UC Berkeley

Epigenetic control of gene expression in children remains poorly understood, but new technologies can help elucidate the relationship between expression and DNA methylation. Here, we utilized the nCounter Analysis System to characterise the expression of 60 genes in 69 9-year-old children from a cohort with a high prevalence of obesity. nCounter expression levels ranged broadly (from 3 to over 10000 messenger RNA counts) and were divided into four categories: high (>2000 counts), moderate (200-1000 counts), low (100-200 counts) and marginal (<100 counts). For a subset of five genes (ADIPOR1, PPARG1, GSTM1, PON1 and ACACA) from different expression level categories, we validated nCounter data using reverse transcription-polymerase chain reaction (RT-PCR), and expanded RT-PCR analysis of ADIPOR1 to include 180 children. Expression data from the two methodologies were correlated for all five genes included in the validation experiment, with estimates ranging from r s = 0.26 (P = 0.02) to r s = 0.88 (P < 5×10(-6)). ADIPOR1 and PPARG1 nCounter expression levels were negatively correlated (r = -0.60, P < 5×10(-5)), and this relationship was stronger in overweight children (r = -0.73, P < 5×10(-5)) than in normal weight children (r = -0.42, P = 0.016). Using methylation data from the Infinium HumanMethylation450 BeadChip (n = 180), we found eight CpG sites in ADIPOR1 and PPARG where methylation level was associated with expression by RT-PCR (P < 0.05). Hypomethylation of PPARG gene body site cg10499651 was associated with increased expression as measured by both RT-PCR and nCounter (P < 0.05). We found no statistically significant relationships between either expression or methylation of ADIPOR1 and PPARG and body mass index or waist circumference. In addition to demonstrating the validity of expression data derived from nCounter, our results illustrate the use of new technologies in assessing epigenetic effects on expression in children.