Background

We recently reported a positive association in ALSPAC between background levels of UVB in the third trimester of pregnancy, derived from meteorological office records, and subsequent bone development of the child based on DXA scans at age nine (1). UVB exposure as analysed in this way provides a measure of vitamin D status in utero (2), which is in turn thought to contribute to programming of development of several systems within the off-spring including the skeleton. Although the mechanisms which mediate these effects are unknown, one possibility is that these involve altered methylation status of as yet unidentified target genes, by virtue of effects of vitamin D on the activity of enzymes involved in DNA methylation. Following recent methodological advances which enable methylation status to be evaluated at multiple sites across the genome (3), proof of concept studies intended to examine the role of methylation are now feasible.

Hypothesis

Our overall hypothesis is that exposure to low levels of vitamin D in utero has adverse effects on subsequent skeletal development as a consequence of altered methylation of key regulatory genes. The present proposal aims to address one component of this hypothesis, namely that vitamin D exposure in utero affects DNA methylation at multiple sites.

Objectives/Design

1. Approximately 50 DNA sites showing the greatest variability in methylation status will be identified in collaboration with Caroline Relton (University of Newcastle), based on methylation status recently measured at 1500 sites (covering 800 genes) in cord blood samples from 174 ALSPAC children.

2. The genes represented by these sites will be classified according to what biological pathway or process they are likely to affect.

3. Relationships will be analysed between maternal UVB and extent of methylation for each of the 50 methylation sites in turn.

Techniques/approaches

Since last trimester UVB exposure and DNA methylation are both continuous variables, analyses will initially involve linear regression. Further analyses will be performed in which UVB exposure is treated as a dichotomous variable, eg by examining differences in methylation status between those in the upper and lower quartile of UVB (equivalent to birth in late summer and early spring respectively). Additional analyses may also be possible between DNA methylation and maternal vitamin D status in the last trimester in ALSPAC, based on direct measurement of vitamin D which is currently in progress. Whether relationships between maternal vitamin D status and DNA methylation vary according to biological pathway will be addressed by comparing these associations between different categories of genes. Positive findings will subsequently be replicated by repeating these analyses in a cohort from North Cumbria where equivalent information on cord blood DNA methylation and birthdate is available from approximately 200 off spring.

References

1. Sayers A, Tobias JH 2009 Estimated maternal ultraviolet B exposure levels in pregnancy influence skeletal development of the child. J Clin Endocrinol Metab 94:765-771

2. Sayers A, Tilling K, Boucher BJ, Noonan K, Tobias JH 2009 Predicting ambient ultraviolet from routine meteorological data; its potential use as an instrumental variable for vitamin D status in pregnancy in a longitudinal birth cohort in the UK. Int J Epidemiol

3. Kaminsky ZA, Tang T, Wang SC, Ptak C, Oh GH, Wong AH, Feldcamp LA, Virtanen C, Halfvarson J, Tysk C, McRae AF, Visscher PM, Montgomery GW, Gottesman, II, Martin NG, Petronis A 2009 DNA methylation profiles in monozygotic and dizygotic twins. Nat Genet 41:240-245.