Abstract
ABSTRACTPoor intrauterine and childhood growth has been linked with the risk of osteoporosis in later life, a relationship that may in part be mediated through altered epigenetic regulation of genes. We previously identified a region within the promoter of the long non‐coding RNA ANRIL encoded by the CDKN2A locus, at which differential DNA methylation at birth showed correlations with offspring adiposity. Given the common lineage of adipocytes and osteoblasts, we investigated the relationship between perinatal CDKN2A methylation and bone mass at ages 4 and 6 years. Using sodium bisulfite pyrosequencing, we measured the methylation status of the 9 CpGs within this region in umbilical cord samples from discovery (n = 332) and replication (n = 337) cohorts of children from the Southampton Women's Survey, whose bone mass was assessed by dual‐energy X‐ray absorptiomietry (DXA; Hologic Discovery). Inverse associations were found between perinatal CDKN2A methylation and whole‐body minus head bone area (BA), bone mineral content (BMC), and areal bone mineral density (BMD). This was confirmed in replication and combined data sets (all p < 0.01), with each 10% increase in methylation being associated with a decrease in BMC of 4 to 9 g at age 4 years (p ≤ 0.001). Relationships were similar with 6‐year bone mass. Functional investigation of the differentially methylated region in the SaOS‐2 osteosarcoma cell line showed that transcription factors bound to the identified CpGs in a methylation‐specific manner and that CpG mutagenesis modulated ANRIL expression. In conclusion, perinatal methylation at CDKN2A is associated with childhood bone development and has significance for cell function. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Highlights
There is evidence of a substantial heritable component to bone mineral density (BMD),(1,2) there is increasing evidence that interactions between environment and genotype, leading to altered gene expression, may contribute to the overall variance in BMD.[3]
Methylation has been implicated in mechanisms of osteoblastic differentiation[18,19,20] and osteoclastogenesis,(21) together with the transition from osteoblast to osteocyte.[22,23,24,25] These experimental findings are complemented by data from genome-wide methylation profiling studies in older patients, demonstrating differential methylation at genes such as the cyclin-dependent kinase inhibitor CKDN1C and cyclin-dependent kinase CDK20 is associated with BMD.[26,27] These findings, indicate the importance of epigenetic processes in bone metabolism, with regard to loci implicated in cellular differentiation, cell cycle regulation, and bone cell function
Single-nucleotide polymorphisms (SNPs) within the CDKN2A locus, those located within ANRIL, have been associated with cardiovascular disease, diabetes, and frailty,(28) and DNA methylation at this locus has recently been demonstrated to vary with age.[29]. Given our previous demonstration of links between perinatal CDKN2A methylation and offspring fat mass,(16) together with the common mesenchymal origin and well-established functional relationships between fat and bone, mediated via both mechanical and endocrine pathways,(30) we carried out a targeted approach to examine DNA methylation at CpG sites within the CDKN2A gene
Summary
There is evidence of a substantial heritable component to bone mineral density (BMD),(1,2) there is increasing evidence that interactions between environment and genotype, leading to altered gene expression, may contribute to the overall variance in BMD.[3]. Single-nucleotide polymorphisms (SNPs) within the CDKN2A locus, those located within ANRIL, have been associated with cardiovascular disease, diabetes, and frailty,(28) and DNA methylation at this locus has recently been demonstrated to vary with age.[29] Given our previous demonstration of links between perinatal CDKN2A methylation and offspring fat mass,(16) together with the common mesenchymal origin and well-established functional relationships between fat and bone, mediated via both mechanical and endocrine pathways,(30) we carried out a targeted approach to examine DNA methylation at CpG sites within the CDKN2A gene. We carried out functional analysis of the region to determine its importance for local gene expression and transcription factor binding
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