Chemical mutagenesis induced two high bone density mouse mutants map to a concordant distal chromosome 4 locus

Phenotype-driven mutagenesis approach in the mouse holds much promise as a method for revealing gene function. Earlier, we have described an N-ethyl-N-nitrosourea (ENU) mutagenesis screen to create genome-wide dominant mutations in the mouse model. Using this approach, we describe identification of two high bone density mutants in C57BL/6J (B6) background. The mutants, named as 12184 and 12137, have been bred more than five generations with wild-type B6 mice, each producing >200 backcross progeny. The average total body areal bone mineral density (aBMD) was 13-17% higher in backcrossed progeny from both mutant lines between 6 and 10 weeks of age, as compared to wild-type (WT) B6 mice (n=60-107). At 3 weeks of age the aBMD of mutant progeny was not significantly affected as compared to WT B6 mice. Data from 10- and 16-week old progeny show that increased aBMD was mainly related to a 14-20% higher bone mineral content, whereas bone size was marginally increased. In addition, the average volumetric BMD (vBMD) was 5-15% higher at the midshaft tibia or femur, as compared to WT mice. Histomorphometric analysis revealed that bone resorption was 23-34% reduced in both mutant mice. Consistent with histomorphometry data, the mRNA expression of genes that regulate osteoclast differentiation and survival were altered in the 12137 mutant mice. To determine the chromosomal location of the ENU mutation, we intercrossed both mutant lines with C3H/HeJ (C3H) mice to generate B6C3H F2 mice (n=164 for line 12137 and n=137 F2 for line 12184). Interval mapping using 60 microsatellite markers and aBMD phenotype revealed only one significant or suggestive linkage on chromosome 4. Since body weight was significantly higher in mutant lines, we also used body weight as additive and interactive covariate for interval mapping; both analyses showed higher LOD scores for both 12137 and 12184 mutants without affecting the chromosomal location. The large phenotype in the mutant mice compared to generally observed QTL effects (<5%) would increase the probability of identifying the mutant gene.     

Identification of genetic loci that regulate bone adaptive response to mechanical loading in C57BL/6J and C3H/HeJ mice intercross

Strain-dependent differences in bone adaptive responses to loading among inbred mouse strains suggest that genetic background contributes significantly to adaptation to exercise. To explore the genetic regulation of response to loading, we performed a genome-wide search for linkage in a cross between two strains, a good responder, C57BL6/J (B6), and a poor responder, C3H/HeJ (C3H). Using a four-point bending model, the right tibia was loaded by applying 9 N force for 36 cycles for 12 days in 10-week-old female B6xC3H F2 mice. Changes in bone density (BMD) and bone size were evaluated in vivo by pQCT. Measurements from non-loaded left tibia were used as an internal control to calculate loading-induced percent increase in BMD and bone size, thus excluding the possibility of identifying background QTL(s) due to natural allelic variation in mapping strains. A genome-wide scan was performed using 111 microsatellite markers in DNA samples collected from 329 F2 mice. Heritability of bone adaptive response to loading was between 70 and 80%. The mean increase, expressed as percent of unloaded tibia, was 5% for BMD, 9% for periosteal circumference (PC), and 14% for cortical thickness in F2 mice (n = 329). All these phenotypes showed normal distributions. Absence of significant correlation between BMD response to four-point bending and body weight or bone size suggested that the bone adaptive response was independent of bone size. Interval mapping revealed that BMD response to four-point bending was influenced by three significant loci on Chrs 1 (log-of-odds ratio score (LOD) 3.4, 91.8 cM), 3 (LOD 3.6, 50.3 cM), and 8 (LOD 4.2, 60.1 cM) and one suggestive QTL on Chr 9 (LOD 2.5, 33.9 cM). Loading-induced increases in PC and Cth were influenced by four significant loci on Chrs 8 (LOD 3.0, 68.9 cM), 9 (LOD 3.0, 13.1 cM), 17 (LOD 3.0, 39.3 cM), and 18 (LOD 3.0, 0 cM) and two suggestive loci on Chr 9 (LOD 2.2, 24 cM) and 11 (LOD 2.1, 69.9 cM). Pairwise analysis showed the presence of several significant and suggestive interactions between loci on Chrs 1, 3, 8, and 13 for BMD trait. This is the first study that provides evidence for the presence of multiple genetic loci regulating bone anabolic responses to loading in the B6xC3H intercross. Knowledge of the genes underlying these loci could provide novel approaches to improve skeletal mass.     

Quantitative trait loci that determine BMD in C57BL/6J and 129S1/SvImJ inbred mice.

BMD is highly heritable; however, little is known about the genes. To identify loci controlling BMD, we conducted a QTL analysis in a (B6 x 129) F2 population of mice. We report on additional QTLs and also narrow one QTL by combining the data from multiple crosses and through haplotype analysis. INTRODUCTION: Previous studies have identified quantitative trait loci (QTL) that determine BMD in mice; however, identification of genes underlying QTLs is impeded by the large size of QTL regions. MATERIALS AND METHODS: To identify loci controlling BMD, we performed a QTL analysis of 291 (B6 x 129) F2 females. Total body and vertebral areal BMD (aBMD) were determined by peripheral DXA when mice were 20 weeks old and had consumed a high-fat diet for 14 weeks. RESULTS AND CONCLUSIONS: Two QTLs were common for both total body and vertebral aBMD: Bmd20 on chromosome (Chr) 6 (total aBMD; peak cM 26, logarithm of odds [LOD] 3.8, and vertebral aBMD; cM 32, LOD 3.6) and Bmd22 on Chr 1 (total aBMD; cM 104, LOD 2.5, and vertebral aBMD; cM 98, LOD 2.6). A QTL on Chr 10 (Bmd21, cM 68, LOD 3.0) affected total body aBMD and a QTL on Chr 7 (Bmd9, cM 44, LOD 2.7) affected vertebral aBMD. A pairwise genome-wide search did not reveal significant gene-gene interactions. Collectively, the QTLs accounted for 21.6% of total aBMD and 17.3% of vertebral aBMD of the F(2) population variances. Bmd9 was previously identified in a cross between C57BL/6J and C3H/HeJ mice, and we narrowed this QTL from 34 to 22 cM by combining the data from these crosses. By examining the Bmd9 region for conservation of ancestral alleles among the low allele strains (129S1/SvImJ and C3H/HeJ) that differed from the high allele strain (C57BL/6J), we further narrowed the region to approximately 9.9 cM, where the low allele strains share a common haplotype. Identifying the genes for these QTLs will enhance our understanding of skeletal biology.     

High bone mass in mice expressing a mutant LRP5 gene.

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease. A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30-55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110-232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34-46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80-140%) and the increased cortical size with significant increases in femoral bending strength (50-130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology.     

Congenic strains of mice for verification and genetic decomposition of quantitative trait loci for femoral bone mineral density.

Peak femoral volumetric bone mineral density (femoral bone mineral density) in C57BL/6J (B6) 4-month-old female mice is 50% lower than in C3H/HeJ (C3H) and 34% lower than in CAST/EiJ (CAST) females. Genome-wide analyses of (B6 x C3H)F2 and (B6 x CAST)F2 4-month-old female progeny demonstrated that peak femoral bone mineral density is a complex quantitative trait associated with genetic loci (QTL) on numerous chromosomes (Chrs) and with trait heritabilities of 83% (C3H) and 57% (CAST). To test the effect of each QTL on femoral bone mineral density, two sets of loci (six each from C3H and CAST) were selected to make congenic strains by repeated backcrossing of donor mice carrying a given QTL-containing chromosomal region to recipient mice of the B6 progenitor strain. At the N6F1 generation, each B6.C3H and B6.CAST congenic strain (statistically 98% B6-like in genomic composition) was intercrossed to obtain N6F2 progeny for testing the effect of each QTL on femoral bone mineral density. In addition, the femoral bone mineral density QTL region on Chr 1 of C3H was selected for congenic subline development to facilitate fine mapping of this strong femoral bone mineral density locus. In 11 of 12 congenic strains, 6 B6.C3H and 5 B6.CAST, femoral bone mineral density in mice carrying c3h or cast alleles in the QTL regions was significantly different from that of littermates carrying b6 alleles. Differences also were observed in body weight, femoral length, and mid-diaphyseal periosteal circumference among these 11 congenic strains when compared with control littermates; however, these latter three phenotypes were not consistently correlated with femoral bone mineral density. Analyses of eight sublines derived from the B6.C3H-1T congenic region revealed two QTLs: one located between 36.9 and 49.7 centiMorgans (cM) and the other located between 73.2 and 100.0 cM distal to the centromere. In conclusion, these congenic strains provide proof of principle that many QTLs identified in the F2 analyses for femoral bone mineral density exert independent effects when transferred and expressed in a common genetic background. Furthermore, significant differences in femoral bone mineral density among the congenic strains were not consistently accompanied by changes in body weight, femur length, or periosteal circumference. Finally, decomposition of QTL regions by congenic sublines can reveal additional loci for phenotypes assigned to a QTL region and can markedly refine genomic locations of quantitative trait loci, providing the opportunity for candidate gene testing.     

Sex-specific quantitative trait loci contribute to normal variation in bone structure at the proximal femur in men

Bone structure is an important determinant of osteoporotic fracture. In women, bone structure is highly heritable, and several quantitative trait loci (QTL) have been reported. There are few comparable data in men. This study in men aimed at establishing the heritability of bone structure at the proximal femur, identifying QTL contributing to normal variation in bone structure, and determining which QTL might be sex-specific. Bone structure at the proximal femur was measured in 205 pairs of brothers age 18-61. Heritability was calculated, and linkage analysis performed on phenotypes at the proximal femur. Heritability estimates ranged from 0.99 to 0.39. A genome wide scan identified suggestive QTL (LOD>2.2) for femoral shaft width on chromosome 14q (LOD=2.69 at position 99 cM), calcar femorale at chromosome 2p (LOD=3.97 at position 194 cM) and at the X chromosome (LOD=3.01 at position 77 cM), femoral neck width on chromosome 5p (LOD=2.28 at position 0 cM), femoral head width on chromosome 11q (LOD=2.30 at position 131 cM) and 15q (LOD=3.11 at position 90 cM), and pelvic axis length on chromosome 4q (LOD=4.16 at position 99 cM) and 17q (LOD=2.80 at position 112 cM). Comparison with published data in 437 pairs of premenopausal sisters from the same geographical region suggested that 3 of the 7 autosomal QTL were male-specific. This study demonstrates that bone structure at the proximal femur in healthy men is highly heritable. The occurrence of sex-specific genes in humans for bone structure has important implications for the pathogenesis and treatment of osteoporosis.     

Generation of a new congenic mouse strain to test the relationships among serum insulin-like growth factor I, bone mineral density, and skeletal morphology in vivo.

Insulin-like growth factor (IGF) I is a critical peptide for skeletal growth and consolidation. However, its regulation is complex and, in part, heritable. We previously indicated that changes in both serum and skeletal IGF-I were related to strain-specific differences in total femoral bone mineral density (BMD) in mice. In addition, we defined four quantitative trait loci (QTLs) that contribute to the heritable determinants of the serum IGF-I phenotype in F2 mice derived from progenitor crosses between C3H/HeJ (C3H; high total femoral BMD and high IGF-I) and C57BL/6J (B6; low total femoral BMD and low IGF-I) strains. The strongest QTL, IGF-I serum level 1 (Igflsl-1; log10 of the odds ratio [LOD] score, approximately 9.0), is located on the middle portion of chromosome (Chr) 6. For this locus, C3H alleles are associated with a significant reduction in serum IGF-I. To test the effect of this QTL in vivo, we generated a new congenic strain (B6.C3H-6T [6T]) by placing the Chr 6 QTL region (D6Mit93 to D6Mit150) from C3H onto the B6 background. We then compared serum and skeletal IGF-I levels, body weight, and several skeletal phenotypes from the N9 generation of 6T congenic mice against B6 control mice. Female 6T congenic mice had 11-21% lower serum IGF-I levels at 6, 8, and 16 weeks of age compared with B6 (p < 0.05 for all). In males, serum IGF-I levels were similar in 6T congenics and B6 controls at 6 weeks and 8 weeks but were lower in 6T congenic mice at 16 weeks (p < 0.02). In vitro, there was a 40% reduction in secreted IGF-I in the conditioned media (CMs) from 6T calvaria osteoblasts compared with B6 cells (p < 0.01). Total femoral BMD as measured by peripheral quantitative computed tomography (pQCT) was lower in both 6T male (-4.8%, p < 0.01) and 6T female (-2.3%, p = 0.06) congenic mice. Geometric features of middiaphyseal cortical bone were reduced in 6T congenic mice compared with control mice. Femoral cancellous bone volume (BV) density and trabecular number (Tb.N) were 50% lower, whereas trabecular separation (Tb.Sp) was 90% higher in 8-week-old female 6T congenic mice compared with B6 control mice (p < 0.01 for all). Similarly, vertebral cancellous BV density and Tb.N were lower (-29% and -19%, respectively), whereas Tb.Sp was higher (+29%) in 16-week-old female 6T congenic mice compared with B6 control mice (p < 0.001 for all). Histomorphometric evaluation of the proximal tibia indicated that 6T congenics had reduced BV fraction, labeled surface, and bone formation rates compared with B6 congenic mice. In summary, we have developed a new congenic mouse strain that confirms the Chr 6 QTL as a major genetic regulatory determinant for serum IGF-I. This locus also influences bone density and morphology, with more dramatic effects in cancellous bone than in cortical bone.     

Novel loci regulating bone anabolic response to loading: expression QTL analysis in C57BL/6JXC3H/HeJ mice cross

Variations in the expression levels of bone marker genes among the inbred strains of mice in response to mechanical loading (ML) are largely determined by genetic factors. To explore this, we performed four-point bending on tibiae of 10-week female F2 mice of B6XC3H cross using 9N at 2 Hz, 36 cycles, once per day for 12 days. We collected tibiae from these mice for RNA extraction. We then measured the expression changes of bone marker genes, bone sialoprotein (BSP), alkaline phosphatase (ALP) and housekeeping genes, beta-actin and peptidylprolyl isomerase A (PPIA), by using real-time PCR in both the loaded and the non-loaded tibiae of F2 mice (n=241). A genome-wide scan was performed using 111 micro satellite markers in DNA sample collected from these mice. Mean increase in gene expression, expressed as fold change, ranges from 2.8 to 3.0 for BSP and 2.7 to 2.8 for ALP. Both showed a skewed distribution with a heritability response of 87 to 91%. Absence of significant correlation between the increased gene expression vs. body weight (BW) and bone size (BS) suggests that bone response to loading is independent of BS or BW. Non-parametric mapping (MapQTL program 5) revealed that BSP and ALP expression in response to bending was regulated by several significant and suggestive QTL: Loci regulating both BSP and ALP were located on Chr 8 (60.1 cM), 16 (45.9 cM), 17 (14.2 cM), 18 (38.0 cM) and Chr 19 (3.3 cM); Loci specific to BSP were found on Chrs 1 (LOD score 10.4 at 91.8 cM), 5 (5.2 at 73.2 cM) and 9 (7.0 at 13.1 cM); Loci regulating only ALP were found on Chrs 1 (7.6 at 46 and 75.4 cM), 3 (8.3 at 47 cM) and 4 (5.6 at 54.6 cM). QTLs on Chrs 1, 3, 8, 9, 17 and 18 correspond to QTLs we previously reported by pQCT measurements, thus validating these findings. In addition, we found that the QTL associated with non-loaded tibiae for BSP and ALP on Chrs 4, 16 and 18 was identical to the QTLs associated with ML. This finding suggests that regions on these chromosomes are responsible for natural variation in expression of BSP and ALP as well as for ML. This is the first expression study to provide evidence for the presence of multiple genetic loci regulating bone anabolic response to loading in the B6XC3H intercross and will lead to a better understanding of how exercise improves the skeletal mass.     

Mapping quantitative trait loci that influence blood levels of alkaline phosphatase in MRL/MpJ and SJL/J mice

To examine the hypothesis that serum alkaline phosphatase (ALP) levels have a heritable component, we analyzed blood from two inbred strains of mice, MRL/MpJ and SJL, which exhibit 90% difference in total serum ALP activity (268+/-26 vs. 140+/-15 U/l, respectively, P<0.001). A genome-wide scan was carried out using 137 polymorphic markers in 518 F2 female mice. Serum ALP activity in the F2 progeny showed a normal distribution with an estimated heritability of 56%. Genome-wide scan for cosegregation of genetic marker data with serum ALP activity revealed three major quantitative trait loci (QTL), one each on chromosomes 2 (LOD score 3.8), chromosome 6 (LOD score 12.0), and chromosome 14 (LOD score 3.7). In addition, there was one suggestive QTL on chromosome 2 (LOD score of 3.3). In aggregate, these QTLs explain 22.5% of variance in serum ALP between these two strains. Serum ALP showed a moderate but significant correlation with body weight adjusted total body bone mineral density (r=0.12, P=0.0108) and periosteal circumference at midshaft tibia (r=0.15, P=0.0006) in F2 mice. The chromosome 6 locus harboring the major serum ALP QTL also contains a major BMD and bone size QTL, identified earlier, between these two strains of mice; in addition, this QTL is also close to the locus that regulates IGF-I levels (LOD score 8-9) in C3HB6 F2 mice. These common QTLs indicate that the observed difference in ALP and BMD or bone size may be regulated by same loci (or genes). Accordingly, the osteoblast cells isolated from femur and tibia of MRL mice showed a significantly higher number of ALP +ve cells/colony and two- to threefold higher ALP activity (P<0.001) as compared to the cells isolated from SJL mice, thus suggesting that differences in serum ALP between MRL and SJL reflect difference in ALP expression from osteoblasts from these strains of mice. These data suggest that serum ALP levels are genetically determined and correlate with cellular mechanisms that differentiate BMD accrual in these two strains of mice. The findings that ALP and BMD traits share the same loci on chromosome 6 suggest a role for genetic determinants of bone formation in overall BMD accretion.     

Genome-wide scan for type 2 diabetes loci in Hong Kong Chinese and confirmation of a susceptibility locus on chromosome 1q21-q25

We conducted an autosomal genome scan to map loci for type 2 diabetes in a Hong Kong Chinese population. We studied 64 families, segregating type 2 diabetes, of which 57 had at least one member with an age at diagnosis of 0.59, P(pointwise) < 0.05): chromosome 1 at 173.9 cM (LOD = 3.09), chromosome 3 at 26.3 cM (LOD = 1.27), chromosome 4 at 135.3 cM (LOD = 2.63), chromosome 5 at 139.3 cM (LOD = 0.84), chromosome 6 at 178.9 cM (LOD = 1.91), chromosome 12 at 48.7 cM (LOD = 1.99), and chromosome 18 at 28.1 cM (LOD = 1.00). Simulation studies showed genome-wide significant evidence for linkage of the chromosome 1 region (P(genome-wide) = 0.036). We have confirmed the results of previous studies for the presence of a susceptibility locus on chromosome 1q21-q25 (173.9 cM) and suggest the locations of other loci that may contribute to the development of type 2 diabetes in Hong Kong Chinese.     

Inbred Strain‐Specific Response to Biglycan Deficiency in the Cortical Bone of C57BL6/129 and C3H/He Mice

Inbred strain‐specific differences in mice exist in bone cross‐sectional geometry, mechanical properties, and indices of bone formation. Inbred strain‐specific responses to external stimuli also exist, but the role of background strain in response to genetic deletion is not fully understood. Biglycan (bgn) deficiency impacts bone through negative regulation of osteoblasts, resulting in extracellular matrix alterations and decreased mechanical properties. Because osteoblasts from C3H/He (C3H) mice are inherently more active versus osteoblasts from other inbred strains, and the bones of C3H mice are less responsive to other insults, it was hypothesized that C3H mice would be relatively more resistant to changes associated with bgn deficiency compared with C57BL6/129 (B6;129) mice. Changes in mRNA expression, tissue composition, mineral density, bone formation rate, cross‐sectional geometry, and mechanical properties were studied at 8 and 11 wk of age in the tibias of male wildtype and bgn‐deficient mice bred on B6;129 and C3H background strains. Bgn deficiency altered collagen cross‐linking and gene expression and the amount and composition of mineral in vivo. In bgn's absence, changes in collagen were independent of mouse strain. Bgn‐deficiency increased the amount of mineral in both strains, but changes in mineral composition, cross‐sectional geometry, and mechanical properties were dependent on genetic background. Bgn deficiency influenced the amount and composition of bone in mice from both strains at 8 wk, but C3H mice were better able to maintain properties close to wildtype (WT) levels. By 11 wk, most properties from C3H knockout (KO) bones were equal to or greater than WT levels, whereas phenotypic differences persisted in B6;129 KO mice. This is the first study into mouse strain‐specific changes in a small leucine‐rich proteoglycan gene disruption model in properties across the bone hierarchy and is also one of the first to relate these changes to mechanical competence. This study supports the importance of genetic factors in determining the response to a gene deletion and defines biglycan's importance to collagen and mineral composition in vivo.     

Increased bone formation in mice lacking apolipoprotein E.

ApoE is a plasma protein that plays a major role in lipoprotein metabolism. Here we describe that ApoE expression is strongly induced on mineralization of primary osteoblast cultures. ApoE-deficient mice display an increased bone formation rate compared with wildtype controls, thereby showing that ApoE has a physiologic function in bone remodeling. INTRODUCTION: Apolipoprotein E (ApoE) is a protein component of lipoproteins and facilitates their clearance from the circulation. This is confirmed by the phenotype of ApoE-deficient mice that have high plasma cholesterol levels and spontaneously develop atherosclerotic lesions. The bone phenotype of these mice has not been analyzed to date, although an association between certain ApoE alleles and BMD has been reported. MATERIALS AND METHODS: Primary osteoblasts were isolated from newborn mouse calvariae and mineralized ex vivo. A genome-wide expression analysis was performed during the course of differentiation using the Affymetrix gene chip system. Bones from ApoE-deficient mice and wildtype controls were analyzed using radiography, micro CT imaging, and undecalcified histology. Cellular activities were assessed using dynamic histomorphometry and by measuring urinary collagen degradation products. Lipoprotein uptake assays were performed with (125)I-labeled triglyceride-rich lipoprotein-remnants (TRL-R) using primary osteoblasts from wildtype and ApoE-deficient mice. Serum concentrations of osteocalcin were determined by radioimmunoassay after hydroxyapatite chromatography. RESULTS: ApoE expression is strongly induced on mineralization of primary osteoblast cultures ex vivo. Mice lacking ApoE display a high bone mass phenotype that is caused by an increased bone formation rate, whereas bone resorption is not affected. This phenotype may be explained by a decreased uptake of triglyceride-rich lipoproteins by osteoblasts, resulting in elevated levels of undercarboxylated osteocalcin in the serum of ApoE-deficient mice. CONCLUSION: The specific induction of ApoE gene expression during osteoblast differentiation along with the increased bone formation rate observed in ApoE-deficient mice shows that ApoE has a physiologic role as a regulator of osteoblast function.     

Quantitative trait loci for femoral and lumbar vertebral bone mineral density in C57BL/6J and C3H/HeJ inbred strains of mice.

Significant differences in vertebral (9%) and femoral (50%) adult bone mineral density (BMD) between the C57BL/6J (B6) and C3H/HeJ (C3H) inbred strains of mice have been subjected to genetic analyses for quantitative trait loci (QTL). Nine hundred eighty-six B6C3F2 females were analyzed to gain insight into the number of genes that regulate peak BMD and their locations. Femurs and lumbar vertebrae were isolated from 4-month-old B6C3F2 females at skeletal maturity and then BMD was determined by peripheral quantitative computed tomography (pQCT). Estimates of BMD heritability were 83% for femurs and 72% for vertebrae. Genomic DNA from F2 progeny was screened for 107 polymerase chain reaction (PCR)-based markers discriminating B6 and C3H alleles on all 19 autosomes. The regression analyses of markers on BMD revealed ten chromosomes (1, 2, 4, 6, 11, 12, 13, 14, 16, and 18) carrying QTLs for femurs and seven chromosomes (1, 4, 7, 9, 11, 14, and 18) carrying QTLs for vertebrae, each with log10 of the odds ratio (LOD) scores of 2.8 or better. The QTLs on chromosomes (Chrs) 2, 6, 12, 13, and 16 were unique to femurs, whereas the QTLs on Chrs 7 and 9 were unique to vertebrae. When the two bone sites had a QTL on the same chromosome, the same marker had the highest, although different, LOD score. A pairwise comparison by analysis of variance (ANOVA) did not reveal significant gene x gene interactions between QTLs for either bone site. BMD variance accounted for by individual QTLs ranged from 1% to 10%. Collectively, the BMD QTLs for femurs accounted for 35.1% and for vertebrae accounted for 23.7 % of the F2 population variances in these bones. When mice were homozygous c3/c3 in the QTL region, 8 of the 10 QTLs increased, while the remaining two QTLs on Chrs 6 and 12 decreased, femoral BMD. Similarly, when mice were homozygous c3/c3 in the QTL region for the vertebrae, five of the seven QTLs increased, while two QTLs on Chrs 7 and 9 decreased, BMD. These findings show the genetic complexity of BMD with multiple genes participating in its regulation. Although 5 of the 12 QTLs are considered to be skeleton-wide loci and commonly affect both femurs and vertebrae, each of the bone sites also exhibited unique QTLs. Thus, the BMD phenotype can be partitioned into its genetic components and the effects of these loci on normal bone biology can be determined. Importantly, the BMD QTLs that we have identified are in regions of the mouse genome that have known human homology, and the QTLs will become useful experimental tools for mechanistic and therapeutic analyses of bone regulatory genes.     

Congenic mapping of the diabetogenic locus Idd4 to a 5.2-cM region of chromosome 11 in NOD mice: identification of two potential candidate subloci.

Twenty diabetes susceptibility loci on 12 mouse chromosomes have been identified to control the development of type 1 diabetes at the level of either initiation of insulitis or progression from insulitis to overt diabetes or both. Previously, we demonstrated that the genetic control of T-cell proliferative unresponsiveness in nonobese diabetic (NOD) mice is linked to Idd4 on mouse chromosome 11. Here, we show by congenic mapping of three newly generated NOD.B6Idd4 diabetes-resistant mouse strains that Idd4 is limited to a 5.2-cM interval of chromosome 11. This B6-derived region expressed in NOD.B6Idd4A mice maps between the D11Nds1 (43.8 cM) and D11Mit38/D11Mit325 (49.0 cM) markers and dramatically reduces the development of both insulitis and type 1 diabetes. NOD.B6Idd4B and NOD.B6Idd4C mice, which carry a smaller B6-derived segment of chromosome 11 that spans <5.2 cM distal to D11Nds1, exhibit protection against type 1 diabetes with the restoration of T-cell proliferation. Our findings suggest that diabetes resistance conferred by Idd4 may be mediated by the Idd4.1 and Idd4.2 subloci. Idd4.1 is localized in the D11Nds1 interval that influences both diabetes and insulitis. Idd4.2 is localized within the D11Mit38/325 interval that mainly influences diabetes incidence and restores T-cell proliferative responsiveness. Three potential candidate genes, platelet activating factor acetylhydrolase Ib1, nitric oxide synthase-2, and CC chemokine genes, are localized in the 5.2-cM interval.     

Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice

Serum insulin-like growth factor-1 (IGF-1) and femoral bone mineral density (BMD) differ between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), by approximately 30% and 50%, respectively. Similarly, skeletal IGF-1 content, bone formation, mineral apposition, and marrow stromal cell numbers are higher in C3H than in B6 mice. Because IGF-1 and several bone parameters cosegregate, we hypothesize that the serum IGF-1 phenotype has a strong heritable component and that genetic determinants for serum IGF-1 are involved in the regulation of bone mass. We intercrossed (B6 x C3H)F1 hybrids and analyzed 682 F2 female offspring at 4 months of age for serum IGF-1 by radioimmunoassay and femoral BMD by peripheral quantitative computerized tomography (pQCT). Genomic DNA was assayed by polymerase chain reaction (PCR) to determine alleles for 114 Mit markers inherited in F2 mice at average distances of 14 centimorgans (cM) along each chromosome (Chr). Serum IGF-1 levels in the F2 progeny were relatively normal in distribution, but showed a greater range than either progenitor, indicating that serum IGF-1 level is a polygenic trait with an estimated heritability of 52%. Serum IGF-1 correlated with femoral length (r = 0.266, p < 0.0001) and femoral BMD (r = 0.267, p < 0.0001). Whole genome scans for main effects associated with serum IGF-1 levels revealed three significant QTLs (in order of significance) on mouse Chrs 6, 15, and 10. The QTL on Chr 6 showed a significant reduction in IGF-1 associated with increasing C3H allele number, whereas the Chr 15 and Chr 10 loci showed additive effects with increasing C3H allele number. A genome-wide search for interacting marker pairs identified a significant interaction between the Chr 6 QTL and a locus on Chr 11. This interactive effect suggested that when the Chr 11 locus was homozygous for C3H, there was no effect of the Chr 6 locus on serum IGF-1; however, the combination of C3H alleles on Chr 6 with B6 alleles on Chr 11 was associated with reduced serum IGF-1 concentrations. To test this in vivo, we tested congenic mice carrying the Chr 6 QTL region from C3H on a B6 background (B6.C3H-6). Both serum IGF-1 and femoral BMD were significantly lower in female congenic than progenitor B6 mice. In summary, we identified three major QTLs on mouse Chrs 6, 10, and 15, and noted a major locus-locus interaction between Chrs 6 and 11. We named these QTLs IGF-1 serum levels (Igf1sl1 to Igf1sl4). Functional isolation of the Igf1sl1 QTL on Chr 6 for IGF-1 in B6.C3H-6 congenic mice demonstrated effects on both the IGF-1 and BMD phenotypes. The genetic determinants of these Igf1sl QTLs will provide much insight into the regulation of IGF-1 and the subsequent acquisition of peak bone mass.     

A chemical mutagenesis screen to identify modifier genes that interact with growth hormone and TGF-beta signaling pathways

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as >3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p<0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.     

In vivo and in vitro evidence that the high osteoblastic activity in C3H/HeJ mice compared to C57BL/6J mice is intrinsic to bone cells

Two inbred mouse strains, C3H/HeJ (C3H) and C57BL/6J (B6), displayed a profound difference in femoral peak bone density. We have previously shown that the difference could be attributed to a greater bone formation rate (BFR) that was due to a higher osteoblastic activity [measured by a mineral apposition rate (MAR)] in the C3H (high density) than B6 (low density) mice. The present study sought to determine (1) whether the BFR/MAR differences between the two mouse strains present in weight-loaded endochondral bones are also seen in less weight-loaded membranous bones and (2) whether the difference in osteoblastic activity was seen in vitro in the absence of systemic factors. To address the first objective, we performed histomorphometric measurements on the weakly loaded membranous bones (i.e., parietal bones of the calvaria) to determine if there were similar differences in MAR and BFR of membranous bones as those of highly loaded, endochondral bones. The parietal bones of adult C3H mice showed similar increases in MAR and BFR as the endochondral bones, compared to B6 mice of same age, suggesting that the differences in the MAR and BFR in the two mouse strains are probably not related to differences in mechanical strain. These findings also suggest that the gene(s) responsible for the difference in MAR between strains may not be a mechanical response gene. With respect to the second objective, we isolated osteoblasts from the parietal bones and determined their differentiation status (i.e., ALP-specific activity) and bone-forming ability (i.e., mineralized nodule formation) in vitro. Consistent with the premise that C3H osteoblasts have an intrinsic, higher differentiation status and bone-forming ability than B6 osteoblasts, osteoblasts isolated from C3H mice as compared with those from B6 mice had a significantly greater ALP-specific activity and a greater ability to form mineralized nodules in vitro in the absence of systemic factors. Because differences in ALP activity, bone-forming ability, cortical bone width, and osteoblastic activity were detected at birth, the different MAR/BFR phenotypes develop at very early life and even perhaps during embryogenesis. In conclusion, we have for the first time provided evidence that the genetic differences responsible for the observed MAR/BFR phenotype in the C3H-B6 strains are intrinsic to osteoblasts and might not depend on responses to mechanical loading and/or alterations in systemic factors.     

Linkage exclusion mapping with bone size in 79 Caucasian pedigrees

Bone size is an important risk factor of osteoporotic fractures and has strong genetic determination. However, genetic studies on bone size variation are relatively rare. In the present study, we conducted a linkage exclusion mapping for bone size variation on chromosomes 1, 4, 6, and 17 in 79 Caucasian pedigrees. For hip bone size variation, several genomic regions were excluded at effect sizes of 10% or greater, including regions of 61–77cM at 1p35-p34, 43–59cM at 4p15-p13, 1–59cM at 6p25-p21, 82–88cM at 17q23-q24, and 113–114cM at 17q25. For spine bone size, at effect sizes of 10% or greater, we excluded regions of 115–122cM at 1p31-p22, 136–141cM at 1p21, 207–260cM at 1q31-q42, 20–89cM at 4p16-q21, 11–21cM at 6p24-p23, and 1–6cM at 17p13. These results suggested that a number of candidate genes located in the excluded regions, such as the growth hormone (GH) gene, tumor necrosis factor-alpha (TNF-α) gene, and bone morphogenetic protein-3 (BMP3) gene, are unlikely to have a substantial effect on bone size variation in this Caucasian population.