Figure legends
FIGURE 1: HLA-DRB1 and HLA-G genes’ chromosomal location, transcripts (dark blue), protein domains (light blue) and CpG islands (green boxes) from GENCODE v.38 (exons are presented as boxes and introns as lines, arrows indicate the 5’-3’ direction). Analyzed genomic areas with CpGs are displayed as purple arrows. Different colors correlate with transcription factors’ binding sites, potentially affected from methylation status of studied CpGs. SNPs and their location across the analyzed regions are displayed as red arrows. rs17878951 is involved in the creation of a new CpG site, while rs75044270 is involved in the deletion of a CpG site withinHLA-DRB1 region. Sequencing primers of each genetic region are also presented.
FIGURE 2: Methylation levels of FOXP3 TSDR and HLA-G promoter. (A) Methylation pattern of reverse (R5) and forward (F5) DNA strand of FOXP3 TSDR among control and FA sample group. Differential methylation profiles were observed between the two DNA strands. (B, C) Methylation status of specific CpG sites lying withinFOXP3 TSDR and HLA-G promoter and total values from all CpGs studied across each region. HLA-G promoter’s CpG site 2 showed statistically significant differences between control and food allergy group (**p=0.004). (D) Effect of rs1233333 on DNA methylation levels of HLA-G promoter region. Homozygous genotype AA showed decreased methylation compared to homozygous GG. Data are presented as mean ± SE. CpG sites across studied region are numbered relative to 5’-3’ direction.
* Indicates p value <0.05, **indicates p value < 0.01
FIGURE 3 : DNA methylation levels of food allergic children and non-allergic controls with and without parental smoking history. (A) Parental smoking habit significantly influenced methylation levels of CpG site 1 (p=0.004), CpG site 5 (p=0.004), CpG site 6 (p=0.011) και CpG site 9 (p=0.023) of FOXP3 TSDR regardless of FA or healthy status. (B) Parental smoking habit in combination with FA significantly influenced methylation levels of specific CpG sites in HLA-Gpromoter: CpG site 3 (p<0.001), CpG site 4 (p=0.028), CpG site 5 (p=0.009), CpG site 6 (p=0.002), CpG site 7 (p=0.036) and whole tested genetic region (p=0.004).
FIGURE 4 : Methylation levels of a 70bp highly polymorphic region within CpG41 of HLA-DRB1 gene. Absence of SNPs is accompanied with complete demethylation (<5%), while the presence of SNPs causes simultaneous high methylation levels (>70%). Data are presented as mean ± SE.