2.1 Research strategy. CpGs and SNPs selection and localization
CpGs of the study were selected by setting specific criteria such as their involvement in immune-related responses in FA in a genetic or epigenetic context. Several studies suggest that DNA methylation profile of the FOXP3 , mainly at Treg-specific demethylated region (TSDR) and HLA locus could mediate genetic susceptibility to immune-mediated disorders such as FA. We thus, evaluated and compared the DNA methylation patterns of immune-related genetic areas among children exhibiting IgE-mediated food allergy versus healthy subjects. We further correlated methylation data with genetic variants residing in the same CpG regions and parental habits.
CpG islands and regulatory CpGs residing as discrete sites were retrieved by the UCSC genome browser (https://genome.ucsc.edu/). Single nucleotide polymorphisms (SNPs) across studied genetic areas were also identified. Characteristic MHCI and MHCII genetic loci with CpGs, including predicted transcription factor binding (TF) motifs (JASPAR CORE 2020), are illustrated in Figure 1. Transcription factors’ recognition sites were detected, aiming to observe sequence alterations ascribed to SNPs or to CG biochemical alterations due to methylation.
Methylation profile at Treg-specific demethylated region (TSDR), located on the 2nd conserved non-coding sequence of FOXP3 gene, was assessed in both DNA strands. PCR and sequencing primers designed (Table 2), encompassed well characterized CpGs of TSDR in either direction. The immediate upstream region of HLA-G CpG96 island was subjected to methylation analysis to uncover its potential role in allergy via demethylation. Genotypic analyses for rs1233333 (G/A), located within studied region was also conducted to reveal its effect in DNA methylation spreading.
Finally, CpG41 and CpG73 within exon 2 of the HLA‐DRB1 andHLA-DQB1 genes respectively, were studied. Also, sequencing of a 70bp core sequence revealed a number of SNPs residing within CpG41 (Figure 1), in a variety of combinations. Methylation status of CpG41 co-occurring with unique SNPs in the extended gene locus region have been involved in multiple sclerosis disease development17,18.