3.2.1. Epigenetic variations influenced by genetic polymorphisms and parents’ smoking habit
We explored the potential moderating role of rs1233333, located withinHLA-G promoter region (Figure 1), on DNA methylation. Genotyping food allergic and control samples indicated only AA and GG homozygotes and statistical analyses performed uncovered a significant association between rs1233333 (A/A) genotype and the lower levels of methylation at CpGs covering the HLA-G promoter region. More specifically, CpG sites 3, 4, 5 and 6, as well as average methylation of the total analysed region were found to differ with statistical significance (p=0.014, p=0.002, p=0.010, p=0.004 and p=0.010 respectively) between homozygous AA and homozygous GG individuals regardless of food allergic or control status (Figure 2D). Furthermore, food allergic children exhibited 2,5 fold elevated frequency in AA genotype compared to control group, implying a contribution of AA genotype both in DNA methylation status and FA development.
Differences in FOXP3 TSDR and HLA-G methylation profiles based on parents’ smoking habit among FA and control groups were also evaluated. A two-way ANOVA analysis was conducted to examine the co-effect of FA and smoking on methylation levels of FOXP3 TSDR. Smoking phenotype was significantly associated with higher methylation at CpG sites 1, 5, 6 and 9, both in control and food allergic groups (p=0.004, p=0.004, p=0.011 and p=0.023 respectively) (Figure 3A), reflecting previously documented conclusions that an exposure-response epigenetic effect appears in the offspring for many smoking-associated DMPs (Differentially Methylated Positions), which increase the risk for allergic reactions development22.
Additionally, a two-way ANOVA analysis was conducted for the co-effect of FA and smoking on methylation profile of HLA-G promoter. Analysis indicated that food allergic children with smoking parents had significantly lower methylation levels than controls at CpG site 3 (F=6.28 df=1, 33 p<0.001), CpG site 4 (F=5.268, df=1, 33 p=0.028), CpG site 5 (F=7.752, df=1, 33 p=0.009), CpG site 6 (F=11.888 df=1, 33 p=0002), CpG site 7 (F=7.773, df=1, 33 p=0.036) and throughout the whole CpG region tested (F=9.597, df=1, 33 p=0.004) (Figure 3B).
Association of breastfeeding with methylation was not possible due to the small sample size of children that were not breastfed.
3.2.2. Methylation status ofHLA-DRB1 CpG41 and co-existence of genetic variants emerge as risk factors for FA development
HLA-DRB1 and HLA-DQB1 (encoding for the β-chains) belong to MHC class II, are closely linked within the genetic loci, share common regulatory features in their promoter regions23 and common CpG islands within exon 218. Also, genetic variants within HLA-DQB1 and HLA-DRB1 sequences have strongly been associated with predisposition to FA15,16, but the linkage between DNA methylation and single-nucleotide polymorphisms (SNPs) across these loci has not yet been elucidated. CpG73 and CpG41 also include DNAse hypersensitive sites, indicating essential trans-acting factor occupancy.
Methylation analyses of CpG73 within exon 2 of the HLA-DQB1 gene clearly showed hypomethylation of the region in both study groups (data not shown), implying a nonculpable epigenetic frame during FA development. In contrast, methylation profile of HLA-DRB1 CpG41 showed highly variable patterns and a strong correlation of hypermethylation and coexistence of sequence polymorphisms. Sequencing of a 70bp hot polymorphic region within CpG41 was preceded to methylation analysis. 25.6 % of controls and 16.9% of food allergic samples were not polymorphic. Polymorphic samples exhibited the following SNPs: rs1059572 (G/A/C/T), rs1059575 (G/C/T), rs3175105 (T/A/C), rs17882300 (A/C/T), rs17882603 (C/A/T), rs11554462 (A/C/G), rs1064664 (A/C/G), rs707957 (G/A/C/T), rs17878951 (A/G), and rs150747106 (C/G/T) in irregular combinations (Figure 1). Absence of SNPs was accompanied with almost complete demethylation of CpG41 (Figure 4).
CpG41 of HLA-DRB1 gene resides within sequence of the antigen-binding groove, explaining the high rate of polymorphisms obtained along this region and its association with autoimmune disorders, such as multiple sclerosis and rheumatoid arthritis17,18,24,25. Accordingly, during FA development certain MHC alleles are possibly more efficient at presenting important food allergic components, leading to increased immune responses and fast progression of allergy symptoms. Failure to correlate any of the genetic variants detected among FA and control samples with CpG41 methylation levels, indicates that other variants located within the extended genetic loci potentially acquire guiding roles for DNA the methylation spreading throughout this genetic area.