Epigenetic mechanisms linking maternal pre-conceptional micronutrient supplementation with offspring health in India and The Gambia

Deficiencies of essential vitamins and minerals (micronutrients) due to poor quality diets are a major public health problem in low-and-middle-income countries (LMICs). In pregnant women they impair fetal development and cause childhood stunting. Recent research pioneered in the UK suggests that they cause long-term problems in the offspring including poor brain and muscle development, increased body fat, and adult heart disease and diabetes. These chronic diseases are rising rapidly in LMICs, where they disable working-age people and require costly treatment. Efforts to prevent them currently focus on promoting healthier adult lifestyles. Research into the developmental origins of health and disease (DOHaD) opens up the potential to both improve child development and prevent adult disease by optimising maternal nutrition.

Until recently, evidence for long-term effects of maternal undernutrition was based on observational studies. The research groups in this application have made a step-change by conducting pre- and peri-conceptional randomised controlled nutrition trials among undernourished mothers in India and The Gambia. These trials provide ideal vehicles to study epigenetic mechanisms by which the 'memory' of an individual's nutritional environment in utero could be retained into later life and influence health. One such mechanism is DNA methylation in which the sequence of genes in DNA is overlaid with chemical (methyl) groups which control whether genes are switched on or off. Methylation patterns ('epigenetic signatures') are laid down in early fetal life and are influenced by the mother's nutritional state. In experimental animals, altering maternal diet peri-conceptionally permanently changes offspring DNA methylation, gene expression and adult metabolism. There is growing indirect evidence of similar effects in humans but there are as yet no comparable studies from specific peri-conceptional nutritional interventions.

The UK, Indian and Gambian groups have already built strong evidence in observational work that maternal deficiencies of 'methyl donor' nutrients such as vitamins B2, B6, B12 and folate impair fetal growth and the child's brain function, increase heart disease and diabetes risk markers, and disrupt the methylation of specific genes. The proposed work will build on these observations using a gold-standard study design. We will analyse DNA methylation in children from two complementary randomised controlled trials in which mothers were supplemented with micronutrients before conception and into early pregnancy. These are: 1) the Mumbai Maternal Nutrition Project, in which women living in Mumbai slums received a daily snack made from micronutrient-rich foods and 2) the Peri-conceptional Multiple Micronutrient Supplementation Trial in rural Gambia, which supplemented women with multiple micronutrient capsules. Fetal growth and newborn size were recorded and the children are being tracked.

The UK-India group is funded by MRC for 3 years to study body composition, risk factors for later heart disease and diabetes, and brain function in the Mumbai children at the age of 5-6 years. DNA is being collected from blood samples and cheek swabs. Children from the Gambian trial have been re-traced at the age of 7-8 years and we will collect the same outcome data. This proposal seeks funds to study DNA methylation in both cohorts of children (Mumbai N=1100, The Gambia N=350). Genome-wide methylation patterns will be compared between intervention groups and between cohorts, and related to the children's body fat, muscle and bone, disease risk factors, and brain function, as well as birth outcomes and stunting. An over-arching objective is to build capacity for epigenetic studies in India and The Gambia. The project will lead to a better understanding of the effects of maternal nutrition on the child, and ultimately to the design of improved nutritional interventions for mothers preparing for pregnancy.

Animal and in vitro studies show that the nutrient milieu of the embryo affects the establishment of epigenetic signatures regulating fetal development, providing a plausible mechanism to explain the developmental origins of health and disease (DOHaD). We have preliminary evidence of such effects in humans and now propose to create an India/Gambia/UK partnership to extend this work by studying children of mothers randomised to receive pre- and peri-conceptional micronutrient supplements or placebo. We will analyse DNA methylation in 5-8 year old children whose mothers took part in the Mumbai Maternal Nutrition Project in India (MMNP; ISRCTN 62811278; n=1100 children) and the Peri-conceptional Multiple Micronutrient Supplementation Trial in rural Gambia (PMMST; ISRCTN 13687662; n=350) to identify differences between intervention and control groups. In an initial discovery phase we will measure genome-wide methylation using the Illumina Human Infinium 450K bead chip employing screens for differentially methylated positions (DMPs), regions (DMRs), and variably methylated regions (VMRs) with validation of top hits by bisulfite pyrosequencing. Candidate loci not present on the 450K array will also be assayed by pyrosequencing. We hope to confirm that maternal micronutrient supplementation reduces disrupted methylation of key genes. We will further correlate differences in methylation with health-related outcomes in the children (body composition, cardiometabolic risk markers and cognitive ability). The project will build capacity for next-generation epigenetic research in India and sub-Saharan Africa. It offers a highly efficient means to explore fundamental questions of human developmental biology. If we show that improving a mother's peri-conceptional diet by micronutrient supplementation minimises epigenetic disruption and improves metabolic health in the child, with potential lifelong benefit, our findings will offer a scalable pathway to impact in mothers worldwide.

The possibility that adjusting a mother's nutrient status before she conceives influences the lifelong health of her children has profound implications that could be of immense importance. Pre-conceptional folic acid supplementation is proven to reduce the incidence of neural tube defects (NTDs) and has been elevated to national policy in many countries worldwide including mandatory food fortification in some countries. By demonstrating that maternal nutrition leaves a lasting 'memory' in the DNA of the offspring in ways that influence their health, human capital and risk of adult chronic disease, this study would have a far-reaching impact. It would suggest that optimising the benefits of pre-pregnancy interventions would require nutrients additional to folic acid and that the possible beneficial outcomes could go well beyond prevention of NTDs. The project therefore has the potential to benefit all mothers, children, and the wider public. This information will benefit global and national agencies responsible for improving maternal and child health and preventing chronic disease by a) demonstrating the importance of maternal nutrition for the health of the offspring and b) identifying the effectiveness of specific scalable nutritional interventions. The research will also build capacity for cutting-edge epigenetic research in the UK, India and The Gambia, especially in the field of bioinformatics, a specialty for which there is currently very limited expertise in low-income countries. It will foster the competitiveness of UK, Indian and Gambian science and research by exploiting the fact that the research groups participating in this project currently lead the world in having conducted randomised nutrition trials covering the peri-conceptional period and in following up the children to study long-term effects. We therefore expect the project to lead to exceptionally high-impact publications.