The effect of prenatal vitamin D supplementation on DNA methylation: Investigation into biomarkers and causal mechanisms of later bone health

Abstract

Osteoporosis is a systemic skeletal disease which affects the ageing population and results in an increased risk of developing fractures, for example at the wrist, hip and spine. Peak bone mass in early adulthood has been shown to predict the risk of developing osteoporosis in later life and studies have shown that the early life environment might influence the bone health trajectory, through epigenetic processes such as DNA methylation. DNA methylation can be measured in suitable candidate genes to potentially act as a biomarker for predicting bone health in later life, and to guide interventions aimed at improving the accrual of bone mass during growth. Studies within the SWS cohort have identified two candidate biomarkers, RXRA and CDKN2A, of which DNA methylation has been shown to be associated with bone measures in later childhood.

Whether DNA methylation is causally involved in bone outcomes is unknown therefore, randomised controlled trials provide an opportunity to investigate these associations further and to determine the possibility of a causal relationship. Within this thesis, DNA methylation of four genes, RXRA, CDKN2A, Osterix and Runx2, were measured within the MAVIDOS trial, a randomised, controlled, double blind study where pregnant mothers were recruited and received either 1000 IU/d cholecalciferol or placebo daily from 14 weeks gestation until delivery. Infants born during the winter months to cholecalciferol supplemented mothers had greater bone measures at birth compared to the placebo group. The results showed that infants born to cholecalciferol supplemented mothers had lower methylation of RXRA and Osterix CpG loci and higher methylation at CDKN2A CpG loci. Furthermore, DNA methylation of Osterix and Runx2 was positively associated with bone measures at birth. Next, the functional importance of the RXRA CpGs of interest was investigated in osteosarcoma cell lines to provide a mechanistic insight into the interactions between vitamin D and RXRA methylation. The results showed that site directed mutagenesis upstream of the RXRA promoter impaired luciferase expression within osteosarcoma cell lines, both in the presence and absence of vitamin D supplementation. In humans, the measurement of DNA methylation in tissues central to the pathogenesis of osteoporosis is difficult, so DNA methylation of several key genes was measured in adult tibiae from mice exposed to a prenatal vitamin D deficient diet. The results showed that a prenatal vitamin D deficient diet altered tibial DNA methylation and suggests a link between prenatal vitamin D deficiency, mechanical loading and DNA methylation within tibiae.

These observations provide insight into candidate biomarkers of bone health that respond accordingly to a dietary intervention of maternal vitamin D supplementation within the MAVIDOS trial, and provide insight into the mechanisms and functional importance that altered DNA methylation of these genes could have on bone phenotype.

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Identifiers

ORCID for Karen Lillycrop: orcid.org/0000-0001-7350-5489

ORCID for Nicholas Harvey: orcid.org/0000-0002-8194-2512

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

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