1 Jackson Laboratory, Bar Harbor, Maine 04609
2 Department of Medicine, Harvard Medical School, Division of Gastroenterology, Brigham and Womens Hospital, Harvard Digestive Diseases Center, Boston, Massachusetts, 02115
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ABSTRACT |
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cholelithiasis; genetics; Castaneus; mouse; Lith genes; Slc21a1; Pparg; Cebpb
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INTRODUCTION |
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Ideally, the identification of the primary genetic determinants that underlie this disorder should allow a true understanding of the pathophysiology of cholesterol gallstone formation. This should lead to novel means of risk assessment, nonsurgical management, and ultimately prevention of this prevalent and costly disorder (44). Quantitative trait locus/loci (QTL) analysis is a powerful, phenotype-driven experimental approach that associates a trait with a genotype, thereby defining genomic regions that harbor genes conferring the given trait in inbred mice (41, 58). The aim of this approach is to identify the entire complement of genes (Lith genes) that carry polymorphisms (i.e., alleles) determining cholesterol gallstone susceptibility (23, 58) in the mouse model. Based on the conservation of the mouse and human genomes, these data will allow for the prediction and evaluation of the corresponding human genes. Inclusive of this report, 14 major QTL for cholesterol gallstone formation, named Lith1 through Lith14 (22, 24, 25, 29, 41, 42, 56, 59), are now known. Of these, pathophysiologically relevant candidate genes were suggested for Lith1 (25), Lith2 (4), Lith6 (29), Lith7, Lith8, and Lith9 (59).
This study comprises the second intercross between the gallstone-susceptible, wild-derived inbred strain CAST/Ei (CAST) and a gallstone-resistant inbred mouse strain, 129S1/SvImJ (strain 129). The first intercross between CAST and DBA/2J was reported previously (29). In the present QTL analysis, we detected one new QTL on proximal chromosome 5 (Chr 5), which we named Lith13. We confirmed a locus, Lith6, previously identified on distal Chr 6 (29). Two additional loci on distal Chr 2 (Wittenburg H, Carey MC, and Paigen B, unpublished observation) and Chr 16 (24), detected below the significance threshold in two previous crosses, were confirmed and named Lith12 and Lith14, respectively.
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MATERIALS AND METHODS |
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Animals and diet.
This study was conducted concurrently with an intercross between strains CAST and DBA/2J. Animals, breeding protocols, and facilities were identical to those described in detail previously (28) except that strain 129 mice were used instead of DBA/2J mice. The male mice displayed greater phenotype variation than female mice, and therefore we evaluated male F2 progeny only because they conferred the greatest statistical power for QTL detection. In addition, the results of our introductory studies (RESULTS, Fig. 2) induced us to prolong the feeding regimen to 10 wk for the F2 mice in an attempt to increase solid gallstone formation. At 68 wk of age, the different populations of animals initiated consumption of the cholesterol gallstone-promoting (lithogenic) diet for periods between 4 and 10 wk. Previously, cholesterol gallstone formation was demonstrated to constitute a distribution among different strains of inbred mice when fed the lithogenic diet (5, 22, 40). Animals were allowed free access to food and water. All animals fasted for 4 h prior to death. The Institutional Animal Care and Use Committees of the Jackson Laboratory and Harvard University approved all experimental protocols.
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Cholesterol gallstone phenotypes.
Mice of the parental strains CAST and 129 (n = 10 per gender and strain) and the reciprocal first filial generation [i.e., (CASTx129)F1 and (129xCAST)F1] (n = 1018 male and n = 68 female per lineage) were fed the lithogenic diet for 8 wk prior to phenotyping for cholesterol gallstones using standard methods in our laboratories (principally based upon microscopic appearance of bile using polarized light), which included gravimetric determination of gallbladder volume (GBV) (55, 56). The second filial (F2 or intercross) generation comprised males only (n = 277; however, 7 lacked gallbladders). Since the parental CAST mice exhibited intermediate prevalence, and neither the CAST nor the combined F1 mice developed solid gallstones, we elected to extend the feeding period of the F2 mice in an attempt to increase the prevalence of solid stones in that population. Thus the F2 mice were phenotyped for gallstones after 10 wk of consumption of the lithogenic diet. Furthermore, cholesterol gallstone prevalence was defined to include both sandy (translucent) and solid (opaque) cholesterol gallstones.
Gallbladder bile, gallstones, and cholesterol crystals from the F2 mice were collected the same way as the parental animals. One investigator (M. A. Lyons) performed all quantifications. When present, opaque solid cholesterol gallstones were counted, air-dried overnight, and weighed (55). Gallstone weight was abbreviated "GSW." A semi-quantitative score designated "Solid" was applied according to a simultaneous evaluation of both the number and size of opaque stones (0 = absence, through 4 = most severe, i.e., largest size, greatest number). As described formerly (56), bile samples were assigned a gallstone "Score" according to absence (Score = 0) or presence (Score = 1) of cholesterol monohydrate crystals (ChMC) and the detection of translucent "sandy" or solid gallstones (Score = 2). It should be appreciated that Score is a composite trait rather than a primary phenotype. Aggregated ChMC (AChMC) were semi-quantified using a 0-to-4 scale (0 = absence, through 4 = most severe). Thus we evaluated four cholesterol gallstone phenotypes: GSW, Solid, Score, and AChMC. We also evaluated the related trait GBV.
QTL Analyses
Genotyping.
DNA was prepared from tail samples and genotyping performed using simple sequence length polymorphisms (SSLP; n = 100; Fig. 1) that discriminate between CAST and strain 129 alleles (MapPairs primers; Research Genetics, Huntsville, AL) as described (28). The entire cohort of F2 mice (n = 277) was genotyped using SSLP markers distributed across the genome (interval range 124.4 cM; Fig. 1). Reported genetic map positions were retrieved from the Mouse Genome Database (http://www.informatics.jax.org).
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Tests for multiple QTL.
To determine the likelihood that a QTL comprised more than one locus linked to the respective phenotype, we fitted models comprising one, two, or three QTL, and a maximum LOD score was calculated for each. Employing permutation testing for an intercross of 277 mice, we determined that increases (LOD) of 2.0, 1.8, and 1.6 in the LOD score between the one- and two-QTL models and between the two- and three-QTL models are the thresholds that define multiple QTL at the 95%, 90%, and 80% confidence levels, respectively. We regard the 95% level as significant, but report the additional thresholds to provide a scale for near-significant results.
QTL nomenclature.
Colocalizing QTL were defined as QTL for which the locus peak fell within the 95% CI of the second QTL. A further criterion was that the 95% CI were substantially overlapping. We interpreted the colocalizing QTL for different gallstone phenotypes to represent identical QTL. Our approach to naming QTL is that significant QTL are named automatically, but suggestive QTL are named only when confirmed by two or more independent breeding crosses. This is in concordance with the recent consensus decision of the Complex Trait Consortium (L. Flaherty et al., unpublished observations).
Allele effects.
For each of the QTL, we determined the "allele effect" by calculating the phenotype mean for each of the three possible genotypes. Using the error bars for each of the three genotypes, we determined which strain contributed the gallstone-susceptibility allele and whether that allele caused dominant, additive, or recessive inheritance of the gallstone susceptibility phenotype. A dominant allele was defined as exhibition of a gallstone-susceptible phenotype by the heterozygous genotype F2 population indistinguishable from the gallstone-susceptible homozygous genotype F2 population, whereas a recessive allele was defined as the heterozygous genotype F2 group being indistinguishable from the gallstone-resistant homozygous genotype F2 group. An additive allele was defined as an allele that produced a gallstone phenotype intermediate between each of the homozygous-susceptible and homozygous-resistant F2 genotype populations.
mRNA expression analyses of candidate genes.
The utility of QTL analysis lies in its ability to detect fundamental genetic differences in genes encoding crucial regulatory proteins (23), which may lie in either regulatory and/or coding regions of the genome. As such, they can affect transcription efficiency, mRNA stability and/or amino acid sequences. From the 95% CI generated in the QTL analyses, we identified positional candidate genes whose products perform direct or indirect roles in lipid metabolism that colocalized with the QTL (Fig. 1). Our objective was to develop a preliminary screening assay to aid the evaluation of candidate genes putative contributions to gallstone formation. We determined hepatic mRNA expression levels of candidate genes in each of the parental strains, CAST and 129. Intercross progeny inherit unique combinations of alleles derived from both parental strains. Therefore, when differential expression of a candidate gene is demonstrated for animals that exhibit a homozygous genotype for one parental strain vs. the other only in the region harboring the candidate gene, it provides strong support for cis-acting elements controlling gene expression rather than trans-acting elements remote from the gene of interest. Hence, we investigated further Lith12, Lith13, and Lith6 in the F2 population. We selected individual samples that were either homozygous CAST or homozygous 129 over these loci on Chrs 2, 5, and 6, respectively. To collect tissue for mRNA expression analyses, male mice of strains CAST and 129 (n = 5 per strain) were fed the lithogenic diet for 4 wk, and livers were harvested as described (28). Livers from F2 mice were removed at the time of phenotyping, frozen immediately in liquid nitrogen, and stored at -80°C. As described elsewhere (28), oligonucleotide primers were designed, verified, and expression analyses performed. (Primers and their sequences are available from the corresponding author.)
DNA sequencing and sequence analysis of candidate genes.
DNA sequencing was performed as described (28). The cDNA and 0.7 to 1.1 kb proximal to the transcription start site of Pparg and Slc21a1, two of the key candidate genes for Lith6, were sequenced from strain 129 to investigate putative polymorphisms that may determine mRNA expression differences and the existence of potential amino acid substitutions. These sequences were reported earlier for strain CAST (28). To investigate potential differences in the proximal putative promoter regions of Pparg1 and Pparg2 from the two strains, we compared the mouse Pparg and human PPARG sequences (ENSEMBL) using VISTA software (7, 33). Second, as necessary, we compared our strain sequences using MATCH software (v1.0, public), a matrix-based tool for searching transcription factor binding sites, and included a liver-specific search criterion (32).
General Statistical Analyses
Data are means ± SE and were analyzed using GraphPad Prism (Windows v3.00; GraphPad Software, San Diego, CA). Students t-test was used to compare the parental strains, CAST and 129, for continuous data (e.g., biliary secretion rates). GBV was analyzed by ANOVA with Bonferroni adjustment for multiple comparisons. Gallstone prevalence rates of the male reciprocal F1 and allele distributions of the F2 mice (i.e., 1:2:1 ratio for homozygous 129:heterozygous:homozygous CAST genotypes) were analyzed using chi-squared analysis. P < 0.05 was considered significant.
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RESULTS |
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Cholesterol Gallstone Prevalence
Figure 2 indicates that male and female CAST mice were gallstone susceptible with intermediate prevalence (both 40%, n = 10 per group), but conversely, both male and female strain 129 mice were gallstone resistant (both 0% prevalence, n = 10 per group). These data are consistent with our previous observations (40). Similar to strain 129 females, both groups of female reciprocal F1 animals (n = 68 per group) were gallstone resistant (both 0% prevalence; Fig. 2). However, male reciprocal F1 mice (n = 1018 per group) were gallstone susceptible [67% (CASTx129)F1 and 90% (129xCAST)F1] (Fig. 2). Since the prevalence rates of the reciprocal male F1 mice did not differ significantly, cholesterol gallstone susceptibility in this cross was not inherited by maternal (e.g., mitochondrial) or imprinted (gene expression predominantly from either a maternal or paternal allele) genetic factors, and QTL analyses could detect autosomal regions carrying Lith genes. The F2 population displayed gallstone prevalence (58%, n = 270) intermediate between CAST and the F1 group (Fig. 2).
Biliary Lipid Analyses
Strain CAST displayed a significantly greater (1.9-fold, P < 0.0001) hepatic bile flow rate compared with strain 129 and displayed a trend (P < 0.06) toward a lower total lipid concentration (Fig. 3A). No difference was observed in bile salt secretion rates between the two strains, but CAST secreted significantly more phospholipid (P < 0.05) and cholesterol (P < 0.001) compared with strain 129 during the 60-min collection period (Fig. 3B). When the hepatic biliary lipid data were expressed proportionally, no difference was observed in the total bile salt composition, but it was confirmed that CAST displayed significantly greater (P < 0.0001) biliary cholesterol content (Fig. 3C). However, the phospholipid proportion of CAST hepatic biles was significantly reduced (P < 0.05) compared with that of strain 129 (Fig. 3C), a phenomenon that also contributed to biliary lithogenicity in strain CAST (53). Bile flow is an osmotic response to solutes secreted by the bile canaliculi and is determined by two factors: 1) bile salt-dependent flow, i.e., bile flow caused by bile salts secreted as anions; 2) bile salt-independent flow, i.e., bile flow caused by all other secreted solutes (14). Therefore, as an approximation (because the sample size was too small and the duration was too brief for accurate measurement), we plotted bile salt secretion against bile flow and, extrapolating to a theoretical bile salt concentration of zero, we determined that CAST very likely displayed greater bile salt-independent bile flow (Fig. 3D). These data are consistent with both the increased bile flow (Fig. 3A) displayed by strain CAST and the lack of difference in bile salt secretion rates (Fig. 3B) and bile salt composition (Fig. 3C) between the two strains. Finally, we determined the cholesterol saturation indices (CSIs) of both hepatic and gallbladder biles. Because of the small volumes available, gallbladder biles from strain CAST were pooled. Strain 129 displayed larger GBV, allowing individual determinations from this strain. Strain CAST displayed CSIs greater than strain 129 in both instances (Fig. 3E). Both strains exhibited CSI > 1 in hepatic bile, reflecting the low total lipid concentration of hepatic bile (53) and the presence of cholesterol-carrying vesicles that are more efficient cholesterol-solubilizing agents than mixed micelles (18). However, in gallbladder bile, CAST displayed CSI >> 1, whereas strain 129 displayed CSI < 1 (Fig. 3E). Despite all efforts to the contrary, the very high CSI of CAST gallbladder bile suggests the admixture of microscopic ChMC in the sample. However, this does not affect the observation that CAST gallbladder bile was cholesterol supersaturated, whereas strain 129 gallbladder bile was not supersaturated. These data provide a physical-chemical explanation for the gallstone susceptibility of strain CAST compared with the gallstone resistance of strain 129.
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Three of the QTL identified here, on Chrs 2, 5, and 6, were detected above the significance threshold. The QTL on distal Chr 2 colocalized with a previously detected suggestive locus for mucin glycoprotein accumulation, a cholesterol gallstone-related phenotype (24), and with a suggestive QTL for the Score phenotype from another intercross (Wittenburg H, Carey MC, and Paigen B, unpublished observation). According to current nomenclature, we named this significant QTL, Lith12. The significant and novel QTL for Solid trait on Chr 5 was named Lith13, but the suggestive QTL for GSW on Chr 5 remains unnamed. The significant QTL for GBV on Chr 5 was named Gbvq1. The QTL on Chr 6 was identified and named Lith6 previously (29). The QTL on Chr 16 was identified previously (24; and Wittenburg H, Carey MC, and Paigen B, unpublished observation) above the suggestive threshold and hence was named Lith14. However, the two remaining QTL, on Chrs 1 and 14, were detected only at the suggestive level and await independent confirmation prior to naming. In summary, using an intercross between CAST and 129 and assuming that colocalizing QTL were identical, we identified seven QTL using traits GSW, Solid, GBV, Score, and AChMC. One QTL was new (Lith13), whereas the remaining QTL were suggestive or confirmed QTL detected previously that were both named and unnamed.
The QTL for GSW and the QTL for Solid (Lith13), both on Chr 5 (Fig. 5, D and E), and the QTL for Score and for AChMC, both on Chr 6 (Lith6, Fig. 6, A and B), were each fitted with models comprising one, two, or three QTL to determine the likelihood that any of the QTL comprised more one than locus. The QTL for GSW was best fit by a two-QTL model, with 90% confidence (LOD = 1.8) that at least two QTL existed on Chr 5, but Solid did not suggest multiple loci. These statistical modeling data, at least for GSW, support the fine mapping data (Fig. 5), which suggested the presence of multiple QTL on Chr 5. For Lith6, neither the QTL for the Score nor the QTL for AChMC suggested statistical evidence for the presence of more than one locus. However, the different localization of the QTL peak for the two phenotypes (Table 1) is consistent with the existence of two loci in close proximity, a conclusion that was also drawn from the analysis of an intercross between CAST and DBA/2J (29).
The second stage of the QTL analysis was used to detect gene-gene interactions (epistasis). Using our strict criteria for significance, we detected no interacting QTL in this intercross. Therefore, only single QTL are presented.
Allele Effects
The QTL for Score on Chr 1 (D1Mit21) was determined by a dominant CAST susceptibility allele (Fig. 7A). Lith12 (Chr 2, D2Mit113), the QTL detected using Score (Fig. 7A) and AChMC (Fig. 7B), was contributed by a recessive CAST allele. In agreement with previous data (29), Lith6 (Chr 6) was conferred by a dominant CAST susceptibility allele (D6Mit44, Fig. 7A; and D6Mit14, Fig. 7B). The QTL for Score (Fig. 7A) and AChMC (Fig. 7B) on Chr 14 (D14Mit98) was contributed by a recessive CAST susceptibility allele. Lith14 (Chr 16, D16Mit65) was determined by an additive CAST susceptibility allele (Fig. 7A). Lith13 (Chr 5) and the QTL for GSW (Chr 5) were conferred by recessive 129 susceptibility alleles (Fig. 7C). Gbvq1 was determined by a recessive CAST allele dictating larger GBV (Fig. 7C). Because cholesterol gallstone susceptibility alleles were contributed by both the gallstone-susceptible (CAST) and gallstone-resistant (129) parental strains, these data are consistent with a complex mode of inheritance of cholesterol gallstone formation. Furthermore, they are consistent with the increased prevalence demonstrated by the male F1 mice compared with CAST, the gallstone-susceptible parental strain. However, because Lith13 and the QTL linked to D5Mit201 were determined by recessive 129 susceptibility alleles, the data suggest the presence of other 129-derived gallstone susceptibility QTL or modifier genes that were not detected using this intercross, but contributed to the increased prevalence of the male F1 mice.
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Consistent with our earlier findings (29), the CAST genotype determined decreased expression of the Lith6 candidate gene Pparg, encoding peroxisome proliferator-activated receptor- (PPAR
), compared with the 129 genotype in both the parental (CAST, 2-fold less; Fig. 7A) and F2 (CAST, 7-fold less; Fig. 7B) populations. However, the second Lith6 candidate gene, solute carrier family 21 (organic anion transporter) member 1 (Slc21a1), was expressed at a higher level by CAST (2-fold), contrary to our prediction based on an intercross between CAST and DBA/2J in which DBA/2J exhibited greater expression than strain CAST (29). Furthermore, differential expression of Slc21a1 was not driven by genotype at Lith6 as determined from expression levels in the F2 population (Fig. 8B).
DNA Sequencing and Sequence Analyses
We aimed to sequence 1 kb of the proximal putative promoter regions and the coding regions of Pparg and Slc21a1 for comparison of the sequences derived from strains 129 and CAST (see Supplemental Table 2, available at the Physiological Genomics web site).1
Pparg1 and Pparg2 represent alternative transcripts of Pparg (62). The two transcripts possess different initiation codons but share six common exons at the 3' end (62). Pparg1 comprises exons A1 and A2, which do not code for amino acids, plus the six common exons. Both PPAR1 and PPAR
2 proteins appear to be expressed in mouse and human liver, although PPAR
2 appears to predominate (3, 16). Pparg2 comprises exon B1 plus the six common exons and encodes 30 amino acids additional to Pparg1. The coding region is comprised entirely by Pparg2 in which sequence variations were observed but with none causing amino acid substitutions (see Supplemental Table 2). The gene structure of mouse Pparg and human PPARG and their corresponding amino acid sequences are highly conserved (99% similarity, 95% identity) (16). However, VISTA (7, 33) sequence comparisons revealed little similarity between species of exon A1, exon A2, and the 5 kb immediately proximal to exon A1 (data not shown). VISTA (7, 33) analyses of the 1.1-kb promoter sequences of Pparg2 from strains 129 and CAST revealed two regions that displayed greater than 75% identity with the human sequence (data not shown); however, no differences in predicted transcription binding sites were observed between the strains CAST and 129 using the MATCH software (data not shown).
Slc21a1, the second candidate gene underlying the dual-QTL Lith6, displayed many sequence variations (see Supplemental Table 2). The coding region exhibited four polymorphisms that caused amino acid substitutions and potentially might affect protein activity. Two of the substitutions [T1769A (nucleotide), C538S (amino acid residue) and C1581G, T475S] resulted in the replacement of conserved residues (19) (see Supplemental Table 2). These data are consistent with the candidacy of Slc21a1 as a genetic determinant of cholesterol gallstone susceptibility but require functional analysis to test our hypothesis directly.
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DISCUSSION |
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The goal of QTL analyses of complex phenotypes is the identification of the genes that carry polymorphisms that determine susceptibility or resistance to a given trait. After performing a breeding cross, this objective is hampered often by the large 95% CI of the QTL that harbor myriad genes. Therefore, we were guided in our identification and assessment of positional candidate genes by knowledge of genes with putative roles in cholesterol gallstone formation (23) and by a rigorous physical-chemical characterization of the parental strains of our intercross (Fig. 3). Biliary lipid analyses of male mice of strains CAST and 129 indicated that CAST gallstone susceptibility was primarily the result of cholesterol hypersecretion (Fig. 3B) and increased biliary cholesterol content (Fig. 3C). CAST gallstone susceptibility also was partly due to decreased biliary phospholipid composition (Fig. 3C), but not bile salt hyposecretion (Fig. 3B) or reduced bile salt composition (Fig. 3C). Cholesterol supersaturation in CAST biles was likely exacerbated by the increased bile salt-independent bile flow (Fig. 3D) and resultant lower total lipid concentration (Fig. 3A, P < 0.06), because low total lipid concentrations (53) and cholesterol-solubilizing vesicles (18) each contribute to increased CSIs typically exhibited by hepatic biles. A preliminary examination of selected genes (Abcc2/Mrp2, Cftr, and Slc4a2/Ae2) that contribute to bile salt-independent bile flow did not elucidate any underlying mechanisms, and in fact, strain 129 exhibited 2-fold greater expression of Abcc2 (data not shown). Strains CAST and 129 both displayed CSI > 1 in hepatic bile (Fig. 3E). However, CAST displayed a significantly greater CSI compared with strain 129, reflecting the cholesterol hypersecretion of strain CAST (Fig. 3E). Analysis of gallbladder bile from parental strains confirmed that CAST exhibited a markedly increased gallbladder CSI compared with 129, which displayed CSI < 1 (Fig. 3E). Therefore, these data provide a physical-chemical explanation of gallstone susceptibility in strain CAST compared with the gallstone resistance of strain 129. Based on this information and the known genetic map positions of the positional candidate genes (Fig. 1, Table 1), we tested for differential mRNA expression between the parental strains among the candidate genes for loci comprising Lith12, Lith13, Lith6, and Lith14. For those putative candidate genes that exhibited differential hepatic expression between the two parental strains (Fig. 8A), mRNA expression levels were examined further in the F2 population (Fig. 8B). Our aim is to complement the genotype and phenotype data such that we can provide a molecular genetic mechanism for the observed gallstone susceptibility traits.
We performed preliminary, prospective evaluations of mRNA expression of positional candidate genes that lay within the 95% CI of our QTL and were involved in lipid metabolism. Two candidate genes demonstrated differential expression between the parental strains, but not between the two genotypic groups of the F2 population (Fig. 8). These findings validate our investigation of mRNA expression in the F2 population. Since the parental strains were identical at all loci, but the F2 mice were identical only at the selected locus, the data suggest that either or both of the parental strains possessed modifier alleles or epistatic interactions that were not present, or were not detected, in the F2 mice. However, this approach and the criteria we employed are consistent with the concept that only highly significant QTL should be reported so that false positives are minimized (26).
We considered three positional candidate genes for Lith12 (Chr 2):Cebpb, Hnf4, and Pltp. We observed no difference in either Hnf4 or Pltp expression (Fig. 8A). Based on our mRNA expression criteria in this study, these were considered poor candidate genes and were not investigated further. The CAST genotype determined increased expression of Cebpb in the parental mice (Fig. 8A) and the F2 progeny (Fig. 8B), which strongly supports the notion that its expression was determined locally and not by an element outside the QTL region. It is not likely that C/EBPß influenced the expression of Pparg in this intercross, because these expression levels were inversely related (Fig. 8). Evaluation of mice possessing targeted mutation of Cebpb revealed little with regard to cholesterol homeostasis (49). Without sequencing the coding regions of these candidate genes, we cannot exclude definitively any of the three alternatives. This locus requires more investigation to elucidate the underlying mechanism that influences cholesterol gallstone formation and to identify the responsible gene, but Cebpb represents an ideal starting point in this endeavor.
Lith13 (Chr 5) was contributed by a recessive 129 susceptibility allele (Fig. 7C) and included Lrpap1, Ppargc1, and Cckar as positional candidate genes (Table 1). We concluded, however, that Lrpap1 and Ppargc1 were unlikely to be responsible for Lith13. Lith13 colocalized with a QTL for GBV (Table 1), whose peak (36 cM) was in close proximity to Cckar (34 cM), an attractive candidate gene for both this phenotype and for cholesterol gallstone formation directly (23). Since the QTL for GBV was determined by a recessive CAST allele that increased volume (Fig. 7C), this locus is consistent with the knowledge that gallbladder stasis contributes to cholesterol crystallization and gallstone formation (41). Although it is not definite that GBV accurately reflects gallbladder stasis, GBV does represent a convenient surrogate indicator of gallbladder contraction, and the QTL for this phenotype provides supporting evidence for Lith13. The gene encoding CCKAR is one of the few examples of genes that were linked to cholesterol gallstone formation in humans (36, 46). In mice, both the CCKAR knockout mouse (38, 45) and the mouse with dysfunctional carboxypeptidase E (fat mutation; the enzyme that hydrolyzes the procholecystokinin to cholecystokinin, the active ligand for the receptor) (6) displayed increased susceptibility to gallstone formation. The pathophysiology of gallstone formation due to dysfunctional cholecystokinin or its receptor most likely involves both noncontraction of the gallbladder and reduced intestinal transit leading to enhanced intestinal cholesterol absorption (6). We did not investigate the candidacy of Cckar in this cross, but we believe that this gene will be interesting to pursue in future as a viable candidate for Lith13. Furthermore, the likely existence of dual QTL on Chr 5 will be investigated.
Previously, we determined that Lith6, detected using an intercross between strains CAST and DBA/2J, comprised two closely linked QTL and that Pparg and Slc21a1 were likely candidates for the two underlying loci (29). Consistent with that conclusion, we detected colocalizing QTL for the Score and AChMC phenotypes (Table 1) whose peaks at 54 cM and 66 cM, respectively, coincided with each of these two candidate genes (Pparg, 52.7 cM; Slc21a1, 67.0 cM). PPAR is a ligand-activated transcription factor. In one study in vivo, PPAR
activation upregulated CYP7A1 activity (37), and in another, it increased Cyp7a1 expression (43). Conversely, we infer that lower Pparg expression and/or PPAR
activation could increase the availability of intracellular free cholesterol for biliary secretion due to decreased catabolism of cholesterol into bile salts via CYP7A1. Consistent with this hypothesis, strain CAST exhibited biliary cholesterol hypersecretion (Fig. 3) and contributed the susceptible Lith6 allele (Fig. 7), and the CAST genotype dictated decreased Pparg expression in the F2 mice (Fig. 8). We detected no DNA sequence variations that would cause amino acid substitutions, thereby eliminating the possibility of altered protein function. Our preliminary analyses of the Pparg promoter regions revealed little in terms of transcriptional regulation. Indeed, this might be predicted since Landers group concluded recently that due to the difficulty in identifying regulatory elements, research efforts would be more productive by searching for regulatory variation rather than for specific regulatory variants (11). It is interesting to note that despite the overall dissimilarity in the 5-kb region upstream of the two orthologs, Pparg1 (62) and PPARG1 (16), each possess substantial promoter activity in the respective 3-kb upstream region and display similar tissue distribution (3, 16).
Slc21a1, the second positional candidate gene for Lith6, is a transporter on the basolateral membrane of hepatocytes responsible for hepatocellular uptake of bile acids and bile salts (51). Since >80% of conjugated taurocholate but <50% of unconjugated cholate uptake is mediated via the Na+-dependent mechanism, i.e., SLC10A1/NTCP (51), it was suggested that unconjugated bile acids might be transported preferentially by the Na+-independent mechanism (2), i.e., SLC21A1. Given that the lithogenic diet included cholic acid (22), Slc21a1 may well be a genetic determinant of cholesterol gallstone susceptibility in the present in vivo model. We hypothesized that higher basolateral uptake of bile acids in CAST mice, due to functional differences or differences in expression, could inhibit (via NR1H4/FXR) cholesterol catabolism to bile salts, thereby increasing the availability of cholesterol for canalicular secretion and thus the lithogenicity of bile. The alignment of sequences from mouse, rat, and human indicated that within the subfamily to which Slc21a1 belongs, valine and isoleucine are interchangeable at residue 8 and phenylalanine is present in one human member at residue 660, suggesting that such amino acid substitutions may not be important. However, threonine and cysteine are conserved at residues 475 and 538, respectively (19), suggesting that these changes might cause a functional variation in protein activity. We speculated that SLC21A1 derived from strain CAST might display heightened transporter activity due to the loss of two conserved amino acids (T475 and C538, also observed between CAST and DBA/2J; Ref. 29). Since the expression profiles of Slc21a1 between the two studies involving strain CAST were inconsistent, and the sequences of CAST and DBA/2J were identical in the promoter region, we infer that the amino acid changes might be important for cholesterol gallstone susceptibility, rather than the nucleotide variations in the regulatory regions. Congenic strains are under construction to confirm and narrow the individual loci comprising Lith6. Biochemical evaluation of SLC21A1 expressed in vitro also appears a powerful approach to further authenticate the contribution of SLC21A1 to cholesterol gallstone formation.
Using an intercross between strains CAST and 129, we generated genetic and molecular data that further support the likelihood that Lith6 represents two QTL in close proximity, and candidate genes include Pparg and Slc21a1. Both genes provide putative molecular explanations for one of the key features of gallstone susceptibility in this cross, biliary hypersecretion of cholesterol via decreased cholesterol degradation into bile salts. The second physical-chemical principle of cholesterol gallstone formation in strain CAST, higher bile salt-independent bile flow, and therefore lower total lipid concentrations that lead to higher CSI values, remains elusive from this analysis. Similar to Lith6, the new QTL for cholesterol gallstone formation on Chr 5 likely represents a complex locus comprising two QTL in close proximity, one of which we named Lith13. Lower expression of Cckar, a candidate gene within this complex region, by strain 129 might contribute to its higher total lipid concentration of gallbladder bile compared with strain CAST, since gallbladder stasis results in water resorption and concentration of gallbladder lipids. QTL on Chrs 2 and 16, detected in previous crosses, were confirmed, thereby allowing us to name these loci Lith12 and Lith14, respectively. From our daisy chain experimental design, it appears that the number of new cholesterol gallstone susceptibility QTL that we detect from each cross is decreasing, but we are confirming many previously detected QTL using both independent crosses and strains. By combining data from different crosses displaying similar QTL, it is probable that we will resolve complex, closely linked QTL, such as those in the vicinity of Lith6 and Lith13, into their individual QTL components (50). Furthermore, at the completion of all eight intercrosses, and combined with previous crosses, it is likely that identification of all Lith loci will be achieved. The focus of such endeavors can then shift entirely to characterizing these primary genetic determinants of cholesterol gallstone formation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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We are indebted to Dr. Jason Stockwell and Jennifer Smith (Jackson Laboratory) for consultation on general statistical methods and assistance with graphics, respectively. We thank David Schultz, Harry Whitmore, and Eric Taylor (Jackson Laboratory) for colony management.
Present address of H. Wittenburg: Department of Medicine II, University of Leipzig, Leipzig, Germany.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Paigen, The Jackson Laboratory, 600 Main St., Bar Harbor, ME 04609 (E-mail: bjp{at}jax.org).
10.1152/physiolgenomics.00073.2003.
1 The Supplementary Material for this article (Table 2, a summary of nucleotide sequence variations in the coding and promoter regions of Pparg and Slc21a1 detected between strains CAST and 129) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00073.2003/DC1.
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