Quantitative trait loci for carbohydrate and total energy intake on mouse chromosome 17: congenic strain confirmation and candidate gene analyses (Glo1, Glp1r)

Quantitative trait loci (QTL) for carbohydrate (Mnic1) and total energy (Kcal2) intake on proximal mouse chromosome 17 were identified previously from a C57BL/6J (B6) X CAST/Ei (CAST) intercross. Here we report that a new congenic strain developed in our laboratory has confirmed this complex locus by recapitulating the original linked phenotypes: B6.CAST-17 homozygous congenic mice consumed more carbohydrate (27%) and total energy (17%) compared with littermate wild-type mice. Positional gene candidates with relevance to carbohydrate metabolism, glyoxalase I (Glo1) and glucagon-like peptide-1 receptor (Glp1r), were evaluated. Glo1 expression was upregulated in liver and hypothalamus of congenic mice when compared with B6 mice. Analyses of Glp1r mRNA and protein expression revealed tissue-specific strain differences in pancreas (congenic>B6) and stomach (B6>congenic). These results suggest the possibility of separate mechanisms for enhanced insulin synthesis and gastric accommodation in the presence of high carbohydrate intake and larger food volume, respectively. Sequence analysis of Glp1r found a G insert at nt position 1349, which results in earlier termination of the open reading frame, thus revealing an error in the public sequence. Consequently, the predicted length of GLP-1R is 463 aa compared with 489 aa, as previously reported. Also, we found a polymorphism in Glp1r between parental strains that alters the amino acid sequence. Variation in Glp1r could influence nutrient intake in this model through changes in the regulatory or protein coding regions of the gene. These congenic mice offer a powerful tool for investigating gene interactions in the control of food intake.     

Genetic variation in Glp1r expression influences the rate of gastric emptying in mice

We demonstrated previously that food intake traits map to a quantitative trait locus (QTL) on proximal chromosome 17, which encompasses Glp1r (glucagon-like peptide 1 receptor), encoding an important modulator of gastric emptying. We then confirmed this QTL in a B6.CAST-17 congenic strain that consumed 27% more carbohydrate and 17% more total calories, yet similar fat calories, per body weight compared with the recipient C57BL/6J. The congenic strain also consumed greater food volume. The current aims were to 1) identify genetic linkage for total food volume in F(2) mice, 2) perform gene expression profiling in stomach of B6.CAST-17 congenic mice using oligonucleotide arrays, 3) test for allelic imbalance in Glp1r expression, 4) evaluate gastric emptying rate in parental and congenic mice, and 5) investigate a possible effect of genetic variation in Glp1r on gastric emptying. A genome scan revealed a single QTL for total food volume (Tfv1) (log of the odds ratio = 7.6), which was confirmed in B6.CAST-17 congenic mice. Glp1r exhibited allelic imbalance in stomach, which correlated with accelerated gastric emptying in parental CAST and congenic B6.CAST-17 mice. Moreover, congenic mice displayed an impaired gastric emptying response to exendin-(9-39). These results suggest that genetic variation in Glp1r contributes to the strain differences in gastric emptying rate.     

A novel mouse Chromosome 2 congenic strain with obesity phenotypes

Linkage studies have identified many chromosomal regions containing obesity genes in mice. However, only a few of these quantitative trait loci (QTLs) have been used to guide the production of congenic mouse strains that retain obesity phenotypes. We seek to identify chromosomal regions containing obesity genes in the BSB model of spontaneous obesity because the BSB model is a multigenic obesity model. Previous studies identified QTLs on Chromosomes (Chrs) 2, 6, 7,12, and 15. BSB mice are made by backcross of lean C57BL/6J × Mus spretus. F₁s were backcrossed to C57BL/6J mice to produce BSB progeny. We have constructed a new BSB cross and produced congenic mice with obesity phenotypes by marker-directed selection called B6.S–D2Mit194–D2Mit311. We found a highly significant QTL for percentage body lipid on Chr 2 just proximal to the Agouti locus. Chr 2 congenics were constructed to determine whether the main effects would be detectable. We observed highly significant linkage of the Chr 2 congenic containing Agouti and containing markers distal to D2Mit311 and proximal to D2Mit194. Thus, this congenic contains approximately 14.6 cM or 30 Mb (about 1.1% of the spretus mouse genome) and several hundred genes. The obesity phenotype of the QTL is retained in the congenic. The congenic can now be used to model the genetic and physiological basis for a relatively simple, perhaps monogenic, obesity.     

A genome-wide set of congenic mouse strains derived from CAST/Ei on a C57BL/6 background

We previously reported the construction of two sets of heterozygous congenic strains spanning the mouse genome. For both sets, C57BL/6J was employed as the background strain while DNA from either DBA/2 or CAST/Ei was introgressed to form the congenic region. We have subsequently bred most of these strains to produce homozygous breeding stocks. Here, we report the characterization of the strain set based on CAST/Ei. CAST/Ei is the most genetically distant strain within the Mus mus species and many trait variations relevant to common diseases have been identified in CAST/Ei mice. Despite breeding difficulties for some congenic regions, presumably due to incompatible allelic variations between CAST/Ei and C57BL/6, the resulting congenic strains cover about 80% of the autosomal chromosomes and will be useful as a resource for the further analysis of quantitative trait loci between the strains.     

Fine mapping reveals sex bias in quantitative trait loci affecting growth, skeletal size and obesity-related traits on mouse chromosomes 2 and 11

Previous speed congenic analysis has suggested that the expression of growth and obesity quantitative trait loci (QTL) on distal mouse chromosomes (MMU) 2 and 11, segregating between the CAST/EiJ (CAST) and C57BL/6J-hg/hg (HG) strains, is dependent on sex. To confirm, fine map, and further evaluate QTL x sex interactions, we constructed congenic by recipient F2 crosses for the HG.CAST-(D2Mit329-D2Mit457)N(6) (HG2D) and HG.CAST-(D11Mit260-D11Mit255)N(6) (HG11) congenic strains. Over 700 F2 mice were densely genotyped and phenotyped for a panel of 40 body and organ weight, skeletal length, and obesity-related traits at 9 weeks of age. Linkage analysis revealed 20 QTL affecting a representative subset of phenotypes in HG2DF2 and HG11F2 mice. The effect of sex was quantified by comparing two linear models: the first model included sex as an additive covariate and the second incorporated sex as an additive and an interactive covariate. Of the 20 QTL, 8 were sex biased, sex specific, or sex antagonistic. Most traits were regulated by single QTL; however, two closely linked loci were identified for five traits in HG2DF2 mice. Additionally, the confidence intervals for most QTL were significantly reduced relative to the original mapping results, setting the stage for quantitative trait gene (QTG) discovery. These results highlight the importance of assessing the contribution of sex in complex trait analyses.     

Genetic studies of B-lymphocyte deficiency and mastocytosis in strain A/WySnJ mice

The A/WySnJ mouse, but not the related A/J strain, has peripheral B-lymphocyte deficiency and mastocytosis. Minimally, two quantitative trait loci (QTLs) control the B-cell deficiency in (A/WySnJ x CAST/Ei)F2 intercross mice; one of them, Bcmd-1, mapped to Chromosome (Chr) 15. Several QTLs controlled the mastocytosis in this intercross, and it was not possible to determine whether any of them co-segregated with Bcmd-1. We have now mapped a second QTL controlling the B-cell deficiency, Bcmd-2, to Chr 4. Furthermore, we narrowed the map position of Bcmd-1 to <2.0 cM. Both QTLs have been confirmed through the construction of AW. Bcmd-1(c), AW. Bcmd-2(c), and AW. Bcmd-1(c)Bcmd-2(c) recombinant congenic strains. The Bcmd-1 locus is the major regulator of B-cell homeostasis, while Bcmd-2 is the minor regulator, and their effects are additive, as shown by splenic B-cells analysis in these congenic strains. In addition, Bcmd-2 or a linked locus controls mastocytosis, while Bcmd-1 does not, as indicated by splenic mast cell analysis in the congenic strains. Thus, the major genetic controls on B-cell homeostasis and mast cell homeostasis in A/WySnJ mice are asserted by distinct genes.     

Increased physical activity cosegregates with higher intake of carbohydrate and total calories in a subcongenic mouse strain

C57BL/6 J (B6) and CAST/EiJ (CAST), the inbred strain derived from M. musculus castaneus, differ in nutrient intake behaviors, including dietary fat and carbohydrate consumption in a two-diet-choice paradigm. Significant quantitative trait loci (QTLs) for carbohydrate (Mnic1) and total energy intake (Kcal2) are present between these strains on chromosome (Chr) 17. Here we report the refinement of the Chr 17 QTL in a subcongenic strain of the B6.CAST- ^{D17Mit19-D17Mit91} congenic mice described previously. This new subcongenic strain possesses CAST Chr 17 donor alleles from 4.8 to 45.4 Mb on a B6 background. Similar to CAST, the subcongenic mice exhibit increased carbohydrate and total calorie intake per body weight, while fat intake remains equivalent. Unexpectedly, this CAST genomic segment also confers two new physical activity phenotypes: 22% higher spontaneous physical activity levels and significantly increased voluntary wheel-running activity compared with the parental B6 strain. Overall, these data suggest that gene(s) involved in carbohydrate preference and increased physical activity are contained within the proximal region of Chr 17. Interval-specific microarray analysis in hypothalamus and skeletal muscle revealed differentially expressed genes within the subcongenic region, including neuropeptide W (Npw); glyoxalase I (Glo1); cytochrome P450, family 4, subfamily f, polypeptide 1 (Cyp4f15); phospholipase A2, group VII (Pla2g7); and phosphodiesterase 9a (Pde9a). This subcongenic strain offers a unique model for dissecting the contributions and possible interactions among genes controlling food intake and physical activity, key components of energy balance.     

Dissection of multigenic obesity traits in congenic mouse strains

Previous quantitative trait locus mapping (QTL) identified multigenic obesity (MOB) loci on mouse Chromosome (Chr) 2 that influence the interrelated phenotypes of obesity, insulin resistance, and dyslipidemia. To better localize and characterize the MOB locus, three congenic mouse strains were created. Overlapping genomic intervals from the lean CAST/Ei (CAST) strain were introgressed onto an obesity-susceptible C57BL/6 (BL6) background to create proximal (15 Mb–73 Mb), middle (63 Mb–165 Mb), and distal (83 Mb–182 Mb) congenic strains. The congenic strains showed differences in obesity, insulin, and lipid traits consistent with the original QTL analysis for the locus. Importantly, characterization of the MOB congenics localized the effects of genes that underlie obesity-related traits to an introgressed interval (73–83 Mb) unique to the middle MOB congenic. Conversely, significant differences between the lipid and insulin profiles of the middle and distal MOB congenics implicated the presence of at least two genes that underlie these traits. When fed an atherogenic diet, several traits associated with metabolic syndrome were observed in the distal MOB congenic, while alterations in plasma lipoproteins were observed in the middle MOB congenic strain.     

Construction of a BAC Contig for a 3 cM Biologically Significant Region of Mouse Chromosome 1

One QTL and genes and phenotypes have been localized in the region between 92 cM and 95cM of mouse chromosome 1. The QTL locus contributes to approximately 40% of the variation of the peak bone density between C57BL/6J (B6) and CAST/EiJ (CAST) strains. Other loci located in this chromosomal region include a neural tube defect mutant loop-tail (Lp), a lymphocyte-stimulating determinant (Lsd), and the Transgelin 2 (Tagln 2). The human chromosome region homologous to this region is 1q21-23, which also contains a QTL locus for high bone mineral density (BMD). Furthermore, it has been reported that this region may have duplicated several times in the mouse genome. Therefore, genomic sequencing of this region will provide important information for mouse genome structure, for positional cloning of mouse genes, and for the study of human homologous genes. In order to provide a suitable template for genomic sequencing by the NIH-sponsored genomic centers, we have constructed a BAC contig of this region using the RPCI-23 library. We have also identified the currently available mouse genomic sequences localized in our BAC contig. Further analysis of these sequences and BAC clones indicated a high frequency of repetitive sequences within this chromosomal area. This region also contains L1 retrotransposon sequences, providing a potential mechanism for the repetitive sequences described in the literature.     

Allelic variation on murine chromosome 11 modifies host inflammatory responses and resistance to Bacillus anthracis

Anthrax is a potentially fatal disease resulting from infection with Bacillus anthracis. The outcome of infection is influenced by pathogen-encoded virulence factors such as lethal toxin (LT), as well as by genetic variation within the host. To identify host genes controlling susceptibility to anthrax, a library of congenic mice consisting of strains with homozygous chromosomal segments from the LT-responsive CAST/Ei strain introgressed on a LT-resistant C57BL/6 (B6) background was screened for response to LT. Three congenic strains containing CAST/Ei regions of chromosome 11 were identified that displayed a rapid inflammatory response to LT similar to, but more severe than that driven by a LT-responsive allele of the inflammasome constituent NRLP1B. Importantly, increased response to LT in congenic mice correlated with greater resistance to infection by the Sterne strain of B. anthracis. The genomic region controlling the inflammatory response to LT was mapped to 66.36-74.67 Mb on chromosome 11, a region that encodes the LT-responsive CAST/Ei allele of Nlrp1b. However, known downstream effects of NLRP1B activation, including macrophage pyroptosis, cytokine release, and leukocyte infiltration could not fully explain the response to LT or the resistance to B. anthracis Sterne in congenic mice. Further, the exacerbated response in congenic mice is inherited in a recessive manner while the Nlrp1b-mediated response to LT is dominant. Finally, congenic mice displayed increased responsiveness in a model of sepsis compared with B6 mice. In total, these data suggest that allelic variation of one or more chromosome 11 genes in addition to Nlrp1b controls the severity of host response to multiple inflammatory stimuli and contributes to resistance to B. anthracis Sterne. Expression quantitative trait locus analysis revealed 25 genes within this region as high priority candidates for contributing to the host response to LT.     

Gene expression profiles of a mouse congenic strain carrying an obesity susceptibility QTL under obesigenic diets

Genetic factors are strongly involved in the development of obesity, likely through the interactions of susceptibility genes with obesigenic environments, such as high-fat, high-sucrose (HFS) diets. Previously, we have established a mouse congenic strain on C57BL/6 J background, carrying an obesity quantitative trait locus (QTL), tabw2, derived from obese diabetic TALLYHO/JngJ mice. The tabw2 congenic mice exhibit increased adiposity and hyperleptinemia, which becomes exacerbated upon feeding HFS diets. In this study, we conducted genome-wide gene expression profiling to evaluate differentially expressed genes between tabw2 and control mice fed HFS diets, which may lead to identification of candidate genes as well as insights into the mechanisms underlying obesity mediated by tabw2. Both tabw2 congenic mice and control mice were fed HFS diets for 10 weeks beginning at 4 weeks of age, and total RNA was isolated from liver and adipose tissue. Whole-genome microarray analysis was performed and verified by real-time quantitative RT–PCR. At False Discovery Rate adjusted P < 0.05, 1026 genes were up-regulated and 308 down-regulated in liver, whereas 393 were up-regulated and 187 down-regulated in adipose tissue in tabw2 congenic mice compared to controls. Within the tabw2 QTL interval, 70 genes exhibited differential expression in either liver or adipose tissue. A comprehensive pathway analysis revealed a number of biological pathways that may be perturbed in the diet-induced obesity mediated by tabw2.     

Gene-environment interaction: a significant diet-dependent obesity locus demonstrated in a congenic segment on mouse Chromosome 7

We have previously reported suggestive evidence for a locus on Chromosome (Chr) 7 that affects adiposity in F₂ mice from a CAST/Ei × C57BL/6J intercross fed a high-fat diet. Here we characterize the effect of a high-fat (32.6 Kcal% fat) diet on male and female congenic mice with a C57BL/6J background and a CAST/Ei-derived segment on Chr 7. Adiposity index (AI) and weights of certain fat pads were approximately 50% lower in both male and female congenic mice than in control C57BL/6J mice, and carcass fat content was significantly reduced. The reduction of fat depot weights was not seen, however, in congenic animals fed a low-fat chow diet (12 Kcal% fat). The congenic segment is approximately 25 cM in length, extending from D7Mit213 to D7Mit41, and includes the tub, Ucp2, and Ucp3, genes, all of which are candidate genes for this effect. Some polymorphisms have been found on comparing c-DNA sequences of the Ucp2 gene from C57BL/6J and CAST/Ei mice. These results suggest that one or more genes present in the congenic segment modulate the susceptibility to fat deposition on feeding a high-fat diet. We were unable to show any significant difference between the energy intakes of the congenic and the control C57BL/6J mice on the high-fat diet. Also, measurements of energy expenditure in male mice at 6 weeks of age, during the first 2 weeks of exposure to the high-fat diet, failed to show any differences between control and congenic animals. Received: 30 September 1998 / Accepted: 22 December 1998     

A large QTL for fear and anxiety mapped using an F2 cross can be dissected into multiple smaller QTLs

Using chromosome substitution strains (CSS), we previously identified a large quantitative trait locus (QTL) for conditioned fear (CF) on mouse chromosome 10. Here, we used an F₂ cross between CSS‐10 and C57BL/6J (B6) to localize that QTL to distal chromosome 10. That QTL accounted for all the difference between CSS‐10 and B6. We then produced congenic strains to fine‐map that interval. We identified two congenic strains that captured some or all the QTL. The larger congenic strain (Line 1: 122.387121–129.068 Mb; build 37) appeared to account for all the difference between CSS‐10 and B6. The smaller congenic strain (Line 2: 127.277–129.068 Mb) was intermediate between CSS‐10 and B6. We used haplotype mapping followed by quantitative polymerase chain reaction to identify one gene that was differentially expressed in both lines relative to B6 (Rnf41) and one that was differentially expressed between only Line 1 and B6 (Shmt2). These cis‐eQTLs may cause the behavioral QTLs; however, further studies are required to validate these candidate genes. More generally, our observation that a large QTL mapped using CSS and F₂ crosses can be dissected into multiple smaller QTLs shows a weaknesses of two‐stage approaches that seek to use coarse mapping to identify large regions followed by fine‐mapping. Indeed, additional dissection of these congenic strains might result in further subdivision of these QTL regions. Despite these limitations, we have successfully fine‐mapped two QTLs to small regions and identified putative candidate genes, showing that the congenic approach can be effective for fine‐mapping QTLs .     

Congenic dissection of a major QTL for methamphetamine sensitivity implicates epistasis

We previously used the C57BL/6J (B6) × A/J mouse chromosome substitution strain (CSS) panel to identify a major quantitative trait locus (QTL) on chromosome 11 influencing methamphetamine (MA)‐induced locomotor activity. We then made an F₂ cross between CSS‐11 and B6 and narrowed the locus (Bayes credible interval: 79–109 Mb) which was inherited dominantly and accounted for 14% of the phenotypic variance in the CSS panel. In the present study, we created congenic and subcongenic lines possessing heterozygous portions of this QTL to narrow the interval. We identified one line (84–96 Mb) that recapitulated the QTL, thus narrowing the region to 12 Mb. This interval also produced a small decrease in locomotor activity following prior saline treatment. When we generated subcongenic lines spanning the entire 12‐Mb region, the phenotypic difference in MA sensitivity abruptly disappeared, suggesting an epistatic mechanism. We also evaluated the rewarding properties of MA (2 mg/kg, i.p.) in the 84‐ to 96‐Mb congenic line using the conditioned place preference (CPP) test. We replicated the locomotor difference in the MA‐paired CPP chamber yet observed no effect of genotype on MA‐CPP, supporting the specificity of this QTL for MA‐induced locomotor activity under these conditions. Lastly, to aid in prioritizing candidate genes responsible for this QTL, we used the Affymetrix GeneChip® Mouse Gene 1.0ST Array to identify genes containing expression QTLs (eQTL) in the striatum of drug‐naÏve, congenic mice. These findings highlight the difficulty of using congenic lines to fine map QTLs and illustrate how epistasis may thwart such efforts.     

Genetics and bone. Using the mouse to understand man

The rationale for use of inbred strains of mice in bone research is well recognized and includes: a) practical factors (economics of scale, rapid development of adult status, pre-existing knowledge, down-sized technologies) and b) proven methodologies for genetic studies (polygenic trait analyses, mapping tools, genomic sequencing, methods for gene manipulation). Initial investigations of inbred strains of mice showed that femoral and lumbar vertebral volumetric bone mineral density (BMD, mg/mm(3)) by pQCT varied in excess of 50% for femurs and 9% in vertebral BMD. Two strains - low BMD C57BL/6J (B6) mice and high BMD C3H/HeJ (C3H) - were investigated for insights to their BMD diversity. B6C3F2 females derived from intercrossing B6C3F1s were raised to adult skeletal status at 4 months, then necropsied for phenotyping of bone and genotyping of genomic DNA. 1000 F2 females were genotyped for PCR product polymorphisms on all 19 autosomes at approximately 15 cM. Genome wide analyses for genotype-phenotype correlations showed 10 chromosomes (Chrs) carried genes for femoral and 7 Chrs for vertebral BMD. LOD scores ranged from 2.90 to 24.4, and percent of F2 variance accounted for ranged from 1 to 10%. Analyses of main effects revealed both dominant-recessive and additive inheritance patterns. Both progenitor strains carried alleles with positive and negative effects on BMD of each bone sites. A remarkable array of additonal skeletal phenotypes (femur and vertebral geometry, strength measures, serum markers) also proved polygenic in nature, with complex segregation patterns. Verification of BMD quantitative trait loci (QTLs) was undertaken by creating congenic B6 strains carrying individual QTL regions from C3H. Following 6 cycles of backcrossing a QTL-containing region from C3H to the B6 strain, N6F2 congenic strain mice were aged to 4 months, then genotyped for the QTL region and phenotyped for skeletal traits. Comparison of mice homozygous for C3H alleles versus homozygous for B6 alleles in the QTL regions showed that femoral BMD increased or decreased significantly in congenic strains, as was predicted from F2 data. Gender differences specific to BMD QTLs have been revealed, as have more than 30 additional phenotypes associated with cortical and trabecular structural parameters and biomechanical properties.     

Variation in Galr1 expression determines susceptibility to exocitotoxin-induced cell death in mice

Inbred strains of mice differ in their susceptibility to excitotoxin-induced cell death, but the genetic basis of individual variation in differential susceptibility is unknown. Previously, we identified a highly significant quantitative trait locus (QTL) on chromosome 18 that influenced susceptibility to kainic acid-induced cell death ( Sicd1 ). Comparison of susceptibility to seizure-induced cell death between reciprocal congenic lines for Sicd1 and parental background mice indicates that genes influencing this trait were captured in both strains. Two positional gene candidates, Galr1 and Mbp , map to 55 cM, where the Sicd1 QTL had been previously mapped. Thus, this study was undertaken to determine if Galr1 and/or Mbp could be considered as candidate genes. Genomic sequence comparison of these two functional candidate genes from the C57BL/6J (resistant at Sicd1 ) and the FVB/NJ (susceptible at Sicd1 ) strains showed no single-nucleotide polymorphisms. However, expression studies confirmed that Galr1 shows significant differential expression in the congenic and parental inbred strains. Galr1 expression was downregulated in the hippocampus of C57BL/6J mice and FVB.B6- Sicd1 congenic mice when compared with FVB/NJ or B6.FVB- Sicd1 congenic mice. A survey of Galr1 expression among other inbred strains showed a significant effect such that 'susceptible' strains showed a reduction in Galr1 expression as compared with 'resistant' strains. In contrast, no differences in Mbp expression were observed. In summary, these results suggest that differential expression of Galr1 may contribute to the differences in susceptibility to seizure-induced cell death between cell death-resistant and cell death-susceptible strains.     

Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68

Seasonal influenza outbreaks and recurrent influenza pandemics present major challenges to public health. By studying immunological responses to influenza in different host species, it may be possible to discover common mechanisms of susceptibility in response to various influenza strains. This could lead to novel therapeutic targets with wide clinical application. Using a mouse-adapted strain of influenza (A/HK/1/68-MA20 [H3N2]), we produced a mouse model of severe influenza that reproduces the hallmark high viral load and overexpression of cytokines associated with susceptibility to severe influenza in humans. We mapped genetic determinants of the host response using a panel of 29 closely related mouse strains (AcB/BcA panel of recombinant congenic strains) created from influenza-susceptible A/J and influenza-resistant C57BL/6J (B6) mice. Combined clinical quantitative trait loci (QTL) and lung expression QTL mapping identified candidate genes for two sex-specific QTL on chromosomes 2 and 17. The former includes the previously described Hc gene, a deficit of which is associated with the susceptibility phenotype in females. The latter includes the phospholipase gene Pla2g7 and Tnfrsf21, a member of the TNFR superfamily. Confirmation of the gene underlying the chromosome 17 QTL may reveal new strategies for influenza treatment.     

Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11^A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11.     

Mechanical loading-induced gene expression and BMD changes are different in two inbred mouse strains.

Our goal is to evaluate skeletal anabolic response to mechanical loading in different age groups of C57B1/6J (B6) and C3H/HeJ (C3H) mice with variable loads using bone size, bone mineral density (BMD), and gene expression changes as end points. Loads of 6-9 N were applied at 2 Hz for 36 cycles for 12 days on the tibia of 10-wk-old female B6 and C3H mice. Effects of a 9-N load on 10-, 16-, and 36-wk-old C3H mice were also studied. Changes in bone parameters were measured using peripheral quantitative computed tomography, and gene expression was determined by real-time PCR. Total volumetric BMD was increased by 5 and 15%, respectively, with 8- and 9-N loads in the B6, but not the C3H, mice. Increases of 20 and 12% in periosteal circumference were reflected by dramatic 44 and 26% increases in total area in B6 and C3H mice, respectively. The bone response to bending showed no difference in the three age groups of B6 and C3H mice. At 2 days, mechanical loading resulted in significant downregulation in expression of bone resorption (BR), but not bone formation (BF) marker genes. At 4 and 8 days of loading, expression of BF marker genes (type I collagen, alkaline phosphatase, osteocalcin, and bone sialoprotein) was increased two- to threefold and expression of BR marker genes (matrix metalloproteinase-9 and thrombin receptor-activating peptide) was decreased two- to fivefold. Although expression of BF marker genes was upregulated four- to eightfold at 12 days of training, expression of BR marker genes was upregulated seven- to ninefold. Four-point bending caused significantly greater changes in expression of BF and BR marker genes in bones of the B6 than the C3H mice. We conclude that mechanical loading-induced molecular pathways are activated to a greater extent in the B6 than in the C3H mice, resulting in a higher anabolic response in the B6 mice.     

Genetic control of HDL levels and composition in an interspecific mouse cross (CAST/Ei x C57BL/6J)

Strain CAST/Ei (CAST) mice exhibit unusually low levels of high density lipoproteins (HDL) as compared with most other strains of mice, including C57BL/6J (B6). This appears to be due in part to a functional deficiency of lecithin:cholesterol acyltransferase (LCAT). LCAT mRNA expression in CAST mice is normal, but the mice exhibit several characteristics consistent with functional deficiency. First, the activity and mass of LCAT in plasma and in HDL of CAST mice were reduced significantly. Second, the HDL of CAST mice were relatively poor in phospholipids and cholesteryl esters, but rich in free cholesterol and apolipoprotein A-I (apoA-I). Third, the adrenals of CAST mice were depleted of cholesteryl esters, a phenotype similar to that observed in LCAT- and acyl-CoA:cholesterol acyltransferase-deficient mice. Fourth, in common with LCAT-deficient mice, CAST mice contained triglyceride-rich lipoproteins with "panhandle"-like protrusions. To examine the genetic bases of these differences, we studied HDL lipid levels in an intercross between strain CAST and the common laboratory strain B6 on a low fat, chow diet as well as a high fat, atherogenic diet. HDL levels exhibited complex inheritance, as 12 quantitative trait loci with significant or suggestive likelihood of observed data scores were identified. Several of the loci occurred over plausible candidate genes and these were investigated.The results indicate that the functional LCAT deficiency is unlikely to be due to variations of the LCAT gene. Our results suggest that novel genes are likely to be important in the control of HDL metabolism, and they provide evidence of genetic factors influencing the interaction of LCAT with HDL.