Stephanie M. Carleton

Variable bone fragility associated with an Amish COL1A2 variant and a knock-in mouse model.

Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z‐scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock‐in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F1 lines of knock‐in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole‐bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock‐in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI.

Role of genetic background in determining phenotypic severity throughout postnatal development and at peak bone mass in Col1a2 deficient mice (oim)

Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disease characterized by extreme bone fragility. Although fracture numbers tend to decrease post-puberty, OI patients can exhibit significant variation in clinical outcome, even among related individuals harboring the same mutation. OI most frequently results from mutations in type I collagen genes, yet how genetic background impacts phenotypic outcome remains unclear. Therefore, we analyzed the phenotypic severity of a known proalpha2(I) collagen gene defect (oim) on two genetic backgrounds (congenic C57BL/6J and outbred B6C3Fe) throughout postnatal development to discern the phenotypic contributions of the Col1a2 locus relative to the contribution of the genetic background. To this end, femora and tibiae were isolated from wildtype (Wt) and homozygous (oim/oim) mice of each strain at 1, 2 and 4 months of age. Femoral geometry was determined via muCT prior to torsional loading to failure to assess bone structural and material biomechanical properties. Changes in mineral composition, collagen content and bone turnover were determined using neutron activation analyses, hydroxyproline content and serum pyridinoline crosslinks. muCT analysis demonstrated genotype-, strain- and age-associated changes in femoral geometry as well as a marked decrease in the amount of bone in oim/oim mice of both strains. Oim/oim mice of both strains, as well as C57BL/6J (B6) mice of all genotypes, had reduced femoral biomechanical strength properties compared to Wt at all ages, although they improved with age. Mineral levels of fluoride, magnesium and sodium were associated with biomechanical strength properties in both strains and all genotypes at all ages. Oim/oim animals also had reduced collagen content as compared to Wt at all ages. Serum pyridinoline crosslinks were highest at two months of age, regardless of strain or genotype. Strain differences in bone parameters exist throughout development, implicating a role for genetic background in determining biomechanical strength. Age-associated improvements indicate that oim/oim animals partially compensate for their weaker bone material, but never attain Wt levels. These studies indicate the importance of genetic background in determining phenotypic severity, but the presence of the proalpha2(I) collagen gene defect and age of the animal are the primary determinants of phenotypic severity.

Gender-Dependence of Bone Structure and Properties in Adult Osteogenesis Imperfecta Murine Model

Osteogenesis imperfecta (OI) is a dominant skeletal disorder characterized by bone fragility and deformities. Though the oim mouse model has been the most widely studied of the OI models, it has only recently been suggested to exhibit gender-dependent differences in bone mineralization. To characterize the impact of gender on the morphometry/ultra-structure, mechanical properties, and biochemical composition of oim bone on the congenic C57BL/J6 background, 4-month-old oim/oim, +/oim, and wild-type (wt) female and male tibiae were evaluated using micro-computed tomography, three-point bending, and Raman spectroscopy. Dramatic gender differences were evident in both cortical and trabecular bone morphological and geometric parameters. Male mice had inherently more bone and increased moment of inertia than genotype-matched female counterparts with corresponding increases in bone biomechanical strength. The primary influence of gender was structure/geometry in bone growth and mechanical properties, whereas the mineral/matrix composition and hydroxyproline content of bone were influenced primarily by the oim collagen mutation. This study provides evidence of the importance of gender in the evaluation and interpretation of potential therapeutic strategies when using mouse models of OI.

Effect of Food Restriction and Leptin Supplementation on Fetal Programming in Mice

Metabolic disease is a significant global health and economic problem. In a phenomenon referred to as fetal programming, offspring of underweight or overweight mothers have an increased incidence of adulthood obesity and metabolic disease. Undernourished individuals have decreased levels of leptin, a regulator of energy balance, whereas obese people develop hyperleptinemia and leptin resistance. We hypothesize that alterations in circulating leptin during pregnancy contribute to programming events caused by maternal nutritional status. To test this hypothesis, pregnant mice were randomly placed in one of three treatment groups: ad libitum feed plus saline injection (control, n = 5), 50% food restriction plus saline injection (restricted, n = 4), or 50% food restriction plus 1 mg/kg · d leptin injection (restricted, leptin treated, n = 4). Mice were treated from 1.5 to 11.5 d after conception and then returned to ad libitum feeding until weaning. At 19 wk after weaning, offspring were placed on a 45% fat diet and then followed up until 26 wk after weaning, at which time they were killed, and samples were collected for further analysis. Our results demonstrate that males are more negatively impacted by high-fat diet than females, regardless of maternal treatment. We provide evidence that differential response to leptin may mediate the sexual dimorphism observed in fetal programming in which male offspring are more affected by maternal undernutrition and female offspring by maternal overnutrition. We show that female offspring born to food-restricted, leptin-supplemented mothers are obese and insulin resistant. This may mimic fetal programming events seen in offspring of overweight women.

Developmental Exposure to Xenoestrogens at Low Doses Alters Femur Length and Tensile Strength in Adult Mice

ABSTRACT Developmental exposure to high doses of the synthetic xenoestrogen diethylstilbestrol (DES) has been reported to alter femur length and strength in adult mice. However, it is not known if developmental exposure to low, environmentally relevant doses of xenoestrogens alters adult bone geometry and strength. In this study we investigated the effects of developmental exposure to low doses of DES, bisphenol A (BPA), or ethinyl estradiol (EE2) on bone geometry and torsional strength. C57BL/6 mice were exposed to DES, 0.1 μg/kg/day, BPA, 10 μg/kg/day, EE2, 0.01, 0.1, or 1.0 μg/kg/day, or vehicle from Gestation Day 11 to Postnatal Day 12 via a mini-osmotic pump in the dam. Developmental Xenoestrogen exposure altered femoral geometry and strength, assessed in adulthood by micro-computed tomography and torsional strength analysis, respectively. Low-dose EE2, DES, or BPA increased adult femur length. Exposure to the highest dose of EE2 did not alter femur length, resulting in a nonmonotonic dose response. Exposure to EE2 and DES but not BPA decreased tensile strength. The combined effect of increased femur length and decreased tensile strength resulted in a trend toward decreased torsional ultimate strength and energy to failure. Taken together, these results suggest that exposure to developmental exposure to environmentally relevant levels of xenoestrogens may negatively impact bone length and strength in adulthood.

Hindlimb Skeletal Muscle Function and Skeletal Quality and Strength in +/G610C Mice With and Without Weight-Bearing Exercise

Osteogenesis imperfecta (OI) is a heterogeneous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared with healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In this study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels before evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function, and their activity levels were not reduced relative to wild‐type mice. The +/G610C mice were also able to complete an 8‐week treadmill regimen. Biomechanical integrity of control and exercised wild‐type and +/G610C femora were analyzed by torsional loading to failure. The greatest skeletal gains in response to exercise were observed in stiffness and the shear modulus of elasticity with alterations in collagen content. Analysis of tibial cortical bone by Raman spectroscopy demonstrated similar crystallinity and mineral/matrix ratios regardless of sex, exercise, and genotype. Together, these findings demonstrate +/G610C OI mice have equivalent muscle function, activity levels, and ability to complete a weight‐bearing exercise regimen as wild‐type mice. The +/G610C mice exhibited increased femoral stiffness and decreased hydroxyproline with exercise, whereas other biomechanical parameters remain unaffected, suggesting a more rigorous exercise regimen or another exercise modality may be required to improve bone quality of OI mice.

Myostatin deficiency partially rescues the bone phenotype of osteogenesis imperfecta model mice

SummaryMice with osteogenesis imperfecta (+/oim), a disorder of bone fragility, were bred to mice with muscle over growth to test whether increasing muscle mass genetically would improve bone quality and strength. The results demonstrate that femora from mice carrying both mutations have greater mechanical integrity than their +/oim littermates.IntroductionOsteogenesis imperfecta is a heritable connective tissue disorder due primarily to mutations in the type I collagen genes resulting in skeletal deformity and fragility. Currently, there is no cure, and therapeutic strategies encompass the use of antiresorptive pharmaceuticals and surgical bracing, with limited success and significant potential for adverse effects. Bone, a mechanosensing organ, can respond to high mechanical loads by increasing new bone formation and altering bone geometry to withstand increased forces. Skeletal muscle is a major source of physiological loading on bone, and bone strength is proportional to muscle mass.MethodsTo test the hypothesis that congenic increases in muscle mass in the osteogenesis imperfecta murine model mouse (oim) will improve their compromised bone quality and strength, heterozygous (+/oim) mice were bred to mice deficient in myostatin (+/mstn), a negative regulator of muscle growth. The resulting adult offspring were evaluated for hindlimb muscle mass, and bone microarchitecture, physiochemistry, and biomechanical integrity.Results+/oim mice deficient in myostatin (+/mstn +/oim) were generated and demonstrated that myostatin deficiency increased body weight, muscle mass, and biomechanical strength in +/mstn +/oim mice as compared to +/oim mice. Additionally, myostatin deficiency altered the physiochemical properties of the +/oim bone but did not alter bone remodeling.ConclusionsMyostatin deficiency partially improved the reduced femoral bone biomechanical strength of adult +/oim mice by increasing muscle mass with concomitant improvements in bone microarchitecture and physiochemical properties.

DNA carrier testing and newborn screening for maple syrup urine disease in Old Order Mennonite communities.

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by mutations in the branched chain alpha-keto acid dehydrogenase complex. Worldwide incidence of MSUD is 1:225,000 live births. However, within Old Order Mennonite communities, the incidence is 1:150 live births and results from a common tyrosine to asparagine substitution (Y438N) in the E1alpha subunit of branched chain alpha-keto acid dehydrogenase. We developed a new DNA diagnostic assay utilizing TaqMan technology and compared its efficacy, sensitivity, and duration with an existing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Carrier testing was performed by both TaqMan technology and PCR-RFLP on DNA isolated from buccal swabs of 160 individuals as well as from buccal swabs and blood spots of nine at-risk newborns; assay time, sensitivity, and reliability were also evaluated. The TaqMan assay, like the PCR-RFLP assay, accurately determined Y438N E1alpha allele status. However, the TaqMan assay appeared (1) more sensitive than the PCR-RFLP assay, requiring 10-fold less DNA (10 ng) to reliably determine genotype status and (2) faster, reducing the assay time required for diagnosis from approximately 12 to 5 h. TaqMan technology allowed more rapid DNA diagnoses of MSUD in the neonate, thereby reducing the likelihood of neurological impairment while enhancing health and prognosis for affected infants.

Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta

Significance Osteogenesis imperfecta (brittle bone disease) is an incurable genetic disorder. We demonstrate that maternal deficiency of myostatin (a negative regulator of muscle growth) can enhance bone biomechanical strength and integrity in control and osteogenesis imperfecta mouse offspring, using three independent approaches. We provide evidence that bone is responsive to developmental programming and that myostatin can mediate these effects. Embryo transfer experiments show that the effects of maternal myostatin deficiency are conferred by the postimplantation environment. These studies represent a paradigm shift in understanding and treating osteogenesis imperfecta—a shift from believing only genetic and postnatal environmental factors control bone health to the inclusion of prenatal/perinatal developmental programming as a modifiable factor controlling adult bone health. During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstntm1Sjl/+) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstntm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2oim), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2oim/+ offspring from natural mating of Mstntm1Sjl/+ dams to Col1a2oim/+sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2oim/+ dams to Col1a2oim/+ sires. Finally, increased bone biomechanical strength of Col1a2oim/+ offspring that had been transferred into Mstntm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

Animal Models of Osteogenesis Imperfecta

Animal models have played major roles in understanding the pathogenesis and etiology of disease and this is no less true for osteogenesis imperfecta (OI). Since 1983, at least 15 animal models have been generated or identified representing the broad range of clinical outcomes and molecular/biochemical mechanisms responsible for OI. In this chapter we describe and summarize those animal models whose molecular defects have been characterized, attempting to subdivide them based on the three primary disease driving mechanisms (1) defects in type I collagen genes (COL1A1 and COL1A2), (2) defects in post-translational hydroxylation of type I procollagen and ER trafficking, and (3) abnormalities in type I procollagen chaperone activity.