Epigenetic events in micronutrient programming during early development

Lead Research Organisation: University of Cambridge
Department Name: Paediatrics

Abstract

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.

Technical Summary

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.

Publications

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Cherney DZ (2012) Urinary markers of renal inflammation in adolescents with Type 1 diabetes mellitus and normoalbuminuria. in Diabetic medicine : a journal of the British Diabetic Association

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Cooper WN (2012) DNA methylation profiling at imprinted loci after periconceptional micronutrient supplementation in humans: results of a pilot randomized controlled trial. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description There is considerable interest in potential impact of early peri-conceptional nutrition on the foetal epigenome and whether this could impact on future childhood and adult disease risk.

Through study of babies born to mothers randomised prior to pregnancy to a micro nutrient supplement or placebo we were able to show that the nutritional exposures did indeed lead to epigenetic changes in cord blood DNA. The differences in the two groups, involved methylation of both imprinted and non-imprinted genes. Some of these changes persisted into childhood, whereas other changes related to early nutritional exposures appeared during childhood. Thus we provide proof of principle that peri-conceptional nutrition does indeed lead to persisting epigenetic changes.

We later expanded these studies to identify epigenetic changes associated with more 'modest' variation in early pregnancy nutrition through study of babies born after foetal growth restraint and gestational diabetes. Interestingly, changes in methylation of genes common to both of these exposures were identified.
Exploitation Route Replication is essential and confirmation that methylation changes observed in cord blood DNA are matched in other tissues (eg buccal smears). Larger studies will be needed and will become possible as the molecular methods come down in costs.
Sectors Healthcare

 
Description Gates Foundation
Amount $623,000 (USD)
Funding ID RG 67603 
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 01/2013 
End 12/2016
 
Title Microarray methylation data (Epigenetic Events study) 
Description The raw data from the genome wide analysis of DNA methylation using the Illumina 27k array in 84 human samples was developed from the study: Epigenetic events in micronutrient programming during early development. 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? Yes  
Impact No known impacts from sharing data 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34257
 
Description Cambridge University -Research News- University Website (BBSRC) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact This research has received a Highlight on the University of Cambridge Research News web site

Dissemination of information about the study.
Year(s) Of Engagement Activity 2012
URL http://www.cam.ac.uk/research/news
 
Description Effects of nutrient supplementation and in utero exposures on newborn epigenetic profiles Pennington Scientific symposium: Nutritional programming and obesity: state-of-the-science, innovation and future directions Symposium Baton Rouge USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Effects of nutrient supplementation and in utero exposures on newborn epigenetic profiles
Pennington Scientific symposium: Nutritional programming and obesity: state-of-the-science, innovation and future directions Symposium Baton Rouge USA
Year(s) Of Engagement Activity 2014
 
Description Poster presentation (Epigenetic Events) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dissemination of information about the study and discussion
Year(s) Of Engagement Activity 2010
 
Description Science Week- University of Cambridge (Wendy Cooper) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact The activity followed after the researcher completed a course in Communicating Science (March 2010 MRC Centre Cambridge). This enabled the researcher to communicate information about the study subject and answer questions from the public.

Communicate information about the study subject and answer questions from the public.
Year(s) Of Engagement Activity 2010
 
Description The Naked Scientists article (BBSRC Epigenetic Events) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Results of the study were discussed

Results of the study were discussed and disseminated widely
Year(s) Of Engagement Activity 2012