sequencer. All PCR reactions and electrophoresis were automated by using the Tecan (Durham, NC), Genesis RSP 100, and Robbins Scientific (Mountain View, CA) Hydra 384 robots and carried out by the Starr Center Genotyping Core Facility at The Rockefeller University. Allele scores were analyzed by using Applied Biosystems GENOTYPE 3.6 NT software. The marker positions in centimorgans correspond to mapping data found in the Mouse Genome Informatics Database at www.informatics.jax.org.
Statistical Analyses.
Differences in plasma plant sterol levels between parentals and F1s and comparisons of plasma plant sterol levels for F2s with the various combinations of genotypes at D14Mit154 and D2Mit405 were analyzed by using one-way ANOVA with Tukey's posttest. 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. r/qtl was also used to permute the actual data sets for F2s to determine the significant logarithm of odds (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 corresponds 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 www.biostat.jhsph.edu/∼kbroman/software. The C57BL/6J and CASA/Rk alleles at chromosome 14 and both chromosome 2 loci were assessed for additivity and dominance by using MAP MANAGER QTXb10 Version 0.19. Differences in food intake, body weight, and food consumption between parentals were analyzed by using a two-tailed Student's t test.
Results
The major plasma plant sterols in the mouse are campesterol and sitosterol. These were measured in parental C57BL/6J, parental CASA/Rk, and F1 males. Table 1 shows the values for plasma plant sterol levels as well as the plasma plant sterol to total cholesterol ratios. Compared with C57BL/6J, campesterol and sitosterol levels were reduced by 50% and 25% in CASA/Rk and F1s, respectively. There was no overlap between the three groups.
Table 1.
Absolute plasma campesterol and sitosterol levels and plasma plant sterols to total cholesterol ratios in C57BL/6J, CASA/Rk, and F1 males
| Group (n)
|
Absolute plasma plant sterol levels, mg/dl (mean ± SD) | Plasma plant sterol/plasma total cholesterol ratio, μg/mg (mean ± SD) | ||
|---|---|---|---|---|
| Campesterol | Sitosterol | Campesterol | Sitosterol | |
| C57BL/6J (5) | 1.39 ± 0.18 | 0.74 ± 0.05 | 19.2 ± 1.3 | 10.3 ± 1.1 |
| CASA/Rk (5) | 0.71 ± 0.09 | 0.33 ± 0.05 | 10.4 ± 0.8 | 4.8 ± 0.4 |
| F1 (8) | 0.96 ± 0.10 | 0.51 ± 0.05 | 15.1 ± 0.9 | 8.0 ± 0.4 |
One-way ANOVA: Overall P < 0.0001; C57 vs. CASA, P < 0.001; C57 vs. F1, P < 0.001; CASA vs. F1, 0.01.
One-way ANOVA: Overall P < 0.0001; C57 vs. CASA, P < 0.001; C57 vs. F1, P < 0.001; CASA vs. F1, 0.001.
One-way ANOVA: Overall P < 0.0001; C57 vs. CASA, P < 0.001; C57 vs. F1, P < 0.001; CASA vs. F1, 0.001.
One-way ANOVA: Overall P < 0.0001; C57 vs. CASA, P < 0.001; C57 vs. F1, P < 0.001; CASA vs. F1, 0.001.
To examine the inheritance of plasma plant sterol levels, F1 mice were intercrossed and plasma plant sterol and total cholesterol levels measured in 201 F2 mice (102 males and 99 females). Fig. 1 shows that the distribution of the ratios of plasma campesterol to total cholesterol in F2 animals is unimodal and the values for individual mice span the parental values. Similar results were obtained when the data were analyzed for the ratios of plasma sitosterol to total cholesterol levels (data not shown).
Fig 1.
Distribution of plasma campesterol to total cholesterol ratio in F2 animals. F2 animals (102 F2 males and 99 F2 females) were fasted and plasma samples were collected and measured for plasma campesterol and total cholesterol levels, as described in Materials and Methods. Plasma plant sterols are expressed as the ratio of plasma campesterol in micrograms per deciliter to total cholesterol in milligrams per deciliter.
To identify loci that control plasma plant sterol levels, a whole genome scan was done on the F2 mice. Quantitative trait locus mapping using r/qtl revealed significant linkages on chromosomes 14 and 2. The interval maps for these chromosomes are shown in Figs. 2 and 3. With regard to chromosome 14, the analysis revealed a peak for linkage with a maximum LOD score of 9.9 at the marker D14Mit18 at 16.5 cM. Thus a strong peak for linkage determining plasma plant sterol levels exists on chromosome 14, designated Plast14. With regard to chromosome 2, the analysis showed a significant peak for linkage with a maximum LOD score of 4.1 at the marker D2Mit296 at 18 cM and another significant peak with a maximum LOD score of 3.7 at 65 cM that fell between the markers D2Mit126 at 48 cM and D2Mit 405 at 69 cM. These loci are designated Plast2a and Plast2b, respectively.
Fig 2.
Chromosome 14 linkage maps of plasma campesterol to total cholesterol ratio and plasma total cholesterol in F2s. Shown are linkage maps of plasma campesterol to total cholesterol ratio (black line) and the linkage of plasma total cholesterol level (red line) in F2 animals. The marker positions correspond to mapping data found in the Mouse Genome Informatics Database. The dashed horizontal line shows the 95% LOD score significance threshold.
Fig 3.
Chromosome 2 linkage maps of plasma campesterol to total cholesterol ratio and plasma total cholesterol in F2s. Shown are linkage maps of plasma campesterol to total cholesterol ratio (black line) and the linkage of plasma total cholesterol level (red line) in F2 animals. The marker positions correspond to mapping data found in the Mouse Genome Informatics Database. The dashed horizontal line shows the 95% LOD score significance threshold.
For the peaks of linkage, the genotypic means of the plasma campesterol to total cholesterol ratios in F2s, represented by the closest markers, are calculated and shown in Table 2. For Plast14, Plast2a, and Plast2b, homozygotes for the C57BL/6J allele had higher plasma plant sterol levels than heterozygotes and homozygotes for the CASA/Rk allele. For Plast14 and Plast2b, the phenotypic effect of the CASA/Rk allele best fit a codominant mode of inheritance, whereas for Past2a, the CASA/Rk allele has a dominant effect. Finally, Plast14, Plast2a, and Plast2b appear to explain 23%, 9%, and 8% of the variance in the ratio of plasma campesterol to total cholesterol, respectively.
Table 2.
Plast loci peaks genotypic values of plasma campesterol to total cholesterol ratio in F2 animals
| Locus
|
Peak, cM
|
Locus closest marker | Plasma campesterol/total cholesterol ratio (mean ± SD) | |||
|---|---|---|---|---|---|---|
| Name | cM | BB | BC | CC | ||
| Plast14 | 17 | D14Mit18 | 16.5 | 18.4 ± 4.7 | 14.1 ± 3.3 | 12.4 ± 3.9 |
| Plast2a | 18 | D2Mit296 | 18 | 16.5 ± 4.2 | 13.8 ± 4.0 | 13.4 ± 4.0 |
| Plast2b | 65 | D2Mit405 | 69 | 16.2 ± 3.6 | 14.2 ± 4.6 | 12.6 ± 3.0 |
One-way ANOVA: Overall P < 0.0001; BB vs. BC, P < 0.001; BB vs. CC, P < 0.001; BC vs. CC, 0.01.
One-way ANOVA: Overall P < 0.0001; BB vs. BC, P < 0.001; BB vs. CC, P < 0.001.
One-way ANOVA: Overall P < 0.0003; BB vs. BC, P < 0.05; BB vs. CC, P < 0.001.
Interactions between Plast loci in determining plasma plant sterol levels were next determined. r/qtl output includes the positions of the interacting loci, the joint LOD score of the interaction, the new LOD score of locus 1 in the presence of locus 2, the new LOD score of locus 2 in the presence of locus 1, and a LOD score of epistasis. As shown in Table 3, significance for the joint LOD score was achieved for the interactions of four pairs of loci, with the strongest joint LOD score of 16.44 for Plast14 and Plast2b. In this particular case, the LOD score of Plast14 increased from 9.9 to 10.7 due to the interaction with Plast2b, and the LOD score of Plast2b increased from 3.65 to 4.14 due to the interaction with Plast14. There was also an epistatic component with a LOD score of 2.75. Thus, in this cross, it appears that there are several gene interactions of a mostly additive nature that influence plasma plant sterol levels. An example of the magnitude of the phenotypic effect of the strongest interaction on the ratio of plasma campesterol to total cholesterol levels for each of the combined genotypes is shown in Fig. 4.
Table 3.
Loci interactions in determining plasma campesterol to total cholesterol ratio
| Locus 1 Chr: cM (Plast) | Locus 2 Chr: cM (Plast) | Joint LOD | Locus 1 LOD | Locus 2 LOD | Epistasis LOD |
|---|---|---|---|---|---|
| 14:24 (14) | 2:60 (2b) | 16.44 | 10.07 | 4.14 | 2.75 |
| 14:24 (14) | 5:71 | 13.63 | 10.92 | 3.57 | 0.50 |
| 14:24 (14) | 7:00 | 13.37 | 9.53 | 0.04 | 3.77 |
| 5:61 | 2:60 (2b) | 9.76 | 3.11 | 4.79 | 3.03 |
Genome-wide 95% significance for joint–LOD and epistasis–LOD thresholds of 8.31 and 6.69, respectively.
Fig 4.
Additive effect of Plast loci interactions on plasma campesterol to total cholesterol ratio in F2s. Shown are loci interactions with the highest joint LOD score, as detailed in Table 4. Displayed are marker genotypes that map closest to interacting loci (D14Mit154 at 22 cM for Plast14 and D2Mit405 at 69 cM for Plast2b, respectively). B, homozygosity for C57BL/6J allele; H, heterozygosity; C, homozygosity for CASA/Rk allele.
The principal trait used in this paper is the ratio of plasma plant sterol levels to total cholesterol levels. To rule out the possibility that the Plast loci identified reflect linkage to plasma total cholesterol rather than plant sterol levels, linkage of the plasma total cholesterol level to markers on chromosomes 14 and 2 was examined and the results indicated in Figs. 2 and 3. There was no suggestion of any linkage of plasma total cholesterol level to chromosome 14. On chromosome 2, there was a suggestive but not significant linkage with a LOD score of 3.1 at the marker D2Mit342 at 83 cM, well distal to the Plast2b locus at 65 cM. In addition, if the cholesterol locus contributed to the Plast2b locus, which is based on the ratio of plasma plant sterol levels to total cholesterol levels, the higher genotypic mean for C57BL/6J alleles would require lower plasma total cholesterol levels. In fact, the opposite is true; the genotypic means for homozygosity for C57BL/6J and CASA/Rk alleles for plasma total cholesterol levels are 40 ± 11 and 34 ± 10 mg/dl, respectively. Thus, it is unlikely that plasma total cholesterol levels influence the linkage at Plast2b.
The ABCG5/ABCG8 locus maps to mouse chromosome 17 at 55 cM (7). The current genome scan used eight markers that map to chromosome 17, including D17Mit122, which maps 3 cM proximal to ABCG5/ABCG8. The maximum LOD for any of these markers was 1.39 at 23 cM (D17Mit52). Furthermore, no significant gene interaction was detected with a locus on chromosome 17. This strongly suggests that the variation in plasma plant sterol levels in the F2s of this cross is not influenced by the ABCG5/ABCG8 locus.
To rule out the possibility that lower levels of plasma plant sterols in CASA/Rk reflect decreased consumption of dietary plant sterols compared with C57BL/6J, we compared the parentals in this cross for total daily food intake and for food intake per gram of body weight. As shown in Table 4, the former did not differ between the strains, and CASA/Rk actually ate 80% more food per gram of body weight than C57BL/6J. Therefore, the lower plasma plant sterol levels in CASA/Rk cannot be explained by decreased dietary intake of plant sterols.
Table 4.
Food intake, body weight, and food consumption in C57BL/6J and CASA/Rk males
| C57BL/6J (n = 5) | CASA/Rk (n = 5) | |
|---|---|---|
| Food intake, g/d | 4.3 ± 0.5 | 4.2 ± 0.6 |
| Body weight, g | 28.7 ± 1.4 | 15.4 ± 0.6 |
| Food consumption† | 0.15 ± 0.01 | 0.27 ± 0.05 |
P < 0.0001.
† Food intake in grams per day/grams body weight.
P < 0.0008 vs. C57BL/6J.
Discussion
In the present study, we describe two inbred mouse strains, C57BL6/J and CASA/Rk, with markedly different plasma plant sterol levels and through a genetic cross have mapped loci in linkage with this phenotype. This analysis revealed a locus on chromosome 14 (Plast14) and two loci on chromosome 2 (Plast2a and b). In addition, four significant additive interactions were demonstrated between pairs of loci, the most significant between Plast14 and Plast2b. Interestingly, there was no significant linkage or gene interaction detected involving the ABCG5/ABCG8 locus on chromosome 17.
The genetic basis of the difference between C57BL/6J and CASA/Rk in plasma plant sterol levels appears to be complex. In the F1 mice, the mean levels of plasma plant sterols are midway between the parentals, suggesting either codominant inheritance of one gene or the average effect of several genes. If it were a single gene, then the F2s would be trimodal, with 25%, 50%, and 25% resembling C57BL/6J, F1s and CASA/Rk, respectively. That the F2s showed a unimodal distribution with levels spanning the parental phenotypes suggests the involvement of two or more genes. This hypothesis is supported by the qtl analysis, revealing a single locus on chromosome 14 and two loci on chromosome 2. The actual number of genes at each of these loci can be determined only by creating and studying subcongenic lines. It is of note that a recent qtl analysis for traits in yeast found that a single locus actually represented the contribution of more than one gene (11).
The Ensembl mouse genome database was searched for known genes in the intervals of chromosome 14 (5–54 cM) and chromosome 2 (10–34 cM for Plast2a and 45–79 cM for Plast2b). The chromosome 14 interval did not contain any genes known to play a prominent role in sterol metabolism. The proximal chromosome 2 interval (Plast2a) contains Abca2, an ABC transporter of unknown function, at 11 cM and Rxrα, an important transcription factor involved in regulating sterol metabolism, at 17 cM. The distal chromosome 2 interval (Plast2b) contains Lxrα, another important transcription factor regulating sterol metabolism, at 49 cM, Ppar interacting protein (PRIP) at 73 cM and Pparγ coactivator 1 at 74 cM. Abca2 and Rxrα are near the peaks for Plast2a at 18 cM and are reasonable candidate genes for regulating plant sterol levels. Lxrα is proximal to the peak for Plast2b at 65 cM, making it a less likely candidate, whereas PRIP and Pparγ cofactor 1 are distal but closer to the peak of Plast2b.
A recent breakthrough in the understanding of the regulation of plasma plant sterol levels has been the identification of the genetic defect in β-sitosterolemia. This is a relatively rare autosomal recessive disorder characterized by xanthomas, premature coronary heart disease, hemolytic episodes, painful arthritis, and 30- to 100-fold elevations of plasma plant sterol levels resulting from increased intestinal absorption and decreased biliary excretion (1). In families with several affecteds, the gene was mapped to the short arm of chromosome 2 (12). Subsequently, an LXRα agonist responsive gene in mouse liver and intestines was identified that mapped to mouse chromosome 17 in the region syntenic to human chromosome 2 that contained the β-sitosterolemia gene (5). On sequencing, this gene turned out to be a new member of the ABCG family of transporters, named ABCG5. ABCG5 codes for a half transporter and is in close proximity to another half transporter ABCG8. These two genes share 5′ regions and are divergently transcribed. Sequencing of affected probands with β-sitosterolemia revealed them to be homozygous or genetic compounds for ABCG8 mutations or to have one mutated ABCG5 or ABCG8 allele with the mutation in the other allele not identified (5). These findings unequivocally established the genetic basis of β-sitosterolemia. In the current mouse cross, we failed to identify any suggestion of linkage of plasma plant sterol levels to mouse chromosome 17 where ABCG5 and ABCG8 reside. Thus the difference in plant sterol levels between C57BL/6J and CASA/Rk cannot be explained by strain differences in ABCG5 and ABCG8. It is possible that the primary genetic difference between the strains could affect ABCG5 and/or ABCG8 function or act by an independent mechanism.
β-Sitosterolemic patients have plasma sitosterol levels of 14–65 mg/dl and campesterol levels of 8–30 mg/dl, whereas normals show a range of plasma sitosterol levels from 0.2 to 1 mg/dl with comparable campesterol levels (1). Genes that influence the variation in plasma plant sterol levels in normal humans have not yet been determined. It is possible that milder mutations in ABCG5/ABCG8 could underlie this variation, or that other genes might be involved. Berge et al. (8) recently showed that plasma plant sterol concentrations were very stable over a 48-wk period, showed a high correlation between midparental and offspring levels, and were more similar in monozygotic than dizygotic twins, all suggesting high heritability of levels in the general population. They found that two sequence variations in ABCG8 were associated with lower plasma plant sterol levels in parents and their offspring. ABCG8 D19H in 10% of the population had a 31% lower genotypic mean campesterol level, and ABCG8 T400K in 36% of the population had a 2% lower genotypic mean. Thus these account for ≈5.5% and 0.1% of the variance in campesterol levels. In the same study, the authors calculate that the genetic contribution to the variance in plasma campesterol concentrations is 59%. Similar calculations for the sitosterol-to-cholesterol ratio indicate that these two polymorphisms account for 5.7% and 5.1% of the variation, respectively, with the genetic contribution to the variance in plasma sitosterol to cholesterol ratio being >80%. Thus it is probable that variation at other loci besides ABCG5/ABCG8 contribute to the variation in plasma plant sterol levels in the general population. We speculate that human loci syntenic to those identified in this paper may contribute to the differences in plasma plant sterol levels between normal humans.
Finally, the question arises whether genes that regulate plasma plant sterol levels have broader significance for the regulation of cholesterol metabolism. It has been observed that the percent dietary cholesterol absorption in patients with β-sitosterolemia is consistently at the upper limits of the normal range. These patients also absorb a high percentage of dietary plant sterols (15–60% in patients vs. 5% in normals). In β-sitosterolemic patients, there is also decreased biliary excretion of plant sterols (1). In addition, in normal individuals, studies have shown a correlation between dietary cholesterol absorption and plasma plant sterol levels (2, 3). Recent studies of mouse models support this relationship. A mouse with a human BAC transgene expressing ABCG5/ABCG8 showed decreased percent dietary cholesterol absorption, decreased plasma plant sterol levels, and increased biliary cholesterol concentration (4). It is also interesting that a recent attempt to map dietary cholesterol absorption genes through a back cross between DBA/2 and AKR.DBA/2F1 reported linkage of dietary cholesterol absorption to D2Mit305 at 60 cM in males (13). This is very close to both the peak LOD in the current cross for Plast2b at 65 cM and the strongest peak LOD for interaction between chromosome 14:24 cM and chromosome 2:60 cM. In an as-yet-unpublished study, C57BL/6J males had higher cholesterol absorption and decreased biliary cholesterol excretion compared with CASA/Rk males (E.S., unpublished data). Taken together, these findings strongly suggest that at least some of the same genes regulate plasma plant sterol levels and cholesterol metabolism. These genes can operate to affect absorption through intestinal epithelial cells and/or excretion from hepatocytes into the bile.
In summary, a cross between two strains that differ in plasma plant sterol levels has revealed loci on chromosomes 14 and 2, distinct from the ABCG5/ABCG8 locus. These loci may provide further insight into molecules that affect plasma plant sterol levels and perhaps even cholesterol absorption and/or biliary excretion. The use of plasma plant sterol levels as a surrogate for these aspects of cholesterol metabolism may provide results that are more easily interpretable, because endogenous synthesis does not complicate the picture as it does for cholesterol itself.
Abbreviations
LOD, logarithm of odds
Plast, plasma plant sterol
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