Demographic characteristics, allelic frequencies, and CDKN2A methylation status
The characteristics and allele frequencies observed among the UC patients enrolled in this study are shown in Table 2. The allele distribution of MIF (rs755622) met the criteria for HWE (p = 1.00). We found no differences with respect to mean age, gender, clinical type, or extent of disease among those with methylated and unmethylated p14ARF or p16INK4a. The minor allele frequency of rs755622 was somewhat higher in the group with p14ARF methylation; of note, the frequency of the C allele carrier was significantly higher (p = 0.01). By contrast, no significant differences in the minor allele frequencies associated with rs755622 were observed when comparing the p16INK4a methylated and unmethylated groups. Similarly, the 7-repeat allele frequency of rs5844572 was significantly higher in the p16INK4a methylated group compared to unmethylated group (p=0.036), but no significant differences were observed when comparing the p14ARF methylated with unmethylated groups.
Table 2 Demographic characteristics, allelic frequencies, and CDKN2A methylation status
|
|
overall UC
|
p14-UM
|
p14-M
|
pa
|
p16-UM
|
p16-M
|
pb
|
number of sample
|
159
|
105
|
54
|
|
89
|
70
|
|
mean age ± SD
|
41.3 ± 13.6
|
40.4 ± 13.4
|
43.0 ± 14.0
|
NS
|
42.1 ± 15.2
|
40.2 ± 11.3
|
NS
|
male : female
|
91 : 68
|
66 : 39
|
25 : 29
|
NS
|
50 : 39
|
41 : 29
|
NS
|
Clinical type
|
|
|
|
NS
|
|
|
NS
|
chronic continuous
|
56
|
38
|
18
|
|
31
|
25
|
|
relapse/remitting
|
103
|
67
|
36
|
|
58
|
45
|
|
Disease extension
|
|
|
|
NS
|
|
|
NS
|
total colitis
|
74
|
49
|
25
|
|
35
|
39
|
|
distal or left side colitis
|
85
|
56
|
29
|
|
54
|
31
|
|
(rs755622 G>C)
|
|
|
|
|
|
|
|
GG
|
93
|
68
|
25
|
0.010
|
57
|
36
|
NS
|
GC
|
57
|
32
|
25
|
|
28
|
29
|
|
CC
|
9
|
5
|
4
|
NS
|
4
|
5
|
NS
|
C allele freqency
|
23.6%
|
20.0%
|
30.6%
|
0.050
|
20.2%
|
27.9%
|
NS
|
(rs5844572 CATT repeat)
|
|
|
|
|
|
|
|
5/5
|
16
|
13
|
3
|
|
9
|
7
|
|
5/6
|
57
|
40
|
17
|
|
31
|
26
|
|
5/7
|
28
|
16
|
12
|
|
12
|
16
|
|
6/6
|
28
|
19
|
9
|
|
23
|
5
|
|
6/7
|
23
|
14
|
9
|
|
12
|
11
|
|
7/7
|
7
|
3
|
4
|
|
2
|
5
|
|
5 repeat freqency
|
36.8%
|
39.0%
|
32.4%
|
|
34.3%
|
40.0%
|
|
6 repeat freqency
|
42.8%
|
43.8%
|
40.7%
|
|
50.0%
|
42.1%
|
|
7 repeat freqency
|
20.4%
|
17.1%
|
26.9%
|
NS
|
15.7%
|
26.4%
|
0.036
|
p14-UM, p14ARF unmethylated; p14-M, p14ARF methylated; p16-UM, p16INK4a unmethylated;
|
p16-M, p16INK4a methylated; a, p14-UM vs. p14-M; b, p16-UM vs. p16-M; NS, not significant;
|
Association between MIF polymorphisms and methylation status of p14ARF or p16INK4a
By a logistic regression analysis after adjusting for confounding factors including age, gender, clinical type, and extent of disease, carrying C allele of rs755622 was significantly associated with CIHM of p14ARF (Table 3; OR, 2.16; 95% CI, 1.08–4.32; p=0.030). By contrast, no significant relationship was found between p16INK4a methylation and the allele frequencies associated with rs755622. The rs755622 CC homozygous was not associated with CIHM of both p14ARF and p16INK4aby a recessive genetic model.
Table 3 Association between MIF rs755622 and CDKN2A methylation
|
|
Genotype
|
GG vs. GC+CC
|
GG+GC vs. CC
|
|
GG
|
GC
|
CC
|
adjusted OR* (95%CI); p value
|
adjusted OR* (95% CI); p value
|
p14-UM (105)
|
68
|
32
|
5
|
reference
|
reference
|
p14-M (54)
|
25
|
25
|
4
|
2.16 (1.08-4.32); p=0.030
|
2.30 (0.52-10.3); p=0.27
|
p16-UM (89)
|
57
|
28
|
4
|
reference
|
reference
|
p16-M (70)
|
36
|
29
|
5
|
1.90 (0.974-3.69); p=0.060
|
2.25 (0.52-9.69); p=0.28
|
*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
|
UM: unmethylated; M: methylated
|
We previously revealed that CATT 7-repeat allele of rs5844572 promotes inflammation. Thus, we assessed the influence of the 7-repeat allele. Our findings indicate that carrying the rs5844572 7-repeat allele was a significant risk factor for p16INK4a methylation by an adjusted logistic regression analysis (Table 4; OR, 2.57; 95% CI, 1.26–5.24; p=0.0094). By contrast, there were no significant relationships between p14ARF methylation and rs5844572 allele frequencies. The homozygous of rs5844572 7-repeat allele was not associated with CIHM of both p14ARF and p16INK4a.
Table 4 Association between MIF rs5844572 and CDKN2A methylation
|
|
Genotype (repeat number)
|
X/X vs. X/7+7/7
|
X/X+X/7 vs. 7/7
|
|
5/5
|
5/6
|
5/7
|
6/6
|
6/7
|
7/7
|
adjusted OR* (95% CI); p value
|
adjusted OR* (95% CI); p value
|
p14-UM (105)
|
13
|
40
|
16
|
19
|
14
|
3
|
reference
|
reference
|
p14-M (54)
|
3
|
17
|
12
|
9
|
9
|
4
|
1.77 (0.864-3.63); p=0.12
|
4.51 (0.79-25.7); p=0.090
|
p16-UM (89)
|
9
|
31
|
12
|
23
|
12
|
2
|
reference
|
reference
|
p16-M (70)
|
7
|
26
|
16
|
5
|
11
|
5
|
2.57 (1.26-5.24); p=0.0094
|
5.51 (0.90-33.9); p=0.066
|
*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
|
UM: unmethylated; M: methylated; X: 5 or 6 repeat allele; 7: 7 repeat allele
|
Demographic characteristics and allele frequencies of subjects demonstrating no methylation or methylation of both p14ARF and p16INK4a
Comparisons among groups demonstrating methylation of both p14ARF and p16INK4a with those in which both were unmethylated are shown in Table 5. The allele distribution of MIF (rs755622) in both methylated and neither methylated groups met the criteria for HWE (p=0.73 and p=0.72, respectively). There were no significant differences with respect to clinicopathological backgrounds between these two groups.
Table 5 Demographic characteristics and allele frequencies of subjects demonstrating no methylation or methylation of both p14ARF and p16INK4a
|
|
|
|
neither methylated
|
both methylated
|
p value
|
|
number of sample
|
77
|
42
|
|
|
mean age ± SD
|
41.6 ± 14.9
|
42.5 ± 12.9
|
NS
|
|
male : female
|
46 : 31
|
21 : 21
|
NS
|
|
Clinical type
|
|
|
NS
|
|
chronic continuous
|
25
|
12
|
|
|
relapse/remitting
|
52
|
30
|
|
|
Extension
|
|
|
NS
|
|
Total colitis
|
31
|
21
|
|
|
distal or left side colitis
|
46
|
21
|
|
|
(rs755622 G>C)
|
|
|
|
|
GG
|
49
|
17
|
0.020
|
|
GC
|
24
|
21
|
|
|
CC
|
4
|
4
|
NS
|
|
C allele freqency
|
20.8%
|
34.5%
|
0.029
|
|
(rs5844572 CATT repeat)
|
|
|
|
|
5/5
|
7
|
1
|
|
|
5/6
|
27
|
13
|
|
|
5/7
|
12
|
12
|
|
|
6/6
|
19
|
5
|
|
|
6/7
|
10
|
7
|
|
|
7/7
|
2
|
4
|
|
|
5 repeat freqency
|
34.4%
|
32.1%
|
|
|
6 repeat freqency
|
48.7%
|
35.7%
|
|
|
7 repeat freqency
|
16.9%
|
32.1%
|
0.0090
|
|
p value: unmethylated vs. both methylated
The minor allele frequencies associated with rs755622 were significantly higher in the group in which both p14ARF and p16INK4a were methylated compared to the fully unmethylated group (p=0.029); the C allele carrier was detected at significantly higher frequency (p=0.020). Similarly, the frequency of the rs5844572 7-repeat allele was significantly higher in the group in which both p14ARF and p16INK4a were methylated compared to the fully unmethylated group (p=0.0090).
Association between MIF polymorphisms and CDKN2A methylation
The results of an analysis in which confounding factors were adjusted revealed that carrying the rs755622 C allele and the rs5844572 7-repeat allele was significantly associated with an increased methylation of both p14ARF and p16INK4a (OR, 2.70; 95% CI, 1.22–6.01; p=0.015 and OR, 2.87; 95% CI, 1.25–6.62; p=0.013, respectively; Table 6). In addition, homozygous of rs5844572 7-repeat allele was significantly associated with CIHM of both genes (OR, 12.0; 95%CI, 1.55-92.2; p=0.017).
Table 6 Association between MIF polymorphisms and CDKN2A methylation
|
rs755622
|
GG vs. GC+CC
|
GG+GC vs. CC
|
|
adjusted OR* (95% CI); p value
|
adjusted OR* (95% CI); p value
|
neither methylated (77)
|
reference
|
reference
|
both methylated (42)
|
2.70 (1.22-6.01); p=0.015
|
3.92 (0.76-20.3); p=0.10
|
|
|
|
rs5844572
|
X/X vs. X/7+7/7
|
X/X+X/7 vs. 7/7
|
|
adjusted OR* (95% C.I.); p value
|
adjusted OR* (95% C.I.); p value
|
neither methylated (77)
|
reference
|
reference
|
both methylated (42)
|
2.87 (1.25-6.62); p=0.013
|
12.0 (1.55-92.2); p=0.017
|
*by logistic regression analysis after adjustment for age, gender, clinical type and disease extension
|
X: 5 or 6 repeat allele; 7: 7 repeat allele
|
Association between MIF polymorphisms and CDKN2A methylation in phenotype of UC
Next, we investigated in what kind of UC phenotype the significant association of MIF polymorphisms with CIHM of CDKN2A was seen (Table 7). Carrying rs755622 C allele was significantly associated with CIHM in chronic continuous of clinical type and total colitis (OR, 25.9; 95% CI, 2.55-262.6; p=0.0059 and OR, 4.38; 95% CI, 1.12-17.2; p=0.034, respectively). Meanwhile, carrying 7-repeat allele of rs5844572 was significantly associated in chronic continuous type (OR, 14.5; 95%CI, 1.46-144.3; p=0.022).
Table 7 Association between MIF polymorphisms and CDKN2A methylation in UC phenotype
|
rs755622
|
genotype
|
adjusted OR (95% C.I.); p value
|
chronic continuous
|
GG
|
GC
|
CC
|
GG vs. GC+CC
|
neither methylated (25)
|
17
|
6
|
2
|
reference
|
both methylated (12)
|
2
|
6
|
4
|
25.9 (2.55-262.6); p=0.0059a
|
relapse/remitting
|
GG
|
GC
|
CC
|
GG vs. GC+CC
|
neither methylated (52)
|
32
|
18
|
2
|
reference
|
both methylated (30)
|
15
|
15
|
0
|
1.93 (0.732-5.07); p=0.18a
|
total colitis
|
GG
|
GC
|
CC
|
GG vs. GC+CC
|
neither methylated (31)
|
21
|
10
|
0
|
reference
|
both methylated (21)
|
9
|
12
|
0
|
4.38 (1.12-17.2); p=0.034b
|
distal or left side colitis
|
GG
|
GC
|
CC
|
GG vs. GC+CC
|
neither methylated (46)
|
28
|
14
|
4
|
reference
|
both methylated (21)
|
8
|
9
|
4
|
2.75 (0.899-8.43); p=0.076b
|
|
|
|
|
|
rs5844572
|
genotype
|
adjusted OR (95% C.I.); p value
|
chronic continuous
|
X/X
|
X/7
|
7/7
|
X/X vs. X/7+7/7
|
neither methylated (25)
|
15
|
8
|
2
|
reference
|
both methylated (12)
|
3
|
5
|
4
|
14.5 (1.46-144.3); p=0.022a
|
relapse/remitting
|
X/X
|
X/7
|
7/7
|
X/X vs. X/7+7/7
|
neither methylated (52)
|
38
|
14
|
0
|
reference
|
both methylated (30)
|
16
|
14
|
0
|
2.64 (0.970-7.17); p=0.058a
|
total colitis
|
X/X
|
X/7
|
7/7
|
X/X vs. X/7+7/7
|
neither methylated (31)
|
21
|
10
|
0
|
reference
|
both methylated (21)
|
10
|
11
|
0
|
3.47 (0.895-13.4); p=0.072b
|
distal or left side colitis
|
X/X
|
X/7
|
7/7
|
X/X vs. X/7+7/7
|
neither methylated (46)
|
32
|
12
|
2
|
reference
|
both methylated (21)
|
9
|
8
|
4
|
2.89 (0.931-8.98); p=0.066b
|
a: adjusted for age, gender and disease extension
|
b: adjusted for age, gender and clinical type
|
X: 5 or 6 repeat allele; 7: 7 repeat allele
|