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.