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.