3.1.1. FOXP3 TSDR methylation profile
CpG sites within TSDR of FOXP3 gene were analysed at both DNA directions (forward and reverse DNA strands) and revealed a clear deviation of methylation pattern between them. Among FA and control samples, FOXP3 TSDR CpGs showed almost 100% methylation across forward strand and a variable demethylation rate in the reverse strand (Figure 2A), indicating that only reverse DNA strand methylation pattern accounts for the gene’s transcriptional regulation.
FOXP3 gene is located on human chromosome X (Xp11.23) and consequently, epigenetic discrepancies between different sexes may arise from the inactivation process of X chromosome in females. To avoid artifacts during methylation data analysis, male and female donors (both controls and food allergic children) were analysed independently, however insignificant gender differences were observed in methylation profiles (data not shown).
Subsequent analysis revealed marginal variations (Figure 2B) in food allergic children compared to controls. Also, the average methylation level of all TSDR CpG sites tested was not statistically significant between food allergic children and control groups, providing poor evidence for the TSDR demethylation.