Demographic data and clinical manifestations
A total of 130 patients with AR (78 boys, 52 girls) and 154 controls (98 boys, 56 girls) were recruited. No statistically significant differences were found between cases and controls in terms of sex, age, weight, and height (all P-value > 0.05). Maternal allergic history (P = 0.0390) and exposed to pet (P = 0.0339) significantly increased the risk of developing AR. However, no effects were found of season of birth or exposed to second-hand smoke on AR risk. The demographic details of the sample are given in Table 1.
Table 1
Characteristics of study participants
Variable | AR | control | P-value |
N | 130 | 154 | |
Male (%) | 78 (60.00) | 98 (63.64) | 0.532 |
Age (SD), month | 53.72 (11.04) | 52.14 (10.11) | 0.209 |
Weight (SD), kg | 19.27 (1.76) | 18.25 (1.77) | 0.0800 |
Height (SD), mm | 105.81 (4.98) | 106.09 (5.07) | 0.581 |
Maternal allergic history (%) | 75 (57.69) | 70 (45.45) | 0.0390 |
Season of birth | | | |
Spring-summer | 62 | 77 | 0.698 |
Autumn-winter | 68 | 77 | |
Exposed to pet (%) | 49 (37.69) | 40 (25.97) | 0.0339 |
Exposed to second-hand smoke (%) | 59 (45.38) | 66 (42.86) | 0.669 |
Serum total eosinophil counts (cells/mL3) | 372.35 (108.02) | | |
Differentially Methylated Sites, Amplicons, And Genes
The result showed that 34 amplicons contained 507 CpG sites in promoter region of the 17 AR-susceptibility genes were sequenced (one to three amplicons for each gene, detailed information can be found in Additional file 1). To better characterize the DNA methylation of the 17 AR-susceptibility genes, differential methylation analyses were performed for the 507 CpG sites, 34 regions and 17 genes, respectively. The results showed that 55 of 520 CpG sites, all located on gene ACE or gene ADAM33, were differently methylated in AR patients compared to controls (all P < 0.05) (Additional file 3). However, the CpG site at the position of 24 bp of the first amplicon of ACE (ACE_1) (Fig. 1) was the only CpG site remained significant after correcting for multiple testing (FDR P = 0.0337).
As shown in Table 2, three amplicons of the CpG regions (GSTP1_1, ADAM33_1 and KCNE4_3) were differently methylated in AR patients compared to healthy controls (all P < 0.05). Locations of each amplicon were shown in Fig. 1. The difference of GSTP1_1 and ADAM33_1 was still significant after correcting for multiple testing (FDR P = 0.04833). In addition, we evaluate the differences between AR cases and controls in the DNA methylation levels of genes. The results found that the DNA methylation levels of ADAM33 and GSTP1 genes were significantly different between AR patients and controls (all P < 0.05). The differences were still significant after correcting for multiple testing (P = 0.0483) (Table 3). Since there were no significant methylation difference for all the CpG sites in GSTP1, ADAM33 was selected for following analysis. The methylation levels of promoter regions in ADAM33 in AR group and control group was shown in Fig. 1.
Table 2
Differentially methylated amplicons of CpG region between AR samples and control samples.
Target/Gene | Mean in AR | Mean in control | MethylDiff | P-value (U test) | FDR P-value (U test) |
GSTP1_1 | 0.30953 | 0.31456 | -0.00502 | 0.00142 | 0.0483 |
ADAM33_1 | 0.37788 | 0.38639 | -0.00852 | 0.00261 | 0.0483 |
KCNE4_3 | 0.91735 | 0.90976 | 0.00759 | 0.04671 | 0.3729 |
Target, the name of the amplicon; Mean in AR, average methylation degree of the AR group; Mean in control, average methylation degree of the control group; MethylDiff, average methylation degree of the AR group minus average methylation degree of the control group; P value (U-test): the U-test model is used to calculate the P value; |
Table 3
Differentially methylated genes between AR samples and control samples.
Gene | Mean in AR | Mean in control | MethylDiff | P-value (U-test) | FDR P-value (U-test) |
ADAM33 | 0.20249 | 0.20783 | -0.00534 | 0.00243 | 0.0483 |
GSTP1 | 0.10541 | 0.10323 | 0.00218 | 0.00424 | 0.0483 |
Mean in AR, average methylation degree of the AR group; Mean in control, average methylation degree of the control group; MethylDiff, average methylation degree of the AR group minus average methylation degree of the control group; P value (U-test): the U-test model is used to calculate the P value. |
Differentially methylated CpG sites in ADAM33
To evaluate the potentiality of the CpG sites as a biomarker for AR, ROC curve analysis was performed on all the CpG sites in ADAM33 gene. Mean methylation level and the AUC of ROC curve of each CpG site was shown in Fig. 3 and Table 4, the highest AUC was 0.6233 for the CG site at position 66 bp of the sequencing region of ADAM33_2. Four CpG sites (CpG sites at position of 45 bp, 85 bp, 87 bp and 89 bp of ADAM33_1 had mean methylation level difference above 0.01 in AR and controls.
Table 4
Methylation sites in promoter region of ADAM33
Target | Position | Type | P-value (Utest) | FDR P-value (Utest) | OR(L95-U95) (Logistic) | AUC (Logistic) | MethylDiff | Mean in AR | Mean in control |
ADAM33_1 | 45 | CG | 0.052 | 0.475 | 0.9761(0.9549–0.9978) | 0.592 | -0.05618 | 0.21179 | 0.26797 |
ADAM33_1 | 85 | CG | 0.040 | 0.456 | 0.9567(0.9024–1.0143) | 0.598 | -0.01416 | 0.73703 | 0.75119 |
ADAM33_1 | 87 | CG | 0.055 | 0.475 | 1.0328(0.995–1.072) | 0.619 | -0.04202 | 0.77197 | 0.81399 |
ADAM33_1 | 89 | CG | 0.049 | 0.475 | 1.009(0.9988–1.0193) | 0.593 | -0.09300 | 0.35000 | 0.44300 |
ADAM33_1 | 109 | CG | 0.057 | 0.475 | 0.8988(0.8054–1.003) | 0.590 | -0.00904 | 0.46346 | 0.47250 |
ADAM33_1 | 131 | CG | 0.047 | 0.462 | 0.9215(0.8309–1.022) | 0.594 | -0.00761 | 0.46077 | 0.46838 |
ADAM33_1 | 148 | CG | 0.046 | 0.462 | 0.9051(0.7932–1.0328) | 0.594 | -0.00564 | 0.33127 | 0.33692 |
ADAM33_1 | 171 | CG | 0.044 | 0.459 | 0.9589(0.8777–1.0477) | 0.595 | -0.00518 | 0.33279 | 0.33797 |
ADAM33_1 | 173 | CG | 0.042 | 0.459 | 0.6178(0.4049–0.9426) | 0.603 | -0.00498 | 0.00858 | 0.01357 |
ADAM33_1 | 176 | CG | 0.053 | 0.475 | 0.8126(0.6548–1.0083) | 0.592 | -0.00456 | 0.05248 | 0.05704 |
ADAM33_2 | 26 | CG | 0.045 | 0.460 | 0.5744(0.3513–0.9392) | 0.601 | -0.00304 | 0.00577 | 0.00881 |
ADAM33_2 | 30 | CG | 0.051 | 0.475 | 0.5615(0.3543–0.8898) | 0.593 | -0.00301 | 0.01210 | 0.01512 |
ADAM33_2 | 38 | CG | 0.040 | 0.456 | 0.7737(0.5536–1.0813) | 0.597 | -0.00231 | 0.01141 | 0.01372 |
ADAM33_2 | 46 | CG | 0.044 | 0.459 | 0.4853(0.243–0.9691) | 0.598 | -0.00222 | 0.00628 | 0.00851 |
ADAM33_2 | 51 | CG | 0.053 | 0.475 | 0.5236(0.2937–0.9335) | 0.592 | -0.00207 | 0.01878 | 0.02086 |
ADAM33_2 | 56 | CG | 0.039 | 0.456 | 0.7242(0.4734–1.108) | 0.597 | -0.00181 | 0.00791 | 0.00972 |
ADAM33_2 | 66 | CG | 0.058 | 0.475 | 0.1676(0.0344–0.8158) | 0.623 | -0.00162 | 0.00684 | 0.00846 |
ADAM33_2 | 69 | CG | 0.053 | 0.475 | 0.8777(0.6093–1.2642) | 0.592 | -0.00098 | 0.00792 | 0.00889 |
ADAM33_2 | 109 | CG | 0.041 | 0.458 | 0.122(0.0229–0.6488) | 0.597 | -0.00087 | 0.00635 | 0.00721 |
ADAM33_2 | 132 | CG | 0.038 | 0.456 | 0.7905(0.4372–1.4293) | 0.601 | -0.00071 | 0.00693 | 0.00764 |
ADAM33_2 | 134 | CG | 0.058 | 0.475 | 0.4438(0.1491–1.3204) | 0.590 | -0.00070 | 0.00511 | 0.00582 |
ADAM33_2 | 137 | CG | 0.055 | 0.475 | 1.5811(1.081–2.3123) | 0.597 | 0.00475 | 0.01382 | 0.00908 |
Target, the name of the amplicon; POS, the specific location of the methylation site in the amplicon; P value (U-test): the U-test model is used to calculate the P value; OR (L95–U95) (Logistic), AUC (Logistic), odds ratio and area under curve was calculated through the logistic regression model; Mean in AR, average methylation degree of the AR group; Mean in control, average methylation degree of the control group; MethylDiff, average methylation degree of the AR group minus average methylation degree of the control group; |
Correlation of ADAM33 methylation with clinical manifestations
The serum eosinophil count (normal range: 50–500 cells/mL3) was 372.35 ± 108.02 cells/mL3 in AR patients. Spearman correlation analysis revealed that the hypermethylation of ADAM33 was significantly associated with lower serum total eosinophil counts (Spearman's ρ: -0.187, P = 0.037; Fig. 4).
Risk factor on ADAM33 promoter methylation levels in the AR and control groups
We compared the mean ADAM33 promoter methylation levels between the AR and control groups stratified by maternal allergic history and exposure to pet, two risk factors identified in this study (Table 5). In the AR group, Children have daily life exposed to pet had significantly lower methylation levels compared to those without pet exposure (P-value = 0.009). The difference in control group was not significant. Impact of Maternal allergic history on methylation level of ADAM33 was not found in both AR and control group.
Table 5
ADAM33 promoter methylation levels between the AR and control groups stratified by environmental risk factors
Variable | AR | Mean ADAM33 promoter methylation levels | Control | Mean ADAM33 promoter methylation levels |
N | 130 | | 154 | |
Maternal allergic history | | | | |
YES | 75 (57.69) | 0.2018 ± 0.008417 | 70 (45.45) | 0.2073 ± 0.01042 |
NO | 55 (42.31) | 0.2029 ± 0.007894 | 84 (54.55) | 0.2081 ± 0.01130 |
P-value | | 0.346 | | 0.897 |
Exposed to pet | | | | |
YES | 49 (37.69) | 0.2012 ± 0.008346 | 40 (25.97) | 0.2098 ± 0.01172 |
NO | 81 (62.31) | 0.2044 ± 0.007349 | 114 (74.03) | 0.2062 ± 0.01015 |
P-value | | 0.009 | | 0.290 |
Values are presented as means ± SD or numbers. a Comparison between AR and control groups by Mann–Whitney U test. Significant results are in bold. |
According to the results of the multiple regression analysis, adjusting for gender, age, height, weight, season of birth and exposed to second-hand smoke, exposed to pet was significantly related to higher risk of developing AR. Furthermore, the interaction of exposed to pet and methylation level of ADAM33 was significantly related to AR risk (OR = 1.423, 95% CI = 0.0290–4.109, P-value = 0.005) (Table 6).
Table 6
Multiple regression analysis for analyzing the relationship among risk factors, the methylation level of ADAM33 and AR, AR as the dependent variable
Risk factor | β | OR (95% CI) | P-value |
Maternal allergic history a | 0.561 | 1.752(0.817–3.761) | 0.150 |
Exposed to peta | 0.721 | 2.057 (1.029–4.109) | 0.001 |
Methylation levelb | -0.225 | 0.799 (0.645–1.006) | 0.114 |
Methylation level × Exposed to pet | 0.353 | 1.423 (1.007–1.639) | 0.005 |
Methylation level × Maternal allergic history | -0.212 | 0.809 (0.699–1.105) | 0.169 |
Note: a 0 = without, 1 = with. B methylation levels were rescaled to rank. |
OR: odds ratio; CI: confidence interval. |
Adjusted for gender, age, height, weight, season of birth and exposure to second-hand smoke. |