Skeletal abnormalities in mice with Dnmt3a missense mutations

AUTHORS

Austin Bell-Hensley, Diana C. Beard, Kathryn Feeney, Hongjun Zheng, Yunhao Jiang, Xiyun Zhang, Jin Liu, Harrison Gabel, Audrey McAlinden

ABSTRACT

Overgrowth and intellectual disability disorders in humans are typified by length/height and/or head circumference ≥ 2 standard deviations above the mean as well as intellectual disability and behavioral comorbidities, including autism and anxiety. Tatton-Brown-Rahman Syndrome is one type of overgrowth and intellectual disability disorder caused by heterozygous missense mutations in the DNA methyltransferase 3A (DNMT3A) gene. Numerous DNMT3A mutations have been identified in Tatton-Brown-Rahman Syndrome patients and may be associated with varying phenotype severities of clinical presentation. Two such mutations are the R882H and P904L mutations which result in severe and mild phenotypes, respectively. Mice with paralogous mutations (Dnmt3aP900L/+ and Dnmt3aR878H/+) exhibit overgrowth in their long bones (e.g., femur, humerus), but the mechanisms responsible for their skeletal overgrowth remain unknown. The goal of this study is to characterize skeletal phenotypes in mouse models of Tatton-Brown-Rahman Syndrome and identify potential cellular mechanisms involved in the skeletal overgrowth phenotype. We report that mature mice with the Dnmt3aP900L/+ or Dnmt3aR878H/+ mutation exhibit tibial overgrowth, cortical bone thinning, and weakened bone mechanical properties. Dnmt3aR878H/+ mutants also contain larger bone marrow adipocytes while Dnmt3aP900L/+ mutants show no adipocyte phenotype compared to control animals. To understand the potential cellular mechanisms regulating these phenotypes, growth plate chondrocytes, osteoblasts, and osteoclasts were assessed in juvenile mutant mice using quantitative static histomorphometry and dynamic histomorphometry. Tibial growth plates appeared thicker in mutant juvenile mice, but no changes were observed in osteoblast activity or osteoclast number in the femoral mid-diaphysis. These studies reveal new skeletal phenotypes associated with Tatton-Brown-Rahman Syndrome in mice and provide a rationale to extend clinical assessments of patients with this condition to include bone density and quality testing. These findings may be also informative for skeletal characterization of other mouse models presenting with overgrowth and intellectual disability phenotypes.

Tmem263 deletion disrupts the GH/IGF-1 axis and causes dwarfism and impairs skeletal acquisition

AUTHORS

Dylan C Sarver, Jean Garcia-Diaz, Muzna Saqib, Ryan C Riddle, G William Wong

ABSTRACT

Genome-wide association studies (GWAS) have identified a large number of candidate genes believed to affect longitudinal bone growth and bone mass. One of these candidate genes, TMEM263, encodes a poorly characterized plasma membrane protein. Single nucleotide polymorphisms in TMEM263 are associated with bone mineral density in humans and mutations are associated with dwarfism in chicken and severe skeletal dysplasia in at least one human fetus. Whether this genotype-phenotype relationship is causal, however, remains unclear. Here, we determine whether and how TMEM263 is required for postnatal growth. Deletion of the Tmem263 gene in mice causes severe postnatal growth failure, proportional dwarfism, and impaired skeletal acquisition. Mice lacking Tmem263 show no differences in body weight within the first 2 weeks of postnatal life. However, by P21 there is a dramatic growth deficit due to a disrupted growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, which is critical for longitudinal bone growth. Tmem263-null mice have low circulating IGF-1 levels and pronounced reductions in bone mass and growth plate length. The low serum IGF-1 in Tmem263-null mice is associated with reduced hepatic GH receptor (GHR) expression and GH-induced JAK2/STAT5 signaling. A deficit in GH signaling dramatically alters GH-regulated genes and feminizes the liver transcriptome of Tmem263-null male mice, with their expression profile resembling wild-type female, hypophysectomized male, and Stat5b-null male mice. Collectively, our data validates the causal role for Tmem263 in regulating postnatal growth and raises the possibility that rare mutations or variants of TMEM263 may potentially cause GH insensitivity and impair linear growth.

Targeted postnatal knockout of Sclerostin using a bone-targeted adeno-associated viral vector increases bone anabolism and decreases canalicular density

AUTHORS

Alexandra K. O'Donohue, Ya Xiao, Lucinda R. Lee, Timothy Schofield, Tegan L. Cheng, Craig Munns, Paul A. Baldock, Aaron Schindeler

ABSTRACT

Purpose

The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice using a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector.

Methods

8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5 × 1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6 weeks, detailed bone analysis performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens.

Results

The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22 % increase in bone volume and 21 % in trabecular bone fraction compared to controls; this translated to a 17 % increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25 % increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation.

Conclusion

This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.

Reduced bone mass in collagen prolyl 4-hydroxylase P4ha1+/-;P4ha2-/- compound mutant mice

AUTHORS

Jussi-Pekka Tolonen, Antti M. Salo, Mikko Finnilä, Ellinoora Aro, Emma Karjalainen, Veli-Pekka Ronkainen, Kati Drushinin, Christophe Merceron, Valerio Izzi, Ernestina Schipani, Johanna Myllyharju

ABSTRACT

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, while inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1+/-;P4ha2-/- mice, we have carried out gene expression analyses at whole tissue and single cell levels, biochemical analyses, microcomputed tomography and histomorphometric analyses and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1+/-;P4ha2-/- tibia and the C-P4H activity in primary P4ha1+/-;P4ha2-/- osteoblasts were reduced, while the population of osteoprogenitor colony forming-unit fibroblasts was increased in the P4ha1+/-;P4ha2-/- marrow. Thus, the P4ha1+/-;P4ha2-/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by bi-allelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele-dose dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix.

In vivo imaging tools for functional assessment of biomaterials implanted bone regeneration

AUTHORS

Subhasis Roy; Prasenjit Mukherjee; Samit Kumar Nandi

ABSTRACT

Since the discovery of X-rays and its first use in imaging of a hand, bone tissue has been the chapter of interest in medical imaging. However, X-ray imaging poses limitations nowadays owing to the augmented complexity of implant scaffolds as well as with the advances in bone engineering. As a result, advanced follow-up imaging techniques are of paramount necessity for effective postoperative characterization. Moreover, it is also needed to search for non-invasive, high-sensitivity, and high-resolution structural, functional, and molecular imaging techniques such as acoustic, optical, magnetic, X-Ray, electron, ultrasound, and nuclear imaging, etc. as an alternative to normally used X-ray computed tomography. Further, enthusiastic preclinical scanners have turned out to be accessible, with sensitivity and resolution even superior to clinical scanners, as a consequence helping a rapid transformation from preclinical to clinical applications. Besides, recently, bone-specific probes and contrast agents are developing for better imaging tools in bone-tissue engineering applications. This review highlights such emerging preclinical imaging tools, each with its individual potencies and flaws, either used only or in combination. In particular, multimodal imaging will significantly add to improve the present understanding in the characterization of bone regenerative processes.