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.     

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.     

Identification of novel genetic loci for bone size and mechanosensitivity in an ENU mutant exhibiting decreased bone size.

Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. We show that mutation results in osteoblasts with reduced proliferation, increased apoptosis, and an impaired response to in vitro mechanical load. The mutation is mapped to a novel locus (LOD score of 7.9 at 10.5 cM) on chromosome 4. INTRODUCTION: Using a dominant ENU mutagenesis screen in C57BL/6J (B6) mice to reveal gene function, we identified a mutant, 917M, with a reduced bone size phenotype, which is expressed only in males. In this report, we show the chromosomal location of this mutation using linkage analysis and cellular characterization of the mutant phenotype. MATERIALS AND METHODS: The mutant mouse was bred to wildtype B6 to produce progeny for characterization of the bone size phenotype. Periosteal osteoblasts isolated from the tibia and femur of mutant and wildtype mice were studied for proliferation, differentiation, and apoptosis potential. To determine the chromosomal location of the mutation, a low-resolution linkage map was established by completing a genome-wide scan in B6C3H F2 male mice generated from intercross breeding of mutant mice. RESULTS AND CONCLUSIONS: Mutant progeny (16 weeks old) displayed a total body bone area that was 10-13% lower and a periosteal circumference that was 5-8% lower at the femur and tibia midshaft compared with wildtype B6 mice. Periosteal osteoblasts from mutant mice showed 17-27% reduced cell proliferation and 23% increased apoptosis compared with wildtype controls. In addition, osteoblasts from mutant mice showed an impaired response to shear stress-induced proliferation rate, an in vitro model for mechanical loading. Interval mapping in B6C3H F2 males (n = 69) indicated two major loci affecting bone size on chromosome 1 at 45 cM (LOD 4.9) and chromosome 4 at 10.5 cM (LOD 7.9, genome-wide p < 0.01). Interval mapping using body weight as covariate revealed only one significant interval at chromosome 4 (LOD 6.8). Alleles of the chromosome 4 interval inherited from the B6 mutant strain contributed to a significantly lower bone size than those inherited from C3H. A pairwise interaction analysis showed evidence for a significant interaction between loci on chromosome 1 with the chromosome 4 quantitative trait loci. The 917M locus on chromosome 4 seems to be novel because it does not correspond with those loci previously associated with bone size on chromosome 4 in B6 and C3H/HeJ mice or other crosses.     

New Variants in the Enpp1 and Ptpn6 Genes Cause Low BMD,Crystal‐Related Arthropathy,and Vascular Calcification

A large genome‐wide, recessive, N‐ethyl‐N‐nitrosourea (ENU)‐induced mutagenesis screen was performed on a mixed C57BL/6J and C3H.SW‐H2/SnJ mouse background to identify genes regulating bone mass. Approximately 6500 male and female G₃ hybrid mice were phenotyped at 8 and 10 wk of age by DXA analysis for evidence of changes in unadjusted or body weight–adjusted BMD or BMC. Phenodeviant lines were identified based on statistical criteria that included a false discovery rate (FDR) <20% and Z‐score >2.8. Genome‐wide mapping scans were initiated on 22 lines, with evidence of high or low BMD or BMC that deviated by approximately ?30% to +50% from the means. Several lines were discontinued as showing lack of heritability, but two heritable lines were identified with narrow chromosomal regions that allowed sequencing of potential mutant candidate genes. Novel mutations were identified in the Enpp1 (C397S) gene on chromosome 10 (line 4482) and the Ptpn6 (I482F) gene on chromosome 6 (line 4489) that were both associated with low bone mass. In addition, the phenotype of the Enpp1 mice showed a striking joint disease and calcification of blood vessels including the aorta, myocardium, and renal arteries and capillaries. These results support a role for the Enpp1 gene in the pathogenesis associated with mineralization of articular cartilage and vascular calcification. This work confirms the utility of the chemical mutagenesis approach for identification of potential disease genes and confirms the role of Enpp1 and Ptpn6 in regulating mineralization and skeletal bone mass.     

Germline Saturation Mutagenesis Induces Skeletal Phenotypes in Mice

Proper embryonic and postnatal skeletal development require coordination of myriad complex molecular mechanisms. Disruption of these processes, through genetic mutation, contributes to variation in skeletal development. We developed a high-throughput N-ethyl-N-nitrosourea (ENU)-induced saturation mutagenesis skeletal screening approach in mice to identify genes required for proper skeletal development. Here, we report initial results from live-animal X-ray and dual-energy X-ray absorptiometry (DXA) imaging of 27,607 G3 mice from 806 pedigrees, testing the effects of 32,198 coding/splicing mutations in 13,020 genes. A total of 39.7% of all autosomal genes were severely damaged or destroyed by mutations tested twice or more in the homozygous state. Results from our study demonstrate the feasibility of in vivo mutagenesis to identify mouse models of skeletal disease. Furthermore, our study demonstrates how ENU mutagenesis provides opportunities to create and characterize putative hypomorphic mutations in developmentally essential genes. Finally, we present a viable mouse model and case report of recessive skeletal disease caused by mutations in FAM20B. Results from this study, including engineered mouse models, are made publicly available via the online Mutagenetix database. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Single amino acid substitution in aquaporin 11 causes renal failure

A screen of recessive mutations generated by the chemical mutagen n-ethyl-n-nitrosourea (ENU) mapped a new mutant locus (5772SB) termed sudden juvenile death syndrome (sjds) to chromosome 7 in mice. These mutant mice, which exhibit severe proximal tubule injury and formation of giant vacuoles in the renal cortex, die from renal failure, a phenotype that resembles aquaporin 11 (Aqp11) knockout mice. In this report, the ENU-induced single-nucleotide variant (sjds mutation) is identified. To determine whether this variant, which causes an amino acid substitution (Cys227Ser) in the predicted E-loop region of aquaporin 11, is responsible for the sjds lethal renal phenotype, Aqp11-/sjds compound heterozygous mice were generated from Aqp11 +/sjds and Aqp11 +/- intercrosses. The compound heterozygous Aqp11 -/sjds offspring exhibited a lethal renal phenotype (renal failure by 2 wk), similar to the Aqp11 sjds/sjds and Aqp11-/- phenotypes. These results demonstrate that the identified mutation causes renal failure in Aqp11 sjds/sjds mutant mice, providing a model for better understanding of the structure and function of aquaporin 11 in renal physiology.     

A new mouse model of type 2 diabetes, produced by N-ethyl-nitrosourea mutagenesis, is the result of a missense mutation in the glucokinase gene

Here we report the first cloned N-ethyl-nitrosourea (ENU)-derived mouse model of diabetes. GENA348 was identified through free-fed plasma glucose measurement, being more than 2 SDs above the population mean of a cohort of >1,201 male ENU mutant mice. The underlying gene was mapped to the maturity-onset diabetes of the young (MODY2) homology region of mouse chromosome 11 (logarithm of odds 6.0). Positional candidate gene analyses revealed an A to T transversion mutation in exon 9 of the glucokinase gene, resulting in an isoleucine to phenylalanine change at amino acid 366 (I366F). Heterozygous mutants have 67% of the enzyme activity of wild-type littermates (P < 0.0012). Homozygous mutants have less enzyme activity (14% of wild-type activity) and are even less glucose tolerant. The GENA348 allele is novel because no mouse or human diabetes studies have described a mutation in the corresponding amino acid position. It is also the first glucokinase missense mutation reported in mice and is homozygous viable, unlike the global knockout mutations. This work demonstrates that ENU mutagenesis screens can be used to generate models of complex phenotypes, such as type 2 diabetes, that are directly relevant to human disease.     

First Mouse Model for Combined Osteogenesis Imperfecta and Ehlers‐Danlos Syndrome

By using a genome‐wide N‐ethyl‐N‐nitrosourea (ENU)‐induced dominant mutagenesis screen in mice, a founder with low bone mineral density (BMD) was identified. Mapping and sequencing revealed a T to C transition in a splice donor of the collagen alpha1 type I (Col1a1) gene, resulting in the skipping of exon 9 and a predicted 18‐amino acid deletion within the N‐terminal region of the triple helical domain of Col1a1. Col1a1^Jrt/+ mice were smaller in size, had lower BMD associated with decreased bone volume/tissue volume (BV/TV) and reduced trabecular number, and furthermore exhibited mechanically weak, brittle, fracture‐prone bones, a hallmark of osteogenesis imperfecta (OI). Several markers of osteoblast differentiation were upregulated in mutant bone, and histomorphometry showed that the proportion of trabecular bone surfaces covered by activated osteoblasts (Ob.S/BS and N.Ob/BS) was elevated, but bone surfaces undergoing resorption (Oc.S/BS and N.Oc/BS) were not. The number of bone marrow stromal osteoprogenitors (CFU‐ALP) was unaffected, but mineralization was decreased in cultures from young Col1a1^Jrt/+ versus +/+ mice. Total collagen and type I collagen content of matrices deposited by Col1a1^Jrt/+ dermal fibroblasts in culture was ～40% and 30%, respectively, that of +/+ cells, suggesting that mutant collagen chains exerted a dominant negative effect on type I collagen biosynthesis. Mutant collagen fibrils were also markedly smaller in diameter than +/+ fibrils in bone, tendon, and extracellular matrices deposited by dermal fibroblasts in vitro. Col1a1^Jrt/+ mice also exhibited traits associated with Ehlers‐Danlos syndrome (EDS): Their skin had reduced tensile properties, tail tendon appeared more frayed, and a third of the young adult mice had noticeable curvature of the spine. Col1a1^Jrt/+ is the first reported model of combined OI/EDS and will be useful for exploring aspects of OI and EDS pathophysiology and treatment. © 2014 American Society for Bone and Mineral Research.     

Detecting Novel Bone Density and Bone Size Quantitative Trait Loci Using a Cross of MRL/MpJ and CAST/EiJ Inbred Mice

Most previous studies to identify loci involved in bone mineral density (BMD) regulation have used inbred strains with high and low BMD in generating F₂ mice. However, differences in BMD may not be a requirement in selecting parental strains for BMD quantitative trait loci (QTL) studies. In this study, we intended to identify novel QTL using a cross of two strains, MRL/MpJ (MRL) and CAST/EiJ (CAST), both of which exhibit relatively high BMD when compared to previously used strains. In addition, CAST was genetically distinct. We generated 328 MRL × CAST F₂ mice of both sexes and measured femur BMD and periosteal circumference (PC) using peripheral quantitative computed tomography. Whole-genome genotyping was performed with 86 microsatellite markers. A new BMD QTL on chromosome 10 and another suggestive one on chromosome 15 were identified. A significant femur PC QTL identified on chromosome 9 and a suggestive one on chromosome 2 were similar to those detected in MRL × SJL. QTL were also identified for other femur and forearm bone density and bone size phenotypes, some of which were colocalized within the same chromosomal positions as those for femur BMD and femur PC. This study demonstrates the utility of crosses involving inbred strains of mice which exhibit a similar phenotype in QTL identification.     

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.     

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.     

Heritability of Bone Mineral Density in a Multivariate Family-Based Study

There is evidence for a genetic contribution to bone mineral density (BMD×). Different loci affecting BMD have been identified by diverse linkage and genome-wide association studies. We studied the heritability of and the correlations among six densitometric phenotypes and four bone mass/fracture phenotypes. For this purpose, we used a family-based study of the genetics of osteoporosis, the Genetic Analysis of Osteoporosis Project. The primary aim of our study was to examine the roles of genetic and environmental factors in determining osteoporosis-related phenotypes. The project consisted of 11 extended families from Spain. All of them were selected through a proband with osteoporosis. BMD was measured using dual-energy X-ray absorptiometry. The proportion of variance of BMD attributable to significant covariates ranged from 25 % (for femoral neck BMD) to 48 % (for whole-body total BMD). The vast majority of the densitometric phenotypes had highly significant heritability, ranging from 0.252 (whole-body total BMD) to 0.537 (trochanteric BMD) after correcting for covariate effects. All of the densitometric phenotypes showed high and significant genetic correlations (from ?0.772 to ?1.000) with a low bone mass/osteopenia condition (Affected 3). Our findings provide additional evidence on the heritability of BMD and a strong genetic correlation between BMD and bone mass/fracture phenotypes in a Spanish population. Our results emphasize the importance of detecting genetic risk factors and the benefit of early diagnosis and especially therapeutic and preventive strategies.     

Mineral density and bone strength are dissociated in long bones of rat osteopetrotic mutations.

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.     

Phenotypic characterization of mice bred for high and low peak bone mass.

In humans, peak bone mineral density (BMD) is a highly heritable trait and a strong determinant of subsequent osteoporotic fracture risk. To identify the genetic factors responsible for variation in peak BMD, investigators have turned to animal models. In this study we examined the heritability of BMD acquisition and characterized differences in skeletal geometry, histomorphometry, and biomechanical competence between two lines of mice artificially selected for extremes of peak whole body BMD. F2 progeny from a cross between C57BL/6 and DBA/2 inbred strains was used as the foundation population to develop lines selected for either high or low BMD. Whole body BMD was measured by dual-energy X-ray absorptiometry (DXA). By the third generation of selection, highest-scoring BMD (HiBMD) mice exhibited 14% greater peak BMD than lowest-scoring BMD (LoBMD) mice. The mean realized heritability of peak BMD was 36%. Femoral shaft cortical area and thickness and vertebral cancellous bone volume (BV) were significantly greater (16-30%) in the HiBMD line compared with the LoBMD line. Mean cancellous bone formation rates (BFRs) were 35% lower in HiBMD mice compared with LoBMD mice. Failure load and stiffness in the femoral shaft, femoral neck, and L6 vertebrae were all substantially greater (by 25-190%) in HiBMD mice. Thus, these divergently selected murine lines serve to illustrate some of the means by which genetic mechanisms can affect skeletal structure and remodeling. Identification of the individual genes influencing peak BMD in this experimental system will likely reveal some of the genetic determinants of overall bone strength.     

Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice

Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1^asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1^asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1^asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1^asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Chromosomal mapping of osteopenia-associated quantitative trait loci using closely related mouse strains.

Peak bone mineral density (BMD) is a highly heritable trait in humans and is currently the best predictor of skeletal fragility underlying osteoporosis. The SAMP6 mouse strain displays unusually low BMD at maturity, and age-dependent osteopenia associated with defective osteoblastogenesis. To identify quantitative trait loci (QTLs) influencing bone density, we constructed crosses between SAMP6 and either AKR/J or SAMP6, two related mouse strains of higher peak BMD. Due to common ancestry of these strains, intercross parents differed at only 39-40% of 227 highly-polymorphic genotyping markers, thus restricting our search to this informative portion of the genome and reducing the number of mice required for QTL significance. Using dual energy X-ray absorptiometry (DEXA), we measured spinal BMD in F2 cross progeny at 4 months of age, and selectively genotyped those in the highest and lowest quartiles for BMD. Based on linear regression of bone density on genotype, including Composite Interval Mapping to enhance mapping precision while adjusting for effects of distal markers, we identified multiple QTLs significantly affecting spinal BMD; these were mapped to regions of chromosomes 2 (two sites, one confirmed in both crosses), 7, 11, 13 and 16. One of these loci had been previously identified as a significant bone-density QTL, while 3 substantiate QTLs suggested by a low-power study of 24 recombinant-inbred mouse lines. Such recurrent appearance of QTLs, especially in crosses involving distantly-related strains, implies that polymorphism at these loci may be favored by evolution and might underlie variation in peak bone density among humans.     

Epistatic effects contribute to variation in BMD in Fischer 344 x Lewis F2 rats.

To further delineate the factors underlying the complex genetic architecture of BMD in the rat model, a genome screen for epistatic interactions was conducted. Several significant interactions were identified, involving both previously identified and novel QTLs. INTRODUCTION: The variation in several of the risk factors for osteoporotic fracture, including BMD, has been shown to be caused largely by genetic differences. However, the genetic architecture of BMD is complex in both humans and in model organisms. We have previously reported quantitative trait locus (QTL) results for BMD from a genome screen of 595 female F(2) progeny of Fischer 344 and Lewis rats. These progeny also provide an excellent opportunity to search for epistatic effects, or interaction between genetic loci, that contribute to fracture risk. MATERIALS AND METHODS: Microsatellite marker data from a 20-cM genome screen was analyzed along with weight-adjusted BMD (DXA and pQCT) phenotypic data using the R/qtl software package. Genotype and phenotype data were permuted to determine a genome-wide significance threshold for the epistasis or interaction LOD score corresponding to an alpha level of 0.01. RESULTS AND CONCLUSIONS: Novel loci on chromosomes 12 and 15 showed a strong epistatic effect on total BMD at the femoral midshaft by pQCT (LOD = 5.4). A previously reported QTL on chromosome 7 was found to interact with a novel locus on chromosome 20 to affect whole lumbar BMD by pQCT (LOD = 6.2). These results provide new information regarding the mode of action of previously identified rat QTLs, as well as identifying novel loci that act in combination with known QTLs or with other novel loci to contribute to the risk factors for osteoporotic fracture.     

Genetic loci for bone architecture determined by three-dimensional CT in crosses with the diabetic GK rat

The F344 rat carries alleles contributing to bone fragility while the GK rat spontaneously develops type-2 diabetes. These characteristics make F344×GK crosses well suited for the identification of genes related to bone size and allow for future investigation on the association with type-2 diabetes. The aim of this study was to identify quantitative trait loci (QTLs) for bone size phenotypes measured by a new application of three-dimensional computed tomography (3DCT) and to investigate the effects of sex- and reciprocal cross.Tibia from male and female GK and F344 rats, representing the parental, F1 and F2 generations, were examined with 3DCT and analyzed for: total and cortical volumetric BMD, straight and curved length, peri- and endosteal area at mid-shaft. F2 progeny (108 male and 98 female) were genotyped with 192 genome-wide microsatellite markers (average distance 10 cM). Sex- and reciprocal cross-separated QTL analyses were performed for the identification of QTLs linked to 3DCT phenotypes and true interactions were confirmed by likelihood ratio analysis in all F2 animals.Several genome-wide significant QTLs were found in the sex- and reciprocal cross-separated progeny on chromosomes (chr) 1, 3, 4, 9, 10, 14, and 17. Overlapping QTLs for both males and females in the (GK×F344)F2 progeny were located on chr 1 (39–67 cM). This region confirms previously reported pQCT QTLs and overlaps loci for fasting glucose. Sex separated linkage analysis confirmed a male specific QTL on chr 9 (67–82 cM) for endosteal area at the fibula site. Analyses separating the F2 population both by sex and reciprocal cross identified cross specific QTLs on chr 14 (males) and chr 3 and 4 (females). Two loci, chr 4 and 6, are unique to 3DCT and separate from pQCT generated loci.The 3DCT method was highly reproducible and provided high precision measurements of bone size in the rat enabling identification of new sex- and cross-specific loci. The QTLs on chr 1 indicate potential genetic association between bone-related phenotypes and traits affecting type-2 diabetes. The results illustrate the complexity of the genetic architecture of bone size phenotypes and demonstrate the importance of complementary methods for bone analysis.     

The V–ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice

A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N‐ethyl‐N‐nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H⁺‐ATPase (V–ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V–ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V–ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V₀ complex has proton‐pumping‐independent functions in mammalian cells. © 2011 American Society for Bone and Mineral Research.     

Genetic and environmental correlations between bone phenotypes and anthropometric indices in Chinese

Height, weight, bone mineral density (BMD), and bone size are all influenced by genetic and environmental factors as well as interactions between them. Height and weight are often used in population studies to adjust the bone phenotypes. However, it is still unknown what proportion of genetic and environmental variability is shared between these anthropometric characteristics and the bone phenotypes. The genetic and environmental correlations between the bone phenotypes and anthropometric indices in Chinese subjects were studied by bivariate quantitative genetic analysis on a sample of 931 healthy subjects from 292 Chinese nuclear families aged from 19 to 79 years. BMD and bone size at the lumbar spine (L1–L4) and the hip of all subjects were measured by dual-energy X-ray absorptiometry. We found significant genetic correlations between weight and spine BMD, hip BMD, spine bone size and hip bone size, which were 0.50 (P<0.01), 0.45 (P<0.01), 0.36 (P=0.02), and 0.38 (P<0.01), respectively. Likewise, significant genetic correlations between height and spine BMD, spine bone size, and hip bone size were 0.30 (P=0.02), 0.54 (P<0.01), and 0.58 (P<0.01), respectively. The environmental correlations were found to be significant only between height and spine bone size (P<0.001) and weight and hip BMD (P=0.02). These results suggest the probability that the same genetic and environmental factors contribute to these different phenotypes. Moreover, when a candidate gene or genomic region is responsible for the variation of both bone phenotypes and anthropometric indices, its true genetic effect on the bone phenotypes may be lost after one has adjusted the phenotypic values with weight and height as random environmental factors. It may have implications for population studies of candidate genes that underlie the complex bone phenotypes and for the development of strategies for therapeutic application.