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Evaluation of Methylation Status of the eNOS Promoter at Birth in Relation to Childhood Bone Mineral Content

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Abstract

Our previous work has shown associations between childhood adiposity and perinatal methylation status of several genes in umbilical cord tissue, including endothelial nitric oxide synthase (eNOS). There is increasing evidence that eNOS is important in bone metabolism; we therefore related the methylation status of the eNOS gene promoter in stored umbilical cord to childhood bone size and density in a group of 9-year-old children. We used Sequenom MassARRAY to assess the methylation status of two CpGs in the eNOS promoter, identified from our previous study, in stored umbilical cords of 66 children who formed part of a Southampton birth cohort and who had measurements of bone size and density at age 9 years (Lunar DPXL DXA instrument). Percentage methylation varied greatly between subjects. For one of the two CpGs, eNOS chr7:150315553 + , after taking account of age and sex, there were strong positive associations between methylation status and the child’s whole-body bone area (r = 0.28, P = 0.02), bone mineral content (r = 0.34, P = 0.005), and areal bone mineral density (r = 0.34, P = 0.005) at age 9 years. These associations were independent of previously documented maternal determinants of offspring bone mass. Our findings suggest an association between methylation status at birth of a specific CpG within the eNOS promoter and bone mineral content in childhood. This supports a role for eNOS in bone growth and metabolism and implies that its contribution may at least in part occur during early skeletal development.

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References

  1. Harvey N, Dennison E, Cooper C (2010) Osteoporosis: impact on health and economics. Nat Rev Rheumatol 6:99–105

    Article  PubMed  Google Scholar 

  2. Harvey N, Cooper C (2004) The developmental origins of osteoporotic fracture. J Br Menopause Soc 10:14–15 29

    PubMed  Google Scholar 

  3. Rivadeneira F, Styrkarsdottir U, Estrada K, Halldorsson BV, Hsu YH, Richards JB et al (2009) Twenty bone-mineral-density loci identified by large-scale meta-analysis of genome-wide association studies. Nat Genet 41:1199–1206

    Article  PubMed  CAS  Google Scholar 

  4. Richards JB, Kavvoura FK, Rivadeneira F, Styrkarsdottir U, Estrada K, Halldorsson BV et al (2009) Collaborative meta-analysis: associations of 150 candidate genes with osteoporosis and osteoporotic fracture. Ann Intern Med 151:528–537

    PubMed  Google Scholar 

  5. Lanham SA, Roberts C, Perry MJ, Cooper C, Oreffo RO (2008) Intrauterine programming of bone. Part 2: alteration of skeletal structure. Osteoporos Int 19:157–167

    Article  PubMed  CAS  Google Scholar 

  6. Lanham SA, Roberts C, Cooper C, Oreffo RO (2008) Intrauterine programming of bone. Part 1: alteration of the osteogenic environment. Osteoporos Int 19:147–156

    Article  PubMed  CAS  Google Scholar 

  7. Lillycrop KA, Slater-Jefferies JL, Hanson MA, Godfrey KM, Jackson AA, Burdge GC (2007) Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications. Br J Nutr 97:1064–1073

    Article  PubMed  CAS  Google Scholar 

  8. Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA, Burdge GC (2008) Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring. Br J Nutr 100:278–282

    Article  PubMed  CAS  Google Scholar 

  9. Gluckman PD, Hanson MA, Cooper C, Thornburg KL (2008) Effect of in utero and early-life conditions on adult health and disease. N Engl J Med 359:61–73

    Article  PubMed  CAS  Google Scholar 

  10. Godfrey KM, Sheppard A, Gluckman PD, Lillycrop KA, Burdge GC, McLean C et al (2011) Epigenetic gene promoter methylation at birth is associated with child’s later adiposity. Diabetes 60:1528–1534

    Article  PubMed  CAS  Google Scholar 

  11. Zaman G, Pitsillides AA, Rawlinson SC, Suswillo RF, Mosley JR, Cheng MZ et al (1999) Mechanical strain stimulates nitric oxide production by rapid activation of endothelial nitric oxide synthase in osteocytes. J Bone Miner Res 14:1123–1131

    Article  PubMed  CAS  Google Scholar 

  12. Sabanai K, Tsutsui M, Sakai A, Hirasawa H, Tanaka S, Nakamura E et al (2008) Genetic disruption of all NO synthase isoforms enhances BMD and bone turnover in mice in vivo: involvement of the renin—angiotensin system. J Bone Miner Res 23:633–643

    Article  PubMed  CAS  Google Scholar 

  13. Nilforoushan D, Gramoun A, Glogauer M, Manolson MF (2009) Nitric oxide enhances osteoclastogenesis possibly by mediating cell fusion. Nitric Oxide 21:27–36

    Article  PubMed  CAS  Google Scholar 

  14. Jamal SA, Browner WS, Bauer DC, Cummings SR (1998) Intermittent use of nitrates increases bone mineral density: the study of osteoporotic fractures. J Bone Miner Res 13:1755–1759

    Article  PubMed  CAS  Google Scholar 

  15. Godfrey K, Robinson S, Barker DJ, Osmond C, Cox V (1996) Maternal nutrition in early and late pregnancy in relation to placental and fetal growth. BMJ 312:410–414

    Article  PubMed  CAS  Google Scholar 

  16. Gale CR, Javaid MK, Robinson SM, Law CM, Godfrey KM, Cooper C (2007) Maternal size in pregnancy and body composition in children. J Clin Endocrinol Metab 92:3904–3911

    Article  PubMed  CAS  Google Scholar 

  17. Gale CR, Jiang B, Robinson SM, Godfrey KM, Law CM, Martyn CN (2006) Maternal diet during pregnancy and carotid intima-media thickness in children. Arterioscler Thromb Vasc Biol 26:1877–1882

    Article  PubMed  CAS  Google Scholar 

  18. Ehrich M, Nelson MR, Stanssens P, Zabeau M, Liloglou T, Xinarianos G et al (2005) Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc Natl Acad Sci USA 102:15785–15790

    Article  PubMed  CAS  Google Scholar 

  19. Li LC, Dahiya R (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18:1427–1431

    Article  PubMed  CAS  Google Scholar 

  20. Godfrey K, Walker-Bone K, Robinson S, Taylor P, Shore S, Wheeler T et al (2001) Neonatal bone mass: influence of parental birthweight, maternal smoking, body composition, and activity during pregnancy. J Bone Miner Res 16:1694–1703

    Article  PubMed  CAS  Google Scholar 

  21. Harvey NC, Javaid MK, Arden NK, Poole JR, Crozier SR, Robinson SM et al (2010) Maternal predictors of neonatal bone size and geometry: the Southampton Women’s Survey. J Dev Origins Health Dis 1:35–41

    Article  Google Scholar 

  22. Talens RP, Boomsma DI, Tobi EW, Kremer D, Jukema JW, Willemsen G et al (2010) Variation, patterns, and temporal stability of DNA methylation: considerations for epigenetic epidemiology. FASEB J 24:3135–3144

    Article  PubMed  CAS  Google Scholar 

  23. Kranz AL, Eils R, Konig R (2011) Enhancers regulate progression of development in mammalian cells. Nucleic Acids Res 39:8689–8702

    Article  PubMed  CAS  Google Scholar 

  24. Park JH, Stoffers DA, Nicholls RD, Simmons RA (2008) Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1. J Clin Invest 118:2316–2324

    Article  PubMed  CAS  Google Scholar 

  25. Champagne FA, Weaver IC, Diorio J, Dymov S, Szyf M, Meaney MJ (2006) Maternal care associated with methylation of the estrogen receptor-alpha 1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring. Endocrinology 147:2909–2915

    Article  PubMed  CAS  Google Scholar 

  26. Brunton JA, Weiler HA, Atkinson SA (1997) Improvement in the accuracy of dual energy X-ray absorptiometry for whole body and regional analysis of body composition: validation using piglets and methodologic considerations in infants. Pediatr Res 41:590–596

    Article  PubMed  CAS  Google Scholar 

  27. Chrusciel M, Andronowska A, Postek A (2009) Expression patterns of endothelial and inducible nitric oxide isoforms in the porcine umbilical cord. Reprod Domest Anim 44:621–630

    Article  PubMed  CAS  Google Scholar 

  28. Hracsko Z, Hermesz E, Ferencz A, Orvos H, Novak Z, Pal A et al (2009) Endothelial nitric oxide synthase is up-regulated in the umbilical cord in pregnancies complicated with intrauterine growth retardation. In Vivo 23:727–732

    PubMed  CAS  Google Scholar 

  29. Zhang MX, Zhang C, Shen YH, Wang J, Li XN, Chen L et al (2008) Effect of 27nt small RNA on endothelial nitric-oxide synthase expression. Mol Biol Cell 19:3997–4005

    Article  PubMed  CAS  Google Scholar 

  30. Fish JE, Matouk CC, Rachlis A, Lin S, Tai SC, D’Abreo C et al (2005) The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code. J Biol Chem 280:24824–24838

    Article  PubMed  CAS  Google Scholar 

  31. Chan Y, Fish JE, D’Abreo C, Lin S, Robb GB, Teichert AM et al (2004) The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation. J Biol Chem 279:35087–35100

    Article  PubMed  CAS  Google Scholar 

  32. Davis TR, Wood MB (1992) Endothelial control of long bone vascular resistance. J Orthop Res 10:344–349

    Article  PubMed  CAS  Google Scholar 

  33. Yashiro Y, Ohhashi T (1997) Flow- and agonist-mediated nitric oxide- and prostaglandin-dependent dilation in spinal arteries. Am J Physiol Heart Circ Physiol 273:H2217–H2223

    CAS  Google Scholar 

  34. Coessens BC, Miller VM, Wood MB (1996) Endothelin induces vasoconstriction in the bone vasculature in vitro: an effect mediated by a single receptor population. J Orthop Res 14:611–617

    Article  PubMed  CAS  Google Scholar 

  35. Marsh N, Marsh A (2000) A short history of nitroglycerine and nitric oxide in pharmacology and physiology. Clin Exp Pharmacol Physiol 27:313–319

    Article  PubMed  CAS  Google Scholar 

  36. Burdan F, Szumilo J, Korobowicz A, Farooquee R, Patel S, Patel A et al (2009) Morphology and physiology of the epiphyseal growth plate. Folia Histochem Cytobiol 47:5–16

    Article  PubMed  Google Scholar 

  37. Teixeira CC, Ischiropoulos H, Leboy PS, Adams SL, Shapiro IM (2005) Nitric oxide–nitric oxide synthase regulates key maturational events during chondrocyte terminal differentiation. Bone 37:37–45

    Article  PubMed  CAS  Google Scholar 

  38. Fish JE, Marsden PA (2006) Endothelial nitric oxide synthase: insight into cell-specific gene regulation in the vascular endothelium. Cell Mol Life Sci 63:144–162

    Article  PubMed  CAS  Google Scholar 

  39. Prisby RD, Ramsey MW, Behnke BJ, Dominguez JM, Donato AJ, Allen MR et al (2007) Aging reduces skeletal blood flow, endothelium-dependent vasodilation, and NO bioavailability in rats. J Bone Miner Res 22:1280–1288

    Article  PubMed  CAS  Google Scholar 

  40. Marshall D, Johnell O, Wedel H (1996) Meta-analysis of how well measures of bone mineral density predict occurrence of osteoporotic fractures. BMJ 312:1254–1259

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

We thank the mothers and their children who gave us their time, and a team of dedicated research nurses and ancillary staff for their assistance. This work was supported by grants from the Medical Research Council; British Heart Foundation; Arthritis Research Campaign; National Osteoporosis Society; International Osteoporosis Foundation; Cohen Trust; Southampton NIHR Biomedical Research Unit in Nutrition, Diet and Lifestyle; and NIHR Musculoskeletal Biomedical Research Unit, University of Oxford. Participants were drawn from a cohort study funded by the Medical Research Council and the Dunhill Medical Trust. We thank Ms. G. Strange for helping to prepare the manuscript.

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Authors and Affiliations

  1. MRC Lifecourse Epidemiology Unit, University of Southampton, Southampton, UK

    Nicholas C. Harvey, Sarah Crozier, Hazel Inskip, Catharine R. Gale, Keith Godfrey & Cyrus Cooper

  2. Southampton Institute of Developmental Sciences, University of Southampton, Southampton, UK

    Karen A. Lillycrop, Emma Garratt, Graham Burdge, Jo Slater-Jefferies, Joanne Rodford, Mark Hanson & Keith Godfrey

  3. Liggins Institute, University of Auckland, Auckland, New Zealand

    Allan Sheppard, Cameron McLean & Peter Gluckman

  4. AgResearch, Ruakura Research Centre, Hamilton, New Zealand

    Allan Sheppard & Cameron McLean

  5. Singapore Institute for Clinical Sciences, Singapore, Singapore

    Peter Gluckman

  6. Southampton NIHR Biomedical Research Unit in Nutrition, Diet and Lifestyle, University of Southampton, Southampton, UK

    Keith Godfrey

  7. Department of Anatomy, Faculty of Medicine and Health Sciences, UAE University, Al ain, United Arab Emirates

    Bright Starling Emerald

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  1. Nicholas C. Harvey
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Correspondence to Cyrus Cooper.

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Nicholas C. Harvey and Karen A. Lillycrop are joint first author.

The authors have stated that they have no conflict of interest.

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Harvey, N.C., Lillycrop, K.A., Garratt, E. et al. Evaluation of Methylation Status of the eNOS Promoter at Birth in Relation to Childhood Bone Mineral Content. Calcif Tissue Int 90, 120–127 (2012). https://doi.org/10.1007/s00223-011-9554-5

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