Characteristics of the cases and controls
This study included 402 female cases with a mean age of 51.75±9.39 and 557 female controls with a mean age of 51.85±10.31. Other demographic information of the cases and controls is listed in Table 1. The definition of variables for environmental factors with ≤5.8% missing data were processed by the multiple imputation method are presented in Table S1.
Table 1. Demographic characteristics of breast cancer patients and controls.
Characteristics
|
No. of
Controls(%)
|
No. of
Cases (%)
|
P Value
|
Age
|
Mean ± SD
|
51.85±10.31
|
51.75±9.39
|
|
< 40
|
82(14.7)
|
41(10.2)
|
0.02
|
40-
|
333(59.8)
|
274(68.2)
|
≥60
|
142(25.5)
|
87(21.6)
|
BMI
|
≤18.5
|
35(6.3)
|
14(3.5)
|
0.12
|
18.5-
|
274(49.2)
|
211(52.5)
|
≥24.0
|
248(44.5)
|
177(44.0)
|
Urban and Rural Status
|
Rural
|
236(42.4)
|
232(57.7)
|
<0.01
|
Urban
|
321(57.6)
|
170(42.3)
|
Education Level
|
Primary School or Below
|
162(29.1)
|
98(24.4)
|
0.27
|
Middle School
|
175(31.4)
|
135(33.6)
|
Senior School and Higher
|
220(39.5)
|
169(42.0)
|
Occupation Type a
|
White Collar
|
273(49.0)
|
233(58.2)
|
0.01
|
Blue Collar
|
284(51.0)
|
169(41.8)
|
Ethnicity
|
Han
|
529(95.0)
|
386(96.0)
|
0.27
|
|
Other
|
28(5.0)
|
16(4.0)
|
a The white collar occupation referred to people work that need mental rather than physical effort, such as office, doctor, accountant, business, teacher, etc.; the blue collar occupation referred to people work as manual labor, such as worker, farmer, cleaner, etc.
Associations between WT1, CA10 methylation and breast cancer risk
WT1 methylation was associated with breast cancer risk both in multivariable and PS adjusted methods with ORs of 2.42 (95% CI: 1.45-4.04, P<0.01) and 3.07 (95% CI: 1.67-5.64, P<0.01), respectively. CA10 methylation was statistically significant associated with breast cancer in the multivariable adjustment with an OR of 1.53 (95% CI: 1.14-2.05, P<0.01), but was only marginally associated with breast cancer after PS adjustment, with an OR of 1.35 (95% CI: 0.97-1.90, P=0.08) (Table 2).
Table 2. The associations between gene methylation and risk of breast cancer and different molecular types of breast cancer.
|
|
Molecular
types a
|
No. of Unmethylation(%)
|
No. of Methylation(%)
|
Crude OR
(95% CI)
|
P
Value
|
ORadjusted b
(95% CI)
|
P
Value
|
ORadjusted c
(95% CI)
|
P
Value
|
WT1
|
Control
|
|
65(11.7)
|
492(88.3)
|
1
|
|
1
|
|
1
|
|
|
Luminal A
|
9(6.4)
|
132(93.6)
|
1.99(0.94-4.23)
|
0.07
|
2.61(1.18-5.74)
|
0.02
|
2.62(1.11-6.20)
|
0.03
|
|
Luminal B
|
8(6.0)
|
125 (94.0)
|
2.12(1.50-2.99)
|
0.07
|
2.49(1.13-5.51)
|
0.02
|
3.23(1.34-7.80)
|
0.01
|
|
HER-2 Enriched
|
5(8.9)
|
51(91.1)
|
1.34(0.51-3.50)
|
0.55
|
1.91(0.69-5.30)
|
0.21
|
1.91(0.66-5.51)
|
0.23
|
|
TNBC
|
1(2.9)
|
33(27.1)
|
4.34(0.58-32.33)
|
0.15
|
5.63(0.73-43.63)
|
0.10
|
6.04(0.76-47.90)
|
0.09
|
All cases
|
|
26(6.5)
|
376(93.5)
|
1.92(1.18-3.13)
|
0.01
|
2.42(1.45-4.04)
|
0.01
|
3.07(1.67-5.64)
|
<0.01
|
CA10
|
Control
|
|
209(37.5)
|
348(62.5)
|
1
|
|
1
|
|
1
|
|
|
Luminal A
|
40(28.4)
|
101(71.6)
|
1.52(1.00-2.26)
|
0.05
|
1.60(1.04-2.45)
|
0.03
|
1.51(0.94-2.41)
|
0.09
|
|
Luminal B
|
34(25.6)
|
99(74.4)
|
1.79(1.17-2.74)
|
0.01
|
2.04(1.30-3.21)
|
<0.01
|
1.80(1.09-2.98)
|
0.02
|
|
HER-2 Enriched
|
18(32.1)
|
38(67.9)
|
1.27(0.71-2.29)
|
0.43
|
1.42(0.76-2.66)
|
0.27
|
1.37(0.71-2.63)
|
0.35
|
|
TNBC
|
14(41.1)
|
20(58.8)
|
0.86(0.43-1.74)
|
0.67
|
0.94(0.45-1.96)
|
0.87
|
1.01(0.46-2.20)
|
0.99
|
All cases
|
|
119(29.6)
|
283(70.4)
|
1.43(1.08-1.88)
|
0.01
|
1.53(1.14-2.05)
|
<0.01
|
1.35(0.97-1.90)
|
0.08
|
a The result excluded 38 breast cancer patients with incomplete immunohistochemical records.
b Adjusted for age, BMI, ethnicity, urban and rural status and family history of breast cancer and cancer.
c Adjusted by propensity score(potential confounder included age, BMI, urban and rural status, ethnicity, education level, mammography, gynecologic surgery, breast disease history, menstrual cycle, menopause, reproduction, abortion, breast feeding, oral contraceptive, female hormone intake, fruit intake, vegetable intake, tomato intake, broccoli intake, bean products, pungent food, pork, beef and lamb consumption, chicken consumption, sea-fish, egg, diary, fungus, pickles, alcohol consumption, tea consumption, cigarette, physical activity, occupation type, family history of breast cancer and cancer).
In the subgroup analyses, after PS adjustment, WT1 methylation was associated with breast cancer risk in both the younger (<60-years-old) and older (≥60-years-old) groups, with ORs of 2.64 (95% CI: 1.31-5.32, P=0.01) and 4.72 (95% CI: 1.31-16.97, P=0.01), respectively. CA10 methylation was associated with breast cancer risk in younger age group (<60-years-old) before PS adjustment, with OR of 1.56 (95% CI: 1.15-2.11, P=0.01); However, the association was not statistically significant after PS adjustment (Table 3). We also analyzed the combination and interaction of age and the methylation of WT1, CA10 on the risk of breast cancer. The P values for the interactions between age and the methylation of WT1 and CA10 on the risk of breast cancer were 0.40 and 0.73, respectively. The results are presented in Table 4.
Table 3. The subgroup analysis of the associations between methylation of genes and the risk of breast cancer based on different age.
|
Crude OR
(95% CI)
|
P Value
|
OR adjusted a
(95% CI)
|
P
Value
|
WT1
|
<60
|
Unmethylation
|
1
|
|
1
|
|
Methylation
|
1.64(0.95-2.84)
|
0.08
|
2.64(1.31-5.32)
|
0.01
|
≥60
|
Unmethylation
|
1
|
|
1
|
|
Methylation
|
3.16(1.05-9.5)
|
0.04
|
4.72(1.31-16.97)
|
0.01
|
CA10
|
<60
|
Unmethylation
|
1
|
|
1
|
|
Methylation
|
1.56(1.15-2.11)
|
0.05
|
1.32(0.90-1.96)
|
0.15
|
≥60
|
Unmethylation
|
1
|
|
1
|
|
Methylation
|
1.20(0.61-2.37)
|
0.60
|
1.52(0.69-3.37)
|
0.30
|
|
|
|
|
|
|
|
|
a Adjusted by propensity score.
Table 4. The interaction between age and gene methylations on the risk of breast cancer.
|
Age
|
≥60
|
<60
|
Interaction
|
P
|
ORegadjusteda (95% CI)
|
ORiadjusteda (95% CI)
|
WT1
|
Unmethylation
|
1
|
1.70(0.40-6.84)
|
1
|
|
Methylation
|
4.90(1.36-17.67)
|
4.44(1.29-15.34)
|
0.53(0.13-2.28)
|
0.40
|
CA10
|
Unmethylation
|
1
|
1.17(0.54-2.54)
|
1
|
|
Methylation
|
1.55(0.70-3.45)
|
1.55(0.74-3.27)
|
0.86(0.35-2.09)
|
0.73
|
a Adjusted for propensity score.
Associations between methylation of WT1, CA10 and risk of different molecular types of breast cancer
WT1 methylation was significantly associated with the risk of luminal A subtype of breast cancer with multivariable adjusted OR of 2.61 (95% CI: 1.18-5.74, P=0.02), and PS adjusted OR of 2.62 (95% CI: 1.11-6.20, P=0.03). WT1 methylation was also significantly associated with the risk of luminal B subtype breast cancer with ORs of 2.49 (95% CI: 1.13-5.51, P=0.02) and 3.23 (95% CI: 1.34-7.80, P=0.01) after multivariable and PS adjustment. However, WT1 methylation was not significantly associated with the risk of HER-2 enriched and TNBC subtypes (Table 2).
The associations between CA10 methylation and the risk of luminal B subtype breast cancer with multivariable adjusted and PS adjusted ORs were 2.04 (95% CI: 1.30-3.21, P P<0.01) and 1.80 (95% CI: 1.09-2.98, P=0.02), respectively. However, CA10 methylation had no significant associations with the risk of luminal A, HER-2 enriched and TNBC subtypes after the adjustment of PS. The association between the methylation of WT1, CA10 and other clinicopathological characteristics of breast cancer patients were analyzed are showed in Table S5.
Association between WT1, CA10 methylation and breast cancer risk in GEO dataset
The GSE51032 dataset is a nested case control study that includes 233 female breast cancer cases and 340 female cancer-free controls. After the data extraction from the 450K array, we identified two CG loci each in our targeted WT1 and CA10 sequences (Figure 1). ROC curves were used to calculate the cut-off values of β, which were 0.057 and 0.226 for average β of probes in WT1 and CA10. The average methylation level of Cg14657517 and Cg19074340, which are located within the WT1 targeted sequence, was associated with breast cancer with OR of 1.88 (95% CI: 1.25-2.83, P=0.03). However, the average methylation level of Cg14054928 and Cg20405017, which are located within the targeted CA10 sequence, was not statistically significant associated breast cancer risk (OR=0.76, 95% CI: 0.54-1.06, P=0.11) (Table 5).
Table 5. The association between gene average CpG sites methylation and risk of female breast cancer in GEO51032.
|
|
Hypomethylation(%)
|
Hypermethylation
(%)
|
Crude OR
(95% CI)
|
P
Value
|
WT1
|
|
|
|
|
|
|
Control
|
285(83.8)
|
55(16.2)
|
1
|
|
|
Case
|
171(73.4)
|
62(26.6)
|
1.88(1.25-2.83)
|
0.03
|
CA10
|
|
|
|
|
|
|
Control
|
146(42.9)
|
194(57.1)
|
1
|
|
|
Case
|
116(49.8)
|
117(50.2)
|
0.76(0.54-1.06)
|
0.11
|