Epigenetic mechanisms linking a mothers’ nutrition to the health of her children in India and Sub-Saharan Africa

The effects of maternal supplementation on the children's DNA methylation:
1. Epigenome wide DNA methylation is measured in the children’s blood DNA samples using Illumina Infinium Methylation EPIC arrays at age 5-7 y (India) and 7-9 y (The Gambia)
2. Locus-specific DNA methylation is measured in blood and buccal DNA samples using pyrosequencing on Pyromark 96 at age 5-7 y (India) and 7-9 y (The Gambia)

The associations of DNA methylation with phenotype in the children:
1. Birth weight and length are measured using digital weighing scales and a neonatal stadiometer respectively, within 10 days (India) or 72 hours (The Gambia) of birth. Smallness for gestational age (SGA) is calculated from measured birth weight using the INTERGROWTH reference.
2. Children’s weight and height are measured using standardised anthropometric methods (body mass index is calculated from these measures) at age 5-7 y (India) and 7-9 y (The Gambia)
3. Children’s total and regional lean mass, fat mass and body fat percent are measured using dual-energy X-ray absorptiometry (DXA, Lunar Prodigy in India, Lunar iDXA in The Gambia) (lean mass index and fat mass index are calculated from these measures) at age 5-7 y (India) and 7-9 y (The Gambia)
4. Children’s fasting plasma glucose concentration, and 30-and 120-minute plasma glucose concentrations after an oral glucose load, are measured using standard enzymic assays by autoanalyser at age 5-7 y (India) and 7-9 y (The Gambia)
5. Children’s fasting and 30-minute plasma insulin concentrations are measured using Mercodia ELISA on a Victor 2 analyser (India) and an SM-chemiluminescence method (The Gambia) at age 5-7 y (India) and 7-9 y (The Gambia). Insulin resistance is calculated from fasting glucose and insulin values using the HOMA-IR Oxford online calculator. Disposition index is calculated from fasting and 30-minute plasma glucose and insulin values using standard formulae.
6. Children’s resting systolic blood pressures is measured using an OMRON automated blood pressure device, after at least 5 minutes seated at rest, at age 5-7 y (India) and 7-9 y (The Gambia)
7. Children’s fasting plasma or serum total, LDL- and HDL-cholesterol, and triglycerides are measured using standard Enzymic assays on an autoanalyser at age 5-7 y (India) and 7-9 y (The Gambia)
8. Children’s cognitive ability is assessed using the raw and combined scores from cognitive tests from the Kaufmann Assessment Battery for Children (Atlantis, pattern reasoning and word order) and from the Wechsler Intelligence Scale for Children (verbal fluency, Kohs block design and coding) at age 5-7 y (India) and 7-9 y (The Gambia)
9. Children’s bone mineral content, bone area and bone mineral apparent density are measured using DXA at age 5-7 y (India) and 7-9 y (The Gambia)

The associations of DNA methylation with phenotype in the children:
1. Newborn head, chest, abdominal and mid-upper-arm circumferences are measured using anthropometric tape within 10 days (India) or 72 hours (The Gambia) of birth
2. Newborn triceps and subscapular skinfolds are measured using Holtain skinfold calipers within 10 days (India) or 72 hours (The Gambia) of birth
3. Newborn gestational age at birth was derived from the mother’s last menstrual period (LMP) date and early pregnancy fetal ultrasound measurements, both recorded prospectively by the research team. The number and percentage of pre-term newborns (<37 weeks completed gestation) are derived using gestational age.
4. Newborn low birth weight (<2500g) is derived from measured birth weight
5. Children’s sitting height, leg length, head circumference, mid-upper arm circumference, chest circumference, waist circumference, hip circumference, and skinfolds are measured using standardised anthropometric methods at age 5-7 y (India) and 7-9 y (The Gambia). Sitting height/leg length ratio, sum of skinfolds and waist/hip ratio are calculated from these measures. Longitudinal indices of growth are derived from these measurements and weight, height and body mass index, using conditional and other modelling techniques.
6. The number and percentage of children stunted, wasted, and underweight are derived from measured weight and height at age 5-7 y (India) and 7-9 y (The Gambia) using the World Health Organization/ Centers for Disease Control child growth reference (www.who.int/childgrowth/en/)
7. Children’s android and gynoid fat mass are measured using DXA at age 5-7 y (India) and 7-9 y (The Gambia)
8. Children’s resting diastolic blood pressures is measured using an OMRON automated blood pressure device, after at least 5 minutes seated at rest at age 5-7 y (India) and 7-9 y (The Gambia)
9. Children’s plasma insulin concentration 30-minutes after an oral glucose load is measured using Mercodia ELISA on a Victor 2 analyser (India) and an SM-chemiluminescence method (The Gambia) at age 5-7 y (India) and 7-9 y (The Gambia). Insulinogenic index is calculated from fasting and 30-minute plasma insulin and glucose values using a standard formula.
10. Metabolic Syndrome is derived from fat mass (DXA), blood pressures, and plasma glucose, insulin, HDL-cholesterol and triglyceride measurements at age 5-7 y (India) and 7-9 y (The Gambia) using values above the age-, sex-, and site-specific upper quartiles for each
11. A category of ‘high blood pressure’ is derived using the systolic and diastolic blood pressure values and reference data at age 5-7 y (India) and 7-9 y (The Gambia)
12. Children’s tibial total and trabecular volumetric bone density and bone area, and diaphyseal bone area, cortical area, thickness, bone mineral content, cortical volumetric bone mineral density and strength (cross-sectional moment of inertia) are measured (in the Gambian children only) using pQCT (Peripheral quantitative computed tomography) at age 7-9 y