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Genetic variability in adult bone density among inbred strains of mice
Bone Vol. 18, No. 5 May 1996:397~-03
ELSEVIER
ORIGINAL ARTICLES
Genetic Variability in Adult Bone Density Among Inbred Strains of Mice W. G. B E A M E R , j L. R. D O N A H U E , I C. J. R O S E N , 2 and D. J. B A Y L I N K 3 1 The Jackson Laborator T, Bar Harbor, ME, USA 2 Maine Centerfiw Osteoporosis Research and Education, Bangor, ME, USA 3 Loma Linda UniversiO' and Jer O" L. Pettis Memorial VA Medical Center, Loma Linda, CA, USA
Key Words: Genetics; Mouse; Bone density; Inbred strains; Development.
More than 70% of the variability in human bone density has been attributed to genetic factors as a result of studies with twins, osteoporotic families, and individuals with rare heritable bone disorders. We have applied the Stratec XCT 960M pQCT, specifically modified for small skeletal specimens, to analyses of bones from 11 inbred strains (AKR/J, BALB/ cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, SJL/BmJ, SWR/BmJ, and 129/.1) of female mice to determine the extent of heritable differences in peak bone density, pQCT scans were taken of femurs from (a) 12month-old inbred strain females and (b) a subset of four strains (C3H/HeJ, DBA/2J, BALB/cByJ, C57BL/6J) at 2, 4, and 8 months. In addition, pQCT scans were also obtained from L5-L6 vertebrae and proximal phalanges from the same subset of four inbred strains at 12 months of age. Comparison of bone parameters among inbred strains revealed significant differences at each of the three sites investigated. Femoral and phalangeal bones differed among strains with respect to total and cortical density, mineral, and volume. Only cortical bone parameters were significantly different among strains at the vertebral site. With respect to strain differences, the highest value for any given bone parameter was found in the C3H/HeJ strain, whereas C57BL/6J values were absolutely, or statistically, the lowest. Similarly, with respect to bone sites, cortical bone density was significantly correlated among strains. On the other hand, we found that none of the femur, vertebral, or phalangeal parameters correlated with body weight, even though body weight varied by 86% among these inbred strains. The developmental studies of femurs conducted at 2, 4, and 8 months of age with C3H/ He J, DBA/2J, BALB/cByJ, and C57BL/6J females showed differences in total density among strains at 2 months and thereafter. Adult peak bone density was typically achieved by 4 months, whereas femurs continued to lengthen for 4 to 8 months thereafter. We conclude that (1) major genetic effects on femoral, vertebral, and phalangeal bone density are detectable among inbred strains of mice; (2) cortical bone density shares common genetic regulation at the three measured sites; and (3) within the femur, genes that regulate length and density are different. (Bone 18:397-403; 1996)
Introduction Peak bone density is considered one of the strongest determinants of subsequent osteoporotic fractures in both men and women. In the adult human skeleton, peak bone density is achieved at the end of the rapid pubertal growth period at approximately 18-22 years of age 9"j8'4°~43 and is under strong genetic regulation. Thus, studies of t w i n s 4'14'16"28"35~36'39 and mother-daughter-grandaughter sets ~3.22,32 have estimated that up to 70% of the variability in bone density is genetically based. Studies are beginning to identify the genes that may be responsible for acquisition of adult peak bone density. Morrison et al. have reported that the vitamin D receptor (VDR) alleles can be associated with (and may be responsible for) genetic variations in bone density. 23 These findings have been reproduced in some patient populations (Japanese, 41 Austrian 12) but not in others ( U S A , 7'14 Japanese, 33 Whites2°). These conflicting results are the likely consequences of genetic heterogeneity of the human population coupled with important environmental differences, especially in the aged. Thus, comparison within twin pairs or among siblings is the only reasonable method of assessing genetic regulation while holding environmental factors reasonably constant. This approach requires large numbers of both dizygotic and monozygotic twin pairs, which are infrequent in the population. Moreover, the genetic variability between pairs of dizygotic twins is as large as in the general population. Animal models are critical for experimentally defining the genetic regulation of bone density. In particular, inbred mice offer unlimited numbers of genetically identical "twins" whose environments can be strictly controlled. These inbred strains offer the opportunity to gain insight into the genetic basis of variation in bone density in phenotypically normal mice. Equally important, each inbred strain is genetically different from every other inbred strain, making possible planned matings to study segregation of genes essential to bone density. In this article are presented bone density data on axial and appendicular sites for adult female mice from I 1 different inbred strains. These strains were selected for analyses because they are progenitors for sets of recombinant inbred strains specifically developed for analyses of heritable traits. ~ The methodology adopted is that of peripheral quantitative computed tomography with sufficient sensitivity and precision to measure small specimens. Among these inbred strains, differences in bone density
Address for correspondence and reprints: Dr. W. G. Beamer, The Jackson
Laboratory, 600 Main St., Bar Harbor, ME 04609.
© 1996 by Elsevier Science Inc. All rights reserved.
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W.G. Bearner et al. Genetic variability in bone density of mice
were identified that could be distributed into subsets, indicative of underlying differences in genetic regulation. Materials and Methods
Mice The inbred strain mice used in these studies were raised and maintained at the Jackson Laboratory. Females from nine strains (AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, and 129/J) were obtained from the Laboratory's Animal Resources production colonies, while strains SWR/ BmJ and SJL/BmJ were obtained from our own research colonies (WGB). Mice were maintained in groups of 3 - 4 in polycarbonate boxes (130 cm 2) on bedding of sterilized White Pine shavings, under conditions of 14 h light: 10 h darkness and ambient temperature of 20 ___2°C. Water (acidified with HC1, pH 2.8-3.2) and autoclaved pelleted diet (Teklad Sterilizable Rodent Diet No. 8656; 24% protein, 4% fat, 1.27% Ca, 0.92% P, trace mineral and vitamin fortified; Madison, WI) were available ad libitum. Mice providing data at ages of 2, 4, and 8 months were obtained at 3-4 weeks of age and maintained on the Teklad diet until necropsy. Mice providing 12 month data were obtained from Animal Resources as retired breeders at 5 - 7 months of age and placed on the Teklad diet until until necropsy at 12 months of age. At the specified ages, groups of 5-10 mice were euthanized by CO 2 and body weights recorded. A partial carcass preparation was derived from each mouse (lumbar spine, pelvis, plus rear limbs) and preserved in 95% ethanol. Subsequently, the left rear limb was carefully dissected free of the iliac acetabulum to preserve the head of the femur. Then, isolated femurs were prepared by dissecting away the tibia, along with remaining muscles and ligaments, and lengths measured by vernier calipers. Hind paws were separated distal to the tibia and stored with isolated femurs and lumbar spines for later measurement of physical dimensions and bone compartmental densities.
Bone Densitometry Isolated femurs were assessed by peripheral quantitative computed tomography (pQCT) with a Stratec XCT 960M (Norland Medical Systems, Ft. Atkinson, WI) specifically modified for use on small bone specimens to measure bone mineral and volume. Routine calibration is performed daily with a defined standard containing hydroxyapatite crystals embedded in lucite. A manufacturer supplied software program, designated "XMICE vl.3," analyzes the attenuation data to generate values for bone parameters including mineral content, volume, density, cortical thickness, periosteal, and endosteal circumferences. The X-ray attenuation data are based upon the XMICE software-defined minimum threshold of 500. Calculation of total bone mineral and volume includes all material with an attenuation of 500 or greater. The authors have empirically determined that a low density threshold of 1300 is necessary to differentiate mouse bone from water, adipose tissue, muscle, and tendon, whereas a high density threshold of 2000 is required to differentiate cortical bone from material of lower density, verified by measuring lamb cortical bone (1.335 g/cm3). The current version of XMICE includes low density material with attenuation values of 500 or greater in algorithms for calculation of trabecular parameters. Thus, the trabecular bone with attenuation values of 1300 to 2000 cannot yet be accurately discriminated from the protein, fat, and water with attenuation values from 500 to 1300. Therefore, in this
Bone Vol. 18, No. 5 May 1996:397-403 article, data are presented for total bone parameters defined by a threshold of 500 or more and on cortical bone parameters defined by a threshold of 2000 or more. In preparation for bone density studies with inbred mice, the XCT 960M was evaluated for precision of measurement. A single femur was replaced eight times in its holder and repetitive measurements taken at the diaphyseal midpoint identified in the XCT 960M scout view. (The scout view is a two dimensional representation of the specimen to be measured by pQCT that permits identification of landmarks and consistent selection of the appropriate site for measurement). The total femur density at the midpoint was 0.622 __ 0.007 _+ 0.002 mg/mm 3 (mean -+ SD _+ SEM), indicating a precision of 1.2% for measurements by the XCT 960M. The values for femur mineral content gathered from the XCT 960M densitometer are similar according to literature reports. Our values ranged from 15.3 to 27.4 mg of mineral per femur, whereas Tyan 45 reported 16.9 to 27.4 mg total mineral per femur gathered by ashing femurs from similarly aged mice. Finally, the resolution of detection is 0.05 ram, and is sufficiently sensitive to detect physiological differences between steroid deficient and normal littermate mice. 6 Isolated femurs from inbred strains were scanned at 2 mm intervals over their entire lengths, and the unit volume within which mineral was measured was set at 0.1 mm 3. The measurements for each bone were derived by summing the data from all scans for a given femur and then, in the case of femurs, multiplying by a factor of 2 in order to include bone not measured in alternate 1 mm scans. Total bone density values were calculated by dividing the total mineral content by the total volume. The coefficient of variation for total femur parameters among the 11 inbreds ranged from (a) 3.5% to 11.4% for density; (b) 8.8% to 15.5% for mineral; and (c) 5.0% to 14.9% for volume. Two additional bone sites in 12 month inbred strain mice were also assessed for density, the L 5 - L 6 vertebrae and the proximal phalanges of the middle three digits of the hind paws. The L 5 - L 6 vertebrae were found to be 4-5 mm in anterior/ posterior length and therefore were scanned at 1 mm intervals in a rostral direction, beginning with the iliac crest. Whichever of the two lumbar vertebrae was completely represented by 4-5 scans was utilized for data analyses. Phalangeal data were collected from tomographs of the middle three toes when the distal tip of the fourth digit was present as a landmark. The coefficient of variation among the four selected inbreds for vertebral density ranged from 5.6% to 11.4%, and for phalangeal density ranged from 6.7% to 16.2%.
Data Analyses Data in this article are presented as means _+ SEM. Statistical analyses were performed with StatView 4.5 software for Macintosh. All data were analyzed first by A N O V A to detect major effects among strains for each measurement. Individual strain means were tested for significant differences by Fisher's Protected LSD test. Differences were judged statistically significant when p < 0.05. Regression analyses were used to assess the relationship between femur parameters and between cortical density at different bone sites (p < 0.05). Results
Total Femoral Density in 12 Month Inbred Strain Females The authors chose to test for differences in bone density among 11 inbred strains at 12 months of age, based on literature reports
Bone Vol. 18, No. 5 May 1996:397-403
W.G. Beamer et al. Genetic variability in bone density of mice
that adult bone density had been established by that time (24, 25). The body weight and femur parameters--length, density, mineral content, and volume---obtained from females of these strains are shown in Table 1. Maximal and minimal means for each measurement are shown in bold print. One-way ANOVA of data for each of the variables revealed highly significant differences among inbred strains (p < 0.0001). Given that bone density is the focus of this report, the inbred strains in Table 1 have been rank ordered for total femoral density. When these data were inspected for possible relationships between femur density and other femur measurements, we found that femur density correlated with femur mineral (r = 0.678, p = 0.02), but not with any other measurement. Although a variety of statistical comparisons may be made among these 11 mouse strains, the authors chose to pursue further analyses of four inbred strains---C3H/HeJ, DBA/2J, BALB/ cByJ, and C57BL/6J--with the latter three strains compared with C3H/HeJ. These four strains are representative of high, middle, and low total femur densities. The greatest difference in femur density was found between C57BL/6J (0.45 mg/mm 3) and C3H/ HeJ (0.69 mg/mm 3) females, with the C3H/HeJ femur density nearly 50% greater. DBA/2J (0.57 mg/mm 3) and BALB/cByJ (0.55 mg/mm 3) also had densities significantly lower than C3H/ HeJ, although the relative differences (17% and 20%, respectively) were not as great as for C57BL/6J. None of the four strains differed in body weight, whereas both DBA/2J and BALB/cByJ had shorter femurs than those of the C57BL/6J females (p < 0.01).
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Figure 1. Mid-diaphyseal pQCT tomograms of femurs from 12-month-
old female mice: (a) C3H/HeJ; (b) DBA/21; (c) BALB/cByJ; and (d) C57BL/6J. High density bone is represented by white and blue-green color, while low density bone is represented by red/orange color. Cortical thickness is greatest for C3H/HeJ and least for C57BL/6J femurs. The DBA/2J and BALB/cByJ are strains with intermediate femur densities and also show intermediate cortical thicknesses.
Midfemoral Diaphyseal Tomograms From the Four Selected Inbred Strains
mean cortical thicknesses of these "slices" were 0.497, 0.379, 0.376, and 0.292 mm, respectively.
Figure 1 presents examples of the mid-diaphyseal scans for femurs from 12 month old females of the strains C3H/HeJ (a), DBA/2J (b), BALB/cByJ (c), and C57BL/6J (d). These scans were obtained simultaneously and demonstrate the volumetric images corresponding to the XCT 960M measurements of bone parameters. The pQCT images showed that the C3H/HeJ femur had a thicker cortex than the C57BL/6J femur, while the DBA/2J and BALB/cBy femurs were similar to each other and clearly intermediate to C3H/HeJ and C57BL/6J femurs. The total densities of the mid-diaphyseal slices for the four strains in the order listed above were: 0.84, 0.72, 0.65, and 0.42 mg/mm3; and the
Femoral Data for the Four Selected Inbred Strains The data for femoral cortical bone are presented in Table 2, with the total density repeated for comparison. Since overall ANOVA for each variable was highly significant across strains, statistical comparisons of mean values were made between C3H/HeJ and each of the other three strains. The cortical densities and mineral contents in C57BL/6J, DBA/2J, and BALB/cByJ femurs were significantly lower than in C3H/HeJ femurs. Cortical volumes for DBA/2J and C57BL/6J were significantly lower than that of C3H/HeJ, whereas BALB/cByJ was not different from C3H/HeJ.
Table 1. Body weight and femur data from 12-month-old female inbred strain mice. Maximal and minimal values for each measure are bolded, and strains are rank ordered by femur density. Mean _ SEM are presented for groups of 5-8 mice Femur Inbred strain (n = 6-13)
Body weight (g)
Length (mm)
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
C3H/HeJ NZB/BINJ 129/J SJL/BmJ SM/J SWR/BmJ DBA/2J AKR/J BALB/cByJ C57L/J C57BL/6J
27.8 __ 1.2 44.5 __2.5
0.69 _+ 0.02
26.6 _+0.6 28.2 -+ 2.1 27.6 -+0.3 31.3 _+1.6 34.4 _+2.2 26.3 _+0.7 27.9 _+ 1.0 30.6 _+0.9
16.37 _+0.20 17.12 _+0.28 15.95 _+0.13 15.80 _+0.07 14.53 _+0.22 16.23 _+0.07 15.99 + 0.13 17.25 __.0.11 17.15 _+0.10 16.23 _+0.12 17.17 _+0.11
0.67 __0.03 0.60 __0.02 0.60 _+0.02 0.58 -+ 0.02 0.58 +- 0.02 0.57 _+0.01 0.57 _+0.01 0.55 _+0.01 0.52 _+0.02 0.45 _+0.01
27.48 _+1.74 26.96 __2.30 22.59 _ 0.98 26.15 __0.94
39.71 _+ 1.69 39.92 __ 1.93 37.88 _+2.00 44.06 _+1.70
ANOVA
p < 0.0001
p < 0.0001
p < 0.0001
23.9 _+ 0.8
17.08 _+ 0.59
29.80 -+ 0.78
20.67 _+1.01 19.32 _+0.62 25.59 _+0.96 23.24 _+0.95 19.69 +__0.77 18.62 _+0.92
35.54 -+ 1.06 33.93 -+ 1.02 45.17 _-21.00 41.99 _ 1.06 38.08 _+0.66 41.33 _+1.46
p < 0.0001
p < 0.0001
400
W.G. Beamer et al. Genetic variability in bone density of mice
Bone Vol. 18, No. 5 May 1996:397-403
Table 2. Comparison of total densities along with cortical densities, volumes, and mineral contents for femurs from females of four inbred strains at 12 months of age. Mean -+ SEM are presented for groups of 5-8 mice Cortical Inbred strain
Total density (rag/ram 3)
C3H/HeJ DBA/2J BALB/cByJ C57BL/6J
0.69 0.58* 0.55* 0.45*
ANOVA
_+0.03 + 0.01 + 0.01 _+0.01
p < 0.001
Density (mg/mm 3)
Mineral (mg)
0.83 _+0.02 0.70* + 0.01 0.70* _+0.01 0.61 * _+0.01
26.53 18.02" 21.62" 16.57"
p < 0.001
Volume (mm 3)
_+ 1.98 _+0.61 _+ 1.08 _+0.92
31.88 -+ 1.97 25.53* _+0.83 30.74 _+ 1.16 27.01 * _+ 1.34
p < 0.0001
p < 0.0001
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test.
Femur Density and Length by Age for the Four Selected Inbred Strains To assure ourselves that data collected from 12 month old females represented adult bone density, we determined the time when adult peak bone density was achieved in these four inbred strains. Femurs from groups o f virgin females o f the four strains were measured at 2, 4, and 8 months o f age, and the resultant femur densities and lengths by age are presented in F i g u r e s 2a and b. For comparison purposes, data from the 12 month old retired breeders (Table 1), collected at least 5 months after the last litter, are included. This is justified by our findings that total femur density for 12 month old virgins versus retired breeders C3IMHeJ (virgin = 0.68 _+ 0.01 vs. RB = 0.69 _+ 0.01) and C57BL/6J (virgins = 0.48 + 0.01 vs. RB = 0.45 _+ 0.01) did not differ. Total femur density by age in Figure 2a revealed that there are strain differences as early as 2 months o f age, with C3H/HeJ possessing the highest density, DBA/2J and B A L B / c B y J intermediate densities, and all three o f these strains showing significantly higher densities than that o f C57BL/6J femurs. With the exception o f B A L B / c B y J strain, the bone density levels acquired in adulthood were consistently maintained through 12 months. The B A L B / c B y J femur density appeared to have peaked at 8 months and then declined by 12 months to the levels observed at 4 months o f age. In the femur length by age data o f Figure 2b, it is clear that for three o f the strains, femurs continued to lengthen through 12 months, while the length o f DBA/2J femurs appeared to plateau at 8 months.
this site measures approximately 0.2 m m in diameter and consists o f a high density bone core with a thin outer shell o f low density periosteal bone. The data are presented in T a b l e 4, with strains rank ordered by total density. Overall strain differences were found for all density, mineral, and volume measures (p < 0.004 to 0.0001). C o m p a r i s o n o f means for total density showed that C3H/HeJ had the highest absolute value, but only C57BL/6J with
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6
8
Age
10
12
14
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Vertebral Data from the Four Selected Inbred Strains
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The lower spine was scanned to determine if the relationship found for femur densities a m o n g the four inbred strains at 12 months o f age also held for the L 5 - 6 vertebrae. The X C T 960M was able to quantify total and cortical bone in the morphologically c o m p l e x vertebrae. These data are presented in T a b l e 3, with strains rank ordered by total density. No overall strain differences were found for total density, mineral or volume. However, overall strain differences were found for cortical density (p < 0.02), and cortical mineral (p < 0.04), but not for cortical volume. The cortical density and cortical mineral in C3H/HeJ vertebrae were significantly h i g h e r than both D B A / 2 J and C57BL/6J, but were not different from BALB/cByJ.
Phalangeal Data From the Four Selected Inbred Strains The proximal phalanges o f the hind paws from the 12 month old females represented the third skeletal site scanned. In the mouse,
17-
16" C3H/HeJ C57BL/6J BALB/cByJ DBA/2J
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Figure 2. Femoral bone (a) density and (b) length as a function of age from 2 through 12 months in groups of 6-10 C3H/HeJ, DBA/2J, BALB/ cByJ, and C57BL/6J mice. Data are presented in terms of mean _+ SEM. The C57BL/6J lemur density was significantly lower than the other strains at all ages examined. Acquisition of adult peak bone density in all four strains was achieved by 4 months of age, while femur length continued to increase through 12 months in 3 of 4 strains•
Bone Vol. 18, No. 5 May 1996:397~-03
W.G. Beamer et al. Genetic variability in bone density of mice
401
Table 3. Bone parameters for lumbar vertebrae L5-6 from females of 4 inbred strains at 12 months of age. Mean +_SEM are presented for groups of 5-8 mice Cortical Inbred strain
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
Density (rag/ram 3)
Mineral (rag)
Volume (mm3)
C3H/HeJ BALB/cByJ C57BL/6J DBA/2J
0.45 -+0.07 0.39 + 0.01 0.38 -+0.01 0.38 -+0.02
4.56 + 0.36 3.88 _+0.29 2.99 _ 0.34 3.08 - 0.86
11.86 -+ 1.06 10.02 -+0.86 8.09 +- 1.09 8.62 -+2.83
0.51 -+0.01 0.48* _+0.01 0.46* _+0.01 0.45* -+0.01
4.20 -+0.30 3.50 -+0.24 2.72* _+0.27 2.47* + 0.65
8.26 _+0.52 7.21 -+0.49 5.83* + 0.51 5.45* - 1.37
ns
ns
ns
p < 0.0001
p < 0.02
p < 0.05
ANOVA
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test. the lowest value was significantly different from C3H/HeJ phalanges. On the other hand, total mineral and volume in BALB/ cBy, DBA/2J, and C57BL/6J were all significantly lower than comparable values in C3H/HeJ phalanges. Comparison of mean values for cortical density again showed the C3H/HeJ phalanges had the highest absolute value, but only the lowest value found in C57BL/6J was significantly different from the C3H/HeJ. Cortical mineral and volume in C3H/HeJ phalanges were significantly higher than BALB/cBy, DBA/2J, and C57BL/6J, with the latter having the lowest values observed for phalanges.
Correlation of Cortical Bone Density Among Bone Sites The high density cortical bone data obtained from each skeletal site for the four selected inbred strains at 12 months of age were tested for possible correlation, as shown in Table 5, When the four strains were taken together, each of the skeletal sites proved to be significantly correlated with each other, with the strongest correlation (r = 0.704) found between femurs and vertebrae. These correlations suggest that for mice, at least some of the genes underlying the strain differences in cortical bone density are acting on the skeleton as a whole and not simply on a specific bone. Discussion We have found that pQCT technology, via the XCT 960M, can be applied with precision to small skeletal bones such as those of the mouse. In this article, femurs, vertebrae, and phalanges proved well within practical limits of this equipment. Measurement of total and cortical mineral and volumes associated with these skeletal sites formed the bases for calculation of bone densities. Furthermore, developmental data confirmed that acquisi-
tion of peak adult femoral density was separable from long bone growth, thus defining these as separable properties of bone. The issue of how well pQCT compares with other methods of assessing bone density has been addressed by Rosen et al. 29'3° Rosen et al. found that in femurs and tibias of orchiectomized male or GHIIGF-I treated female rats, (a) histomorphometry (BV/TV%), (b) areal bone density of trabecular-rich bone regions by dual energy X-ray absorptiometry (DXA; BMC/area), and (c) trabecular bone density by pQCT (BMC/vol) each detected treatment differences. However, histomorphometry and pQCT data correlated best with each other and showed the largest treatment effects as compared with DXA-based data. Other comparisons of data gathered via pQCT, DXA, and SPA methodologies based on X-ray absorptiometry also have found excellent correlation of treatment effects on tibias from aged and ovariectomized rats. s'3 ~'42 The data presented in this article clearly show that phenotypically normal inbred strains of mice harbor remarkable differences in bone density, and in its components--mineral and volume. These differences exist during the prime reproductive period for these stains and are not attributable to senescent changes. The variability for density and mineral measurements was significantly correlated, whereas length and volume were not correlated with density. Since these genetically distinct strains of mice were raised in a controlled environment (diet, living space, light exposure, ambient temperature range, epizooites), the differences observed in bone parameters are primarily the result of genetic variation. We found that femur volume correlated with length and mineral, whereas mineral and length showed no correlation. These analyses suggest that length and volume are likely to share some genetic regulation, and that mineral and length are likely to have independent genetic regulation. Evidence for genetic variation in phenotypically normal bone
Table 4. Bone parameters for proximal phalanges from females of 4 inbred strains at 12 months of age. Mean _+SEM are presented for groups of 5-8 mice Cortical Inbred strain C3H/HeJ BALB/cByJ DBA/2J C57BL/6J ANOVA
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
Density (mg/mm 3)
Mineral (rag)
Volume (mm3)
0.51 _ 0.01 0.48 +_0.01 0.46 _+0.02 0.43* _+0.02
0.75 -+0.04 0.61" -+ 0.01 0.59* +- 0.02 0.49* -+0.02
1.47 + 0.09 1.27" -+0.04 1.28* - 0.02 1.14" _+0.04
0.66 _ 0.02 0.63 _+0.01 0.62 _ 0.02 0.55* _+0.02
0.62 +-0.03 0.48* -+0.01 0.45* -+0.03 0.36* -+0.03
0.94 - 0.06 0.77* -+0.01 0.72* _+0.03 0.65* -+0.03
p < 0.006
p < 0.0001
p < 0.004
p < 0.0003
p < 0.0001
p < 0.0002
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test.
402
W.G. Beamer et al. Genetic variability in bone density of mice
Table 5. Correlations of cortical bone density among skeletal sites in 12 month old females from the inbred strains C3H/HeJ, DBA/2J, BALB/cByJ, and C57BL/6J
Skeletal sites compared
Correlation of densities
Femurs vs. phalanges Femurs vs. vertebrae Vertebrae vs. phalanges
r = 0.593, p = 0.004 r = 0.704, p = 0.0003 r = 0.475, p = 0.025
density is readily available in the scientific literature. For example, among humans, in addition to twins and mother-daughtergranddaughter studies noted in the Introduction, racial group differences between blacks, whites and Asians are well known. Recent illustrative reports include that of Patel et a l Y showing that enhanced radial bone density of blacks vs. whites develops during pubertal maturation, as well as that of Sugimoto et al. TM documenting subtle differences in bone mineral content between adult Japanese, Koreans, and Taiwanese people. In the mouse, population-based data on bone size and mineral content have been reported for some genetically homogeneous inbred strains. Perkins et al. 2v compared 6 and 24 month old C57BL/6N and with BALB/cN male mice and found significant differences in femoral cortical bone thicknesses. Murray et al. 24 reported that C57BL/6J mice had lower vertebral mass, % ash, and lacked coupling of resorption and formation when compared with SENCAR strain mice. Matsushita et al. 2j reported differences in peak adult femoral cortical thickness indices in several A K R / J - r e l a t e d s u b l i n e s of mice, d e s i g n a t e d s e n e s c e n c e accelerated mice (SAM). Low bone density in one of the SAM models, SAM-P/6, has been suggested to result from a small number of genes. 44 Mapping data have not yet been reported. Kaye and Kusy ~5 have recently reported differences in tibial ash for young adult mice of five strains including three strains in this article--BALB/cByJ, C57BL/6J, and DBA/2J. Most provocative were their findings that increased quadriceps muscle mass and home cage activity levels of C57BL/6J mice compared with A/J mice correlated with differences in tibial ash, indicating a possible biomechanical aspect underlying the strain differences in bone mineral. Our article expands the knowledge base on bone for these widely used inbred strains and points to the cortical bone as a target for strong genetic effects on density of phenotypically normal bones. In contrast to the modest data on bone density among strains of mice, there is a large array of reports documenting specific gene effects on mouse bone morphogenesis. For example, short ear (se/se) mice, a mutation in the Bmp-5 gene, j7 have reduced bone turnover without a decrease in bone density when compared with normal littermates. 3 Bailey used bilineal congenic inbred strains derived from C57BL/6By and BALB/cBy to show that a large number of genes governed mandibular shape. 2 In fact, more than 140 spontaneous mouse mutations affecting bone, particularly bone morphology, have been summarized by Green. m Furthermore, an expanding list of induced mutations in m i c e - transgenes and genes disrupted by homologous recombination-affect the skeleton. Examples include mice carrying the human transgene COLI A I that causes fetal rib and long bone fractures 26 or lck-IL-4 that causes reduced bone formation by osteoblasts, j9 and mice with null alleles at specific loci, such as c-fos that causes osteopetrosis through reduced osteoclast differentiation, j j IGF-II that disrupts fetal and neonatal skeletal growth in addition to other tissues, 5 or c-src that also causes osteoclast dysfunction and osteopetrosis. 37 These models underscore the complexity of genetic loci that participate in the development of the skeleton from the time of earliest embryonic differentiation to adulthood.
Bone Vol. 18, No. 5 May 1996:397-403 Each of these classes of genetic information has different uses. Mutations--spontaneous or induced--typically cause recognizable bone pathology as a consequence of their actions and, thus, reveal the existence of genes important to development of specific bones or bone components. Such genetic events offer salient opportunities to identify relevant cell types and investigate cellular mechanisms critical to specific facets of bone biology. On the other hand, differences in bone density among inbred strains also demonstrate genetic regulation, but in this instance the consequence of gene action yields normal variation in the adult bone phenotype. Such strain differences in bone density should be clearly amenable to modern genetic analyses for mapping of loci critical to acquiring adult bone density. Identification of such genes and the particulars of their biological functions will provide a more rationale basis for therapies intended to improve on the quality of existing bone by maximizing peak bone density.
Acknowledgments: The authors thank A. Kirley and K. Kovalsky, participants in the Jackson Laboratory's Summer College Student Training Program, and K. Chapman for assistance with bone measurements. The authors are additionally indebted to Dr. D. Bailey, Dr. T. CunliffeBeamer, and Dr. D. Serreze for review of this manuscript. This work was supported by NIH RR-09174 (WGB, LRD, CJR), CA-34196 (WGB), AG-10942 (CJR), and by Veterans Administration Merit Review Grant (DJB). The Jackson Laboratory is fully accredited by the American Association for Laboratory Animal Care.
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14. Hustmyer, F. G., Peacock, M., Hui, S., Johnston, C. C., and Christian, ,i. Bone mineral density in relation to polymorphism at the vitamin D receptor gene locus. J Clin Invest 94:2130-2134; 1994. 15. Kaye, M. and Kusy, R. P. Genetic lineage, bone mass, and physical activity in mice. Bone 17:131-135; 1995. 16. Kelly, P. J., Nguyen, T., Hooper, J., Pocock, N., Sambrook, P., and Eisman, ,i. Changes in axial bone density with age. J Bone Miner Res 8:11-17; 1993. 17. King, J. A., Marker, P. C., Seung, K. ,i., and Kingsley, D. M. BMP5 and the molecular, skeletal, and soft-tissue alterations in short ear mice. Dev Biol 166:112-122; 1994. 18. Kroger, H., Kotaniemi, E., Kroger, L., and Alhava, E. Development of bone mass and bone density of the spine and femoral neck: A prospective study of 65 children and adolescents. Bone Miner 23:171-182; 1993. 19. Lewis, D. B., Liggitt, H. D., Effmann, E. L., Motley, S. T., Teitelbaum, S. L., ,iepsen, K. ,i., Goldstein, S. A., Bonadio, J., Carpenter, J., and Perlmutter, R. M. Osteoporosis induced in mice by overproduction of interleukin 4. Proc Natl Acad Sci USA 90:11618-11622; 1993. 20. Looney, J. E., Yoon, H. K., Fischer, M., Farley, S. M., Farley, ,i. R., Wergedal, `i. E., and Baylink, D. Lack of a high prevalence of the BB Vitamin D receptor genotype in severely osteoporotic women. ,i Clin Endocrinol Metab. In press. 21. Matsushita, M., Tsuboyama, T., Kasai, R., Higucbi, K., Higuchi, K., Kohno, A., Yonezu, T., Utani, A., Umezawa, M., and Takeda, T. Age-related changes in bone mass in the senescence-accelerated mouse (SAM). Am J Pathol 125:276-283; 1986. 22. McKay, H. A., Bailey, D. A., Wilkinson, A. A., and Houston, C. S. Familial comparison of bone mineral density at the proximal femur and lumbar spine. Bone Miner 24:95-107; 1994. 23. Morrison, N. A., Qi, J. C., Tokita, A., Kelly, P. J., Crofts, L., Nguyen, T. V., Sambrook, P. N., and Eisman, J. A. Prediction of bone density from vitamin D receptor alleles. Nature 367:284-287; 1994. 24. Murray, E. ,i. B., Song, M. K., Laird, E. C., and Murray, S. Strain-dependent differences in vertebral bone mass, serum osteocalcin, and calcitonin in calcium-replete and -deficient mice. Proc Soc Exp Biol Med 203:64-73; 1993. 25. Patel, D. N., Pettifor, J. M., Becker, P. J., Grieve, C., and Leschner, K. The effect of ethnic group on appendicular bone mass in children. J Bone Miner Res 7:263-272; 1992. 26. Pereira, R., Khillan, J. S., Helminen, H.J., Hume, E. L., and Prockop, D.J. Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral. J Clin Invest 91:709-716; 1993. 27. Perkins, S. L., Gibbons, R., Kling, S., and Kahn, A. J. Age-related bone loss in mice is associated with an increased osteoclast progenitor pool. Bone 15:6572; 1994. 28. Pocock, N. A., Eisman, J. A., Hopper, J. L., Yeates, M. G., Sambrook, P. N., and Eberl, S. Genetic determinants of bone mass in adults: A twin study. ,i Clin Invest 80:706-710; 1987. 29. Rosen, H. N., Cben, V., Citadini, A., Greenspan, S. L., Douglas, P. S., Moses, A. C., and Beamer, W. G. Treatment with growth hormone and IGF-1 in growing rats increases bone mineral content but not bone mineral density. J Bone Miner Res 10:1352-1358; 1995. 30. Rosen, H. N., Tollin, S., Balena, R., Middlebrooks, V. L., Beamer, W., Donahue, L. R., Rosen, C. ,i., Turner, A., Holick, M., and Greenspan, S. Differentiating between orchiectomized rats and controls using measurements of trabecular bone density: A comparison among DXA, histomorphometry, and peripheral quantitative computerized tomography. Calcif Tissue lnt 57:35-39; 1995.
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31. Sato, M., Kim, J., and Bryant, H. Comparative X-ray densitometry of bones from ovariectomized rats. Bone 17(Suppl):157S-162S; 1995. 32. Seeman, E., Hopper, J. L., Bach, L. A., Cooper, M. E., Parkinson, E., McKay, J., and Jerums, G. Reduced bone mass in daughters of women with osteoporosis. N Engl J Med 320:554-558; 1989. 33. Shiraki, M., Eguchi, H., Aoki, C., and Shiraki, Y. Can allelic variations in vitamin D receptor gene predict bone densities and serum osteocalcin level in Japanese women? Bone 16(Suppl. 1):84S; 1995 (abstract 18). 34. Silberberg, M. and Silberberg, R. Age changes of bones and joints in various strains of mice. Am J Anat 68:69-95; 1941. 35. Slemenda, C. W., Christian, J. C., Williams, C. ,i., Norton, ,i. A., and Johnston, C. C. J. Genetic determinants of bone mass in adult women: A reevaluation of the twin model and the potential importance of gene interaction on heritability estimates. J Bone Miner Res 6:561-567; 1991. 36. Smith, D. M., Nance, W. E., Kang, K. W., Christian, ,i. C., and Johnston, C. C. Genetic factors in determining bone mass. ,i Clin Invest 52:2800-2808; 1973. 37. Soriano, P., Montgomery, C., Geske, R., and Bradley, A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693-702; 1991. 38. Sugimoto, T., Tsutsumi, M., Fujii, Y., Kawakatsu, M., Negishi, H., Lee, M. C., Tsai, K. S., Fukase, M., and Fujita, T. Comparison of bone mineral content among Japanese, Koreans, and Taiwanese assessed by dual-photon absorptiometry. J. Bone Miner Res 7:153-159; 1992. 39. Takeshita, T., Yamagata, Z., Iijima, S., Nakamura, T., Ouchi, Y., Orimo, H., and Asaka, A. Genetic and environmental factors of bone mineral density indicated in Japanese twins. Gerontology. 38(Suppl. 1):43-49; 1992. 40. Teegarden, D., Proulx, W. R., Martin, B. R., Zhao, J., McCabe, G. P., Lyle, R. M., Peacock, M., Slemenda, C., Johnston, C. C., and Weaver, C. M. Peak bone mass in young women. J Bone Miner Res 10:711-715; 1995. 41. Tokita, A., Watanabe, T., Miura, Y., Yabuta, K., Mathumoto, H., Yamamori, S., Morrison, N. A., and Eisman, J. A. Vitamin D receptor RFLP and bone mineral density in japanese. Bone 16(Suppl. 1):83S; 1995 (abstract 17). 42. Tom, H., Tressler, D. L., and McOsker, J. E. Validation of the Norland pQCT bone densitometer for bone mineral density measurement of rat tibiae., Int Conf. Animal Models in The Prevention and Treatment of Osteopenia. Cairns, Australia. Bone 17 (Suppl):45; 1995. (abstract 13). 43. Theintz, G., Buchs, B., Rizzoli, R., Slosman, D., Clavien, H., Sizonenki, P. C., and Bonjour, J.P. Longitudinal monitoring of bone mass accumulation in healthy adolescents: Evidence for a marked reductio after 16 years of age at the levels of lumbar spine and femoral neck in female subjects. J Clin Endocrinol Metab 75:1060-1065; 1992. 44. Tsuboyama, T., Takahashi, K., Yamamuro, T., Hosokawa, M., and Takeda, T. Cross-mating study on bone mass in the spontaneously osteoporotic mouse (SAM-P/6). Bone Miner 23:57-64; 1993. 45. Tyan, M. L. Femur mass: Modulation by marrow cells from young and old donors. Proc Soc Exp Biol Med 164:89-92; 1980.
Date Received: October 17, 1995 Date Revised: January 30, 1996 Date Accepted: January 31, 1996
ELSEVIER
ORIGINAL ARTICLES
Genetic Variability in Adult Bone Density Among Inbred Strains of Mice W. G. B E A M E R , j L. R. D O N A H U E , I C. J. R O S E N , 2 and D. J. B A Y L I N K 3 1 The Jackson Laborator T, Bar Harbor, ME, USA 2 Maine Centerfiw Osteoporosis Research and Education, Bangor, ME, USA 3 Loma Linda UniversiO' and Jer O" L. Pettis Memorial VA Medical Center, Loma Linda, CA, USA
Key Words: Genetics; Mouse; Bone density; Inbred strains; Development.
More than 70% of the variability in human bone density has been attributed to genetic factors as a result of studies with twins, osteoporotic families, and individuals with rare heritable bone disorders. We have applied the Stratec XCT 960M pQCT, specifically modified for small skeletal specimens, to analyses of bones from 11 inbred strains (AKR/J, BALB/ cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, SJL/BmJ, SWR/BmJ, and 129/.1) of female mice to determine the extent of heritable differences in peak bone density, pQCT scans were taken of femurs from (a) 12month-old inbred strain females and (b) a subset of four strains (C3H/HeJ, DBA/2J, BALB/cByJ, C57BL/6J) at 2, 4, and 8 months. In addition, pQCT scans were also obtained from L5-L6 vertebrae and proximal phalanges from the same subset of four inbred strains at 12 months of age. Comparison of bone parameters among inbred strains revealed significant differences at each of the three sites investigated. Femoral and phalangeal bones differed among strains with respect to total and cortical density, mineral, and volume. Only cortical bone parameters were significantly different among strains at the vertebral site. With respect to strain differences, the highest value for any given bone parameter was found in the C3H/HeJ strain, whereas C57BL/6J values were absolutely, or statistically, the lowest. Similarly, with respect to bone sites, cortical bone density was significantly correlated among strains. On the other hand, we found that none of the femur, vertebral, or phalangeal parameters correlated with body weight, even though body weight varied by 86% among these inbred strains. The developmental studies of femurs conducted at 2, 4, and 8 months of age with C3H/ He J, DBA/2J, BALB/cByJ, and C57BL/6J females showed differences in total density among strains at 2 months and thereafter. Adult peak bone density was typically achieved by 4 months, whereas femurs continued to lengthen for 4 to 8 months thereafter. We conclude that (1) major genetic effects on femoral, vertebral, and phalangeal bone density are detectable among inbred strains of mice; (2) cortical bone density shares common genetic regulation at the three measured sites; and (3) within the femur, genes that regulate length and density are different. (Bone 18:397-403; 1996)
Introduction Peak bone density is considered one of the strongest determinants of subsequent osteoporotic fractures in both men and women. In the adult human skeleton, peak bone density is achieved at the end of the rapid pubertal growth period at approximately 18-22 years of age 9"j8'4°~43 and is under strong genetic regulation. Thus, studies of t w i n s 4'14'16"28"35~36'39 and mother-daughter-grandaughter sets ~3.22,32 have estimated that up to 70% of the variability in bone density is genetically based. Studies are beginning to identify the genes that may be responsible for acquisition of adult peak bone density. Morrison et al. have reported that the vitamin D receptor (VDR) alleles can be associated with (and may be responsible for) genetic variations in bone density. 23 These findings have been reproduced in some patient populations (Japanese, 41 Austrian 12) but not in others ( U S A , 7'14 Japanese, 33 Whites2°). These conflicting results are the likely consequences of genetic heterogeneity of the human population coupled with important environmental differences, especially in the aged. Thus, comparison within twin pairs or among siblings is the only reasonable method of assessing genetic regulation while holding environmental factors reasonably constant. This approach requires large numbers of both dizygotic and monozygotic twin pairs, which are infrequent in the population. Moreover, the genetic variability between pairs of dizygotic twins is as large as in the general population. Animal models are critical for experimentally defining the genetic regulation of bone density. In particular, inbred mice offer unlimited numbers of genetically identical "twins" whose environments can be strictly controlled. These inbred strains offer the opportunity to gain insight into the genetic basis of variation in bone density in phenotypically normal mice. Equally important, each inbred strain is genetically different from every other inbred strain, making possible planned matings to study segregation of genes essential to bone density. In this article are presented bone density data on axial and appendicular sites for adult female mice from I 1 different inbred strains. These strains were selected for analyses because they are progenitors for sets of recombinant inbred strains specifically developed for analyses of heritable traits. ~ The methodology adopted is that of peripheral quantitative computed tomography with sufficient sensitivity and precision to measure small specimens. Among these inbred strains, differences in bone density
Address for correspondence and reprints: Dr. W. G. Beamer, The Jackson
Laboratory, 600 Main St., Bar Harbor, ME 04609.
© 1996 by Elsevier Science Inc. All rights reserved.
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were identified that could be distributed into subsets, indicative of underlying differences in genetic regulation. Materials and Methods
Mice The inbred strain mice used in these studies were raised and maintained at the Jackson Laboratory. Females from nine strains (AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, NZB/B1NJ, SM/J, and 129/J) were obtained from the Laboratory's Animal Resources production colonies, while strains SWR/ BmJ and SJL/BmJ were obtained from our own research colonies (WGB). Mice were maintained in groups of 3 - 4 in polycarbonate boxes (130 cm 2) on bedding of sterilized White Pine shavings, under conditions of 14 h light: 10 h darkness and ambient temperature of 20 ___2°C. Water (acidified with HC1, pH 2.8-3.2) and autoclaved pelleted diet (Teklad Sterilizable Rodent Diet No. 8656; 24% protein, 4% fat, 1.27% Ca, 0.92% P, trace mineral and vitamin fortified; Madison, WI) were available ad libitum. Mice providing data at ages of 2, 4, and 8 months were obtained at 3-4 weeks of age and maintained on the Teklad diet until necropsy. Mice providing 12 month data were obtained from Animal Resources as retired breeders at 5 - 7 months of age and placed on the Teklad diet until until necropsy at 12 months of age. At the specified ages, groups of 5-10 mice were euthanized by CO 2 and body weights recorded. A partial carcass preparation was derived from each mouse (lumbar spine, pelvis, plus rear limbs) and preserved in 95% ethanol. Subsequently, the left rear limb was carefully dissected free of the iliac acetabulum to preserve the head of the femur. Then, isolated femurs were prepared by dissecting away the tibia, along with remaining muscles and ligaments, and lengths measured by vernier calipers. Hind paws were separated distal to the tibia and stored with isolated femurs and lumbar spines for later measurement of physical dimensions and bone compartmental densities.
Bone Densitometry Isolated femurs were assessed by peripheral quantitative computed tomography (pQCT) with a Stratec XCT 960M (Norland Medical Systems, Ft. Atkinson, WI) specifically modified for use on small bone specimens to measure bone mineral and volume. Routine calibration is performed daily with a defined standard containing hydroxyapatite crystals embedded in lucite. A manufacturer supplied software program, designated "XMICE vl.3," analyzes the attenuation data to generate values for bone parameters including mineral content, volume, density, cortical thickness, periosteal, and endosteal circumferences. The X-ray attenuation data are based upon the XMICE software-defined minimum threshold of 500. Calculation of total bone mineral and volume includes all material with an attenuation of 500 or greater. The authors have empirically determined that a low density threshold of 1300 is necessary to differentiate mouse bone from water, adipose tissue, muscle, and tendon, whereas a high density threshold of 2000 is required to differentiate cortical bone from material of lower density, verified by measuring lamb cortical bone (1.335 g/cm3). The current version of XMICE includes low density material with attenuation values of 500 or greater in algorithms for calculation of trabecular parameters. Thus, the trabecular bone with attenuation values of 1300 to 2000 cannot yet be accurately discriminated from the protein, fat, and water with attenuation values from 500 to 1300. Therefore, in this
Bone Vol. 18, No. 5 May 1996:397-403 article, data are presented for total bone parameters defined by a threshold of 500 or more and on cortical bone parameters defined by a threshold of 2000 or more. In preparation for bone density studies with inbred mice, the XCT 960M was evaluated for precision of measurement. A single femur was replaced eight times in its holder and repetitive measurements taken at the diaphyseal midpoint identified in the XCT 960M scout view. (The scout view is a two dimensional representation of the specimen to be measured by pQCT that permits identification of landmarks and consistent selection of the appropriate site for measurement). The total femur density at the midpoint was 0.622 __ 0.007 _+ 0.002 mg/mm 3 (mean -+ SD _+ SEM), indicating a precision of 1.2% for measurements by the XCT 960M. The values for femur mineral content gathered from the XCT 960M densitometer are similar according to literature reports. Our values ranged from 15.3 to 27.4 mg of mineral per femur, whereas Tyan 45 reported 16.9 to 27.4 mg total mineral per femur gathered by ashing femurs from similarly aged mice. Finally, the resolution of detection is 0.05 ram, and is sufficiently sensitive to detect physiological differences between steroid deficient and normal littermate mice. 6 Isolated femurs from inbred strains were scanned at 2 mm intervals over their entire lengths, and the unit volume within which mineral was measured was set at 0.1 mm 3. The measurements for each bone were derived by summing the data from all scans for a given femur and then, in the case of femurs, multiplying by a factor of 2 in order to include bone not measured in alternate 1 mm scans. Total bone density values were calculated by dividing the total mineral content by the total volume. The coefficient of variation for total femur parameters among the 11 inbreds ranged from (a) 3.5% to 11.4% for density; (b) 8.8% to 15.5% for mineral; and (c) 5.0% to 14.9% for volume. Two additional bone sites in 12 month inbred strain mice were also assessed for density, the L 5 - L 6 vertebrae and the proximal phalanges of the middle three digits of the hind paws. The L 5 - L 6 vertebrae were found to be 4-5 mm in anterior/ posterior length and therefore were scanned at 1 mm intervals in a rostral direction, beginning with the iliac crest. Whichever of the two lumbar vertebrae was completely represented by 4-5 scans was utilized for data analyses. Phalangeal data were collected from tomographs of the middle three toes when the distal tip of the fourth digit was present as a landmark. The coefficient of variation among the four selected inbreds for vertebral density ranged from 5.6% to 11.4%, and for phalangeal density ranged from 6.7% to 16.2%.
Data Analyses Data in this article are presented as means _+ SEM. Statistical analyses were performed with StatView 4.5 software for Macintosh. All data were analyzed first by A N O V A to detect major effects among strains for each measurement. Individual strain means were tested for significant differences by Fisher's Protected LSD test. Differences were judged statistically significant when p < 0.05. Regression analyses were used to assess the relationship between femur parameters and between cortical density at different bone sites (p < 0.05). Results
Total Femoral Density in 12 Month Inbred Strain Females The authors chose to test for differences in bone density among 11 inbred strains at 12 months of age, based on literature reports
Bone Vol. 18, No. 5 May 1996:397-403
W.G. Beamer et al. Genetic variability in bone density of mice
that adult bone density had been established by that time (24, 25). The body weight and femur parameters--length, density, mineral content, and volume---obtained from females of these strains are shown in Table 1. Maximal and minimal means for each measurement are shown in bold print. One-way ANOVA of data for each of the variables revealed highly significant differences among inbred strains (p < 0.0001). Given that bone density is the focus of this report, the inbred strains in Table 1 have been rank ordered for total femoral density. When these data were inspected for possible relationships between femur density and other femur measurements, we found that femur density correlated with femur mineral (r = 0.678, p = 0.02), but not with any other measurement. Although a variety of statistical comparisons may be made among these 11 mouse strains, the authors chose to pursue further analyses of four inbred strains---C3H/HeJ, DBA/2J, BALB/ cByJ, and C57BL/6J--with the latter three strains compared with C3H/HeJ. These four strains are representative of high, middle, and low total femur densities. The greatest difference in femur density was found between C57BL/6J (0.45 mg/mm 3) and C3H/ HeJ (0.69 mg/mm 3) females, with the C3H/HeJ femur density nearly 50% greater. DBA/2J (0.57 mg/mm 3) and BALB/cByJ (0.55 mg/mm 3) also had densities significantly lower than C3H/ HeJ, although the relative differences (17% and 20%, respectively) were not as great as for C57BL/6J. None of the four strains differed in body weight, whereas both DBA/2J and BALB/cByJ had shorter femurs than those of the C57BL/6J females (p < 0.01).
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ct
Figure 1. Mid-diaphyseal pQCT tomograms of femurs from 12-month-
old female mice: (a) C3H/HeJ; (b) DBA/21; (c) BALB/cByJ; and (d) C57BL/6J. High density bone is represented by white and blue-green color, while low density bone is represented by red/orange color. Cortical thickness is greatest for C3H/HeJ and least for C57BL/6J femurs. The DBA/2J and BALB/cByJ are strains with intermediate femur densities and also show intermediate cortical thicknesses.
Midfemoral Diaphyseal Tomograms From the Four Selected Inbred Strains
mean cortical thicknesses of these "slices" were 0.497, 0.379, 0.376, and 0.292 mm, respectively.
Figure 1 presents examples of the mid-diaphyseal scans for femurs from 12 month old females of the strains C3H/HeJ (a), DBA/2J (b), BALB/cByJ (c), and C57BL/6J (d). These scans were obtained simultaneously and demonstrate the volumetric images corresponding to the XCT 960M measurements of bone parameters. The pQCT images showed that the C3H/HeJ femur had a thicker cortex than the C57BL/6J femur, while the DBA/2J and BALB/cBy femurs were similar to each other and clearly intermediate to C3H/HeJ and C57BL/6J femurs. The total densities of the mid-diaphyseal slices for the four strains in the order listed above were: 0.84, 0.72, 0.65, and 0.42 mg/mm3; and the
Femoral Data for the Four Selected Inbred Strains The data for femoral cortical bone are presented in Table 2, with the total density repeated for comparison. Since overall ANOVA for each variable was highly significant across strains, statistical comparisons of mean values were made between C3H/HeJ and each of the other three strains. The cortical densities and mineral contents in C57BL/6J, DBA/2J, and BALB/cByJ femurs were significantly lower than in C3H/HeJ femurs. Cortical volumes for DBA/2J and C57BL/6J were significantly lower than that of C3H/HeJ, whereas BALB/cByJ was not different from C3H/HeJ.
Table 1. Body weight and femur data from 12-month-old female inbred strain mice. Maximal and minimal values for each measure are bolded, and strains are rank ordered by femur density. Mean _ SEM are presented for groups of 5-8 mice Femur Inbred strain (n = 6-13)
Body weight (g)
Length (mm)
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
C3H/HeJ NZB/BINJ 129/J SJL/BmJ SM/J SWR/BmJ DBA/2J AKR/J BALB/cByJ C57L/J C57BL/6J
27.8 __ 1.2 44.5 __2.5
0.69 _+ 0.02
26.6 _+0.6 28.2 -+ 2.1 27.6 -+0.3 31.3 _+1.6 34.4 _+2.2 26.3 _+0.7 27.9 _+ 1.0 30.6 _+0.9
16.37 _+0.20 17.12 _+0.28 15.95 _+0.13 15.80 _+0.07 14.53 _+0.22 16.23 _+0.07 15.99 + 0.13 17.25 __.0.11 17.15 _+0.10 16.23 _+0.12 17.17 _+0.11
0.67 __0.03 0.60 __0.02 0.60 _+0.02 0.58 -+ 0.02 0.58 +- 0.02 0.57 _+0.01 0.57 _+0.01 0.55 _+0.01 0.52 _+0.02 0.45 _+0.01
27.48 _+1.74 26.96 __2.30 22.59 _ 0.98 26.15 __0.94
39.71 _+ 1.69 39.92 __ 1.93 37.88 _+2.00 44.06 _+1.70
ANOVA
p < 0.0001
p < 0.0001
p < 0.0001
23.9 _+ 0.8
17.08 _+ 0.59
29.80 -+ 0.78
20.67 _+1.01 19.32 _+0.62 25.59 _+0.96 23.24 _+0.95 19.69 +__0.77 18.62 _+0.92
35.54 -+ 1.06 33.93 -+ 1.02 45.17 _-21.00 41.99 _ 1.06 38.08 _+0.66 41.33 _+1.46
p < 0.0001
p < 0.0001
400
W.G. Beamer et al. Genetic variability in bone density of mice
Bone Vol. 18, No. 5 May 1996:397-403
Table 2. Comparison of total densities along with cortical densities, volumes, and mineral contents for femurs from females of four inbred strains at 12 months of age. Mean -+ SEM are presented for groups of 5-8 mice Cortical Inbred strain
Total density (rag/ram 3)
C3H/HeJ DBA/2J BALB/cByJ C57BL/6J
0.69 0.58* 0.55* 0.45*
ANOVA
_+0.03 + 0.01 + 0.01 _+0.01
p < 0.001
Density (mg/mm 3)
Mineral (mg)
0.83 _+0.02 0.70* + 0.01 0.70* _+0.01 0.61 * _+0.01
26.53 18.02" 21.62" 16.57"
p < 0.001
Volume (mm 3)
_+ 1.98 _+0.61 _+ 1.08 _+0.92
31.88 -+ 1.97 25.53* _+0.83 30.74 _+ 1.16 27.01 * _+ 1.34
p < 0.0001
p < 0.0001
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test.
Femur Density and Length by Age for the Four Selected Inbred Strains To assure ourselves that data collected from 12 month old females represented adult bone density, we determined the time when adult peak bone density was achieved in these four inbred strains. Femurs from groups o f virgin females o f the four strains were measured at 2, 4, and 8 months o f age, and the resultant femur densities and lengths by age are presented in F i g u r e s 2a and b. For comparison purposes, data from the 12 month old retired breeders (Table 1), collected at least 5 months after the last litter, are included. This is justified by our findings that total femur density for 12 month old virgins versus retired breeders C3IMHeJ (virgin = 0.68 _+ 0.01 vs. RB = 0.69 _+ 0.01) and C57BL/6J (virgins = 0.48 + 0.01 vs. RB = 0.45 _+ 0.01) did not differ. Total femur density by age in Figure 2a revealed that there are strain differences as early as 2 months o f age, with C3H/HeJ possessing the highest density, DBA/2J and B A L B / c B y J intermediate densities, and all three o f these strains showing significantly higher densities than that o f C57BL/6J femurs. With the exception o f B A L B / c B y J strain, the bone density levels acquired in adulthood were consistently maintained through 12 months. The B A L B / c B y J femur density appeared to have peaked at 8 months and then declined by 12 months to the levels observed at 4 months o f age. In the femur length by age data o f Figure 2b, it is clear that for three o f the strains, femurs continued to lengthen through 12 months, while the length o f DBA/2J femurs appeared to plateau at 8 months.
this site measures approximately 0.2 m m in diameter and consists o f a high density bone core with a thin outer shell o f low density periosteal bone. The data are presented in T a b l e 4, with strains rank ordered by total density. Overall strain differences were found for all density, mineral, and volume measures (p < 0.004 to 0.0001). C o m p a r i s o n o f means for total density showed that C3H/HeJ had the highest absolute value, but only C57BL/6J with
A
E E
0.8
O~
0.7
E
0.6 C
m
E Ll.
0.5 0.4 DBN2J C57BI../6J
,I,,
0.3 2
4
6
8
Age
10
12
14
(too)
18" A
Vertebral Data from the Four Selected Inbred Strains
E E •, - . ,
The lower spine was scanned to determine if the relationship found for femur densities a m o n g the four inbred strains at 12 months o f age also held for the L 5 - 6 vertebrae. The X C T 960M was able to quantify total and cortical bone in the morphologically c o m p l e x vertebrae. These data are presented in T a b l e 3, with strains rank ordered by total density. No overall strain differences were found for total density, mineral or volume. However, overall strain differences were found for cortical density (p < 0.02), and cortical mineral (p < 0.04), but not for cortical volume. The cortical density and cortical mineral in C3H/HeJ vertebrae were significantly h i g h e r than both D B A / 2 J and C57BL/6J, but were not different from BALB/cByJ.
Phalangeal Data From the Four Selected Inbred Strains The proximal phalanges o f the hind paws from the 12 month old females represented the third skeletal site scanned. In the mouse,
17-
16" C3H/HeJ C57BL/6J BALB/cByJ DBA/2J
E is. 1,1.
14
•
0
i
2
,
i
4
,
i
6 Age
,
i
-
i
8 10 (mo)
•
i
1")
,
,
14
Figure 2. Femoral bone (a) density and (b) length as a function of age from 2 through 12 months in groups of 6-10 C3H/HeJ, DBA/2J, BALB/ cByJ, and C57BL/6J mice. Data are presented in terms of mean _+ SEM. The C57BL/6J lemur density was significantly lower than the other strains at all ages examined. Acquisition of adult peak bone density in all four strains was achieved by 4 months of age, while femur length continued to increase through 12 months in 3 of 4 strains•
Bone Vol. 18, No. 5 May 1996:397~-03
W.G. Beamer et al. Genetic variability in bone density of mice
401
Table 3. Bone parameters for lumbar vertebrae L5-6 from females of 4 inbred strains at 12 months of age. Mean +_SEM are presented for groups of 5-8 mice Cortical Inbred strain
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
Density (rag/ram 3)
Mineral (rag)
Volume (mm3)
C3H/HeJ BALB/cByJ C57BL/6J DBA/2J
0.45 -+0.07 0.39 + 0.01 0.38 -+0.01 0.38 -+0.02
4.56 + 0.36 3.88 _+0.29 2.99 _ 0.34 3.08 - 0.86
11.86 -+ 1.06 10.02 -+0.86 8.09 +- 1.09 8.62 -+2.83
0.51 -+0.01 0.48* _+0.01 0.46* _+0.01 0.45* -+0.01
4.20 -+0.30 3.50 -+0.24 2.72* _+0.27 2.47* + 0.65
8.26 _+0.52 7.21 -+0.49 5.83* + 0.51 5.45* - 1.37
ns
ns
ns
p < 0.0001
p < 0.02
p < 0.05
ANOVA
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test. the lowest value was significantly different from C3H/HeJ phalanges. On the other hand, total mineral and volume in BALB/ cBy, DBA/2J, and C57BL/6J were all significantly lower than comparable values in C3H/HeJ phalanges. Comparison of mean values for cortical density again showed the C3H/HeJ phalanges had the highest absolute value, but only the lowest value found in C57BL/6J was significantly different from the C3H/HeJ. Cortical mineral and volume in C3H/HeJ phalanges were significantly higher than BALB/cBy, DBA/2J, and C57BL/6J, with the latter having the lowest values observed for phalanges.
Correlation of Cortical Bone Density Among Bone Sites The high density cortical bone data obtained from each skeletal site for the four selected inbred strains at 12 months of age were tested for possible correlation, as shown in Table 5, When the four strains were taken together, each of the skeletal sites proved to be significantly correlated with each other, with the strongest correlation (r = 0.704) found between femurs and vertebrae. These correlations suggest that for mice, at least some of the genes underlying the strain differences in cortical bone density are acting on the skeleton as a whole and not simply on a specific bone. Discussion We have found that pQCT technology, via the XCT 960M, can be applied with precision to small skeletal bones such as those of the mouse. In this article, femurs, vertebrae, and phalanges proved well within practical limits of this equipment. Measurement of total and cortical mineral and volumes associated with these skeletal sites formed the bases for calculation of bone densities. Furthermore, developmental data confirmed that acquisi-
tion of peak adult femoral density was separable from long bone growth, thus defining these as separable properties of bone. The issue of how well pQCT compares with other methods of assessing bone density has been addressed by Rosen et al. 29'3° Rosen et al. found that in femurs and tibias of orchiectomized male or GHIIGF-I treated female rats, (a) histomorphometry (BV/TV%), (b) areal bone density of trabecular-rich bone regions by dual energy X-ray absorptiometry (DXA; BMC/area), and (c) trabecular bone density by pQCT (BMC/vol) each detected treatment differences. However, histomorphometry and pQCT data correlated best with each other and showed the largest treatment effects as compared with DXA-based data. Other comparisons of data gathered via pQCT, DXA, and SPA methodologies based on X-ray absorptiometry also have found excellent correlation of treatment effects on tibias from aged and ovariectomized rats. s'3 ~'42 The data presented in this article clearly show that phenotypically normal inbred strains of mice harbor remarkable differences in bone density, and in its components--mineral and volume. These differences exist during the prime reproductive period for these stains and are not attributable to senescent changes. The variability for density and mineral measurements was significantly correlated, whereas length and volume were not correlated with density. Since these genetically distinct strains of mice were raised in a controlled environment (diet, living space, light exposure, ambient temperature range, epizooites), the differences observed in bone parameters are primarily the result of genetic variation. We found that femur volume correlated with length and mineral, whereas mineral and length showed no correlation. These analyses suggest that length and volume are likely to share some genetic regulation, and that mineral and length are likely to have independent genetic regulation. Evidence for genetic variation in phenotypically normal bone
Table 4. Bone parameters for proximal phalanges from females of 4 inbred strains at 12 months of age. Mean _+SEM are presented for groups of 5-8 mice Cortical Inbred strain C3H/HeJ BALB/cByJ DBA/2J C57BL/6J ANOVA
Density (mg/mm 3)
Mineral (mg)
Volume (mm3)
Density (mg/mm 3)
Mineral (rag)
Volume (mm3)
0.51 _ 0.01 0.48 +_0.01 0.46 _+0.02 0.43* _+0.02
0.75 -+0.04 0.61" -+ 0.01 0.59* +- 0.02 0.49* -+0.02
1.47 + 0.09 1.27" -+0.04 1.28* - 0.02 1.14" _+0.04
0.66 _ 0.02 0.63 _+0.01 0.62 _ 0.02 0.55* _+0.02
0.62 +-0.03 0.48* -+0.01 0.45* -+0.03 0.36* -+0.03
0.94 - 0.06 0.77* -+0.01 0.72* _+0.03 0.65* -+0.03
p < 0.006
p < 0.0001
p < 0.004
p < 0.0003
p < 0.0001
p < 0.0002
* indicate significant difference (p < 0.01) when compared with value for C3H/HeJ by Fisher's PLSD test.
402
W.G. Beamer et al. Genetic variability in bone density of mice
Table 5. Correlations of cortical bone density among skeletal sites in 12 month old females from the inbred strains C3H/HeJ, DBA/2J, BALB/cByJ, and C57BL/6J
Skeletal sites compared
Correlation of densities
Femurs vs. phalanges Femurs vs. vertebrae Vertebrae vs. phalanges
r = 0.593, p = 0.004 r = 0.704, p = 0.0003 r = 0.475, p = 0.025
density is readily available in the scientific literature. For example, among humans, in addition to twins and mother-daughtergranddaughter studies noted in the Introduction, racial group differences between blacks, whites and Asians are well known. Recent illustrative reports include that of Patel et a l Y showing that enhanced radial bone density of blacks vs. whites develops during pubertal maturation, as well as that of Sugimoto et al. TM documenting subtle differences in bone mineral content between adult Japanese, Koreans, and Taiwanese people. In the mouse, population-based data on bone size and mineral content have been reported for some genetically homogeneous inbred strains. Perkins et al. 2v compared 6 and 24 month old C57BL/6N and with BALB/cN male mice and found significant differences in femoral cortical bone thicknesses. Murray et al. 24 reported that C57BL/6J mice had lower vertebral mass, % ash, and lacked coupling of resorption and formation when compared with SENCAR strain mice. Matsushita et al. 2j reported differences in peak adult femoral cortical thickness indices in several A K R / J - r e l a t e d s u b l i n e s of mice, d e s i g n a t e d s e n e s c e n c e accelerated mice (SAM). Low bone density in one of the SAM models, SAM-P/6, has been suggested to result from a small number of genes. 44 Mapping data have not yet been reported. Kaye and Kusy ~5 have recently reported differences in tibial ash for young adult mice of five strains including three strains in this article--BALB/cByJ, C57BL/6J, and DBA/2J. Most provocative were their findings that increased quadriceps muscle mass and home cage activity levels of C57BL/6J mice compared with A/J mice correlated with differences in tibial ash, indicating a possible biomechanical aspect underlying the strain differences in bone mineral. Our article expands the knowledge base on bone for these widely used inbred strains and points to the cortical bone as a target for strong genetic effects on density of phenotypically normal bones. In contrast to the modest data on bone density among strains of mice, there is a large array of reports documenting specific gene effects on mouse bone morphogenesis. For example, short ear (se/se) mice, a mutation in the Bmp-5 gene, j7 have reduced bone turnover without a decrease in bone density when compared with normal littermates. 3 Bailey used bilineal congenic inbred strains derived from C57BL/6By and BALB/cBy to show that a large number of genes governed mandibular shape. 2 In fact, more than 140 spontaneous mouse mutations affecting bone, particularly bone morphology, have been summarized by Green. m Furthermore, an expanding list of induced mutations in m i c e - transgenes and genes disrupted by homologous recombination-affect the skeleton. Examples include mice carrying the human transgene COLI A I that causes fetal rib and long bone fractures 26 or lck-IL-4 that causes reduced bone formation by osteoblasts, j9 and mice with null alleles at specific loci, such as c-fos that causes osteopetrosis through reduced osteoclast differentiation, j j IGF-II that disrupts fetal and neonatal skeletal growth in addition to other tissues, 5 or c-src that also causes osteoclast dysfunction and osteopetrosis. 37 These models underscore the complexity of genetic loci that participate in the development of the skeleton from the time of earliest embryonic differentiation to adulthood.
Bone Vol. 18, No. 5 May 1996:397-403 Each of these classes of genetic information has different uses. Mutations--spontaneous or induced--typically cause recognizable bone pathology as a consequence of their actions and, thus, reveal the existence of genes important to development of specific bones or bone components. Such genetic events offer salient opportunities to identify relevant cell types and investigate cellular mechanisms critical to specific facets of bone biology. On the other hand, differences in bone density among inbred strains also demonstrate genetic regulation, but in this instance the consequence of gene action yields normal variation in the adult bone phenotype. Such strain differences in bone density should be clearly amenable to modern genetic analyses for mapping of loci critical to acquiring adult bone density. Identification of such genes and the particulars of their biological functions will provide a more rationale basis for therapies intended to improve on the quality of existing bone by maximizing peak bone density.
Acknowledgments: The authors thank A. Kirley and K. Kovalsky, participants in the Jackson Laboratory's Summer College Student Training Program, and K. Chapman for assistance with bone measurements. The authors are additionally indebted to Dr. D. Bailey, Dr. T. CunliffeBeamer, and Dr. D. Serreze for review of this manuscript. This work was supported by NIH RR-09174 (WGB, LRD, CJR), CA-34196 (WGB), AG-10942 (CJR), and by Veterans Administration Merit Review Grant (DJB). The Jackson Laboratory is fully accredited by the American Association for Laboratory Animal Care.
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Date Received: October 17, 1995 Date Revised: January 30, 1996 Date Accepted: January 31, 1996