Epigenetic events in micronutrient programming during early development

Although the information content of the DNA is determined primarily by its nucleotide sequence, further modifications of the DNA itself or the proteins binding to it are important to determine which genes are turned on and off. These modifications are influenced by environmental factors. One of them, methylation of the DNA molecule is dependent on the supply and metabolism of several vitamins and trace elements, such as folic acid, vitamins B6 and B12 and zinc. If pregnant mice are given food deficient in these factors, DNA methylation in the offspring is altered with resultant changes in gene regulation. A similar mechanism is possible in humans and could account for permanent consequences of micronutrient (that is, vitamin and trace element) deficiencies, however, data supporting this hyposthesis are lacking. Our aim is to study whether the micronutrient supply of the pregnant mother during early human embryonic development influences DNA methylation in the offspring. Of particular interest is the DNA methylation of genes which could potentially effect growth and development of the embryo and placenta. We will take advantage of an ongoing trial run by the MRC Keneba Field Station in the Gambia. This is a double blind, randomised control trial food supplementation containing multiple micronutrients given to women of childbearing age living in rural Gambia. The major endpoints of the study are fetal growth, placental development, weight, length and head circumference of the newborn infant. We will study DNA from children whose mothers received micronutrient supplementation and from those whose mothers received placebo only. Using this design we will study whether micronutrients supplementation has an effect on total methylation of the genome using a simple biochemical assay. We will also use methods that assess methylation at large numbers of specific points throughout the human DNA at the sametime in order to identify genes which are methylated in one group of subjects but not in the other group. The latter method includes the use of microarray (DNA chip) technology that allows for genome-wide methylation analysis in one experiment. We will also investigate DNA methylation of individual 'imprinted' genes. These are particularly interesting candidates for epigenetic modulation as any one of the two copies, either the maternal or the paternal switched on and this is mainly regulated by DNA methylation of sequences called 'imprinting centres'. We will investigate and compare DNA methylation of 9 imprinting centres controlling 8 groups of such imprinted genes. Many of these genes are thought to be important in the control of growth of the unborn child. We will, therefore, assess whether DNA methylation of these 'imprinting centres' is different in individuals with or without maternal micronutrient supplementation. If the trial shows that micronutrient supplementation does indeed improve the growth of the fetus, the weight of the placenta, the size of the newborn then our data could provide supportive evidence that this may be related to altered methylation of these imprinted genes. Evidence in humans that periconceptual variation in nutrients can result in permanent epigenetic changes would have profound effects on the direction of future human developmental biology. It would initiate follow up studies to investigate the consequence of such changes in terms of gene function and could open up new avenues for drug development. For health care professionals and the general public, it would lead to more detailed examination of the importance of maternal diet both before and during pregancy.

Epigenetic regulation of gene expression plays a major role in antenatal development. Epigenetic marks are more sensitive to the environment than the nucleotide sequence; yet epigenetic patterns can be transmitted through generations of cells and even through the germ line. Therefore environmental stimuli may result in permanent phenotypes. Normal DNA methylation is dependent on several micronutrients such as vitamins B6, B12 and folic acid and we aim to explore the hypothesis that variation in micronutrient supply at conception and during pregnancy could lead to changes in genomic DNA methylation. Genomic DNA samples will be analysed from individuals whose mothers received multiple and micro nutrient supplementation or from individuals whose mothers received placebo. These samples are being collected as part of a randomised double blind placebo controlled trial being carried out at the MRC field station in the Gambia. The mothers are recruited from a population which naturally undergoes cyclical patterns of maternal under nutrition and are randomised to receive either nutrient supplements or placebo prior to pregnancy. We will assess and compare total DNA cytosine methylation of the two groups using a cytosine extension assay. We will also use microarray-based methylation detection and subtractive hybridisation to compare CpG methylation profiles at a genome-wide level. In addition we will study 9 imprinting centres (ICRs) regulating gene expression in the 8 human imprinted clusters (11p15.5, 6q24, 7q12, 7q32, 14q32, 15q24, 19q13 and 20q13). Bisulphite conversion and pyrosequencing will be used to quatitatively analyse DNA methylation. DNA methylation in the two groups of individuals will be compared. DNA methylation will also be correlated with fetal and neonatal anthropometric measures as well as placental weight.