Bone Abstracts

Objectives: Maternal vitamin D status in pregnancy is associated with neonatal bone mass, and altered DNA methylation. Mice exposed to early life vitamin D deficiency have lower bone mass and reduced bone accrual in response to mechanical loading. Using tibias from these mice we assessed DNA methylation of promoters of genetic loci important for bone growth and development.

Methods: C57/BL6 mice received a vitamin D replete or deplete diet for 6 weeks periconceptionally until weaning of offspring at 3 weeks. Non-invasive tibial axial loading with a peak 11N dynamic load was applied to offsprings’ left tibiae 3x weekly for 2 weeks at age 8 and 16 weeks. Tibias were collected at 18 weeks and mechanically crushed in liquid nitrogen. DNA was extracted from bone powder using the QiAMP DNA mini kit, and 1ug DNA was bisulfite converted using the EZ DNA methylation kit. Bisulfite pyrosequencing was used to measure DNA methylation of individual CpGs at single base pair resolution within the promoters of Retinoid X receptor (RXRA), Vitamin D receptor, Osterix and Runx2 in loaded and non-loaded tibias. Independent t-tests were used to compare the effect of treatment and/or loading on CpG methylation.

Results: ‘Deplete’ vs ‘Replete’ groups

In non-loaded tibias, DNA methylation was lower at CpG site −2148 (wrt the transcription start site) in the Runx2 promoter (mean difference −3.44%, 95% CI −0.27 to −6.61, P=0.036).

‘Deplete’ group only

In loaded (vs non-loaded) tibias, DNA methylation was lower at CpG −396 in the Osterix promoter (mean difference −1.54%, 95% CI −0.12 to −2.96, P=0.037), and at CpG site −503 in the RXRA promoter (mean difference −4.75%, 95% CI −8.80 to 0.69, P=0.026).

‘Replete’ group only

There was no change in RXRA methylation status at CpG site −503 in the early life vitamin D ‘replete’ group with loading.

Discussion: These data suggest Vitamin D deficiency induces persistent epigenetic changes at specific gene loci within RUNX2 and Osterix in the offspring. Mechanical loading induced very dynamic changes in DNA methylation within RXRA. Epigenetic mechanisms may contribute to early life nutritional programming of bone mass and the response to mechanical loading.

Disclosure: NJB consults for Alexion, Mereo, UCB and Amgen, and receives grant support for clinical studies from Alexion and Amgen.