To evaluate the association between genetic polymorphisms in enzymes involved in folate metabolism with cervical carcinogenesis, we analyzed samples of 106 women with remission or persistence of cytological abnormalities. We also evaluated sociodemographic and behavioral characteristics such as age, education, use of alcohol, smoking, marital status, information about sexual life, pregnancies, and use of hormonal contraceptives. These characteristics can be considered cofactors of cervical cancer (5, 8, 10, 40).
The mean age of participants was 39.7±11.4 years, ranging from 19 to 71 years, and 32.1% (n=34) were between 35 and 44 years. Most women resided in urban areas (n=97, 91.5%), had family financial income <US$250/month (n=70, 74.5%), high school education (n=46, 47.9%), were non-smokers (n=81, 84.4%), and ingested alcoholic beverages (n=57, 59.4%).
Most of the women reported being married or having a fixed partner (n=77, 80.2%), having had the first sexual intercourse at the age of 18 years or older (n=53, 56.4%), and three or more sexual partners (n=55, 57.3%). Also, most of them did not use hormonal contraceptives (n=61, 63.5%) and had already been pregnant (n=81, 84.4%) (Table 2). We observed these characteristics at a similar rate and with no significant association (p<0.05) among the Remission and Persistence groups (Table 2).
Concerning HPV, the main factor for cervical carcinogenesis (3), 50.9% (n=54) of the participants were infected. The infection rate was higher in the Persistence group (n=31, 67.4%) than in the Remission group (n=23, 38.3%) (p=0.003). We obtained similar results with the analysis of hr-HPV infection alone (p=0.000) (data not shown).
TABLE 2: Sociodemographic and behavioral characteristics
|
|
Cytological abnormality
|
|
Characteristics
|
Total
n (%)
|
Remission
n (%)
|
Persistence
n (%)
|
p
|
Age (years)
|
|
|
|
|
<25
|
12 (11.3)
|
7 (11.7)
|
5 (10.9)
|
0.302
|
25-34
|
21 (19.8)
|
9 (15.0)
|
12 (26.1)
|
35-44
|
34 (32.1)
|
18 (30.0)
|
16 (34.8)
|
45-54
|
28 (26.4)
|
17 (28.3)
|
11 (23.9)
|
≥55
|
11 (10.4)
|
9 (15.0)
|
2 (4.3)
|
Area
|
|
|
|
|
Urban
|
97 (91.5)
|
54 (90.0)
|
43 (93.5)
|
0.524
|
Countryside
|
9 (8.5)
|
6 (10.0)
|
3 (6.5)
|
Income per person (US$/month)1
|
|
|
|
|
<250
|
70 (74.5)
|
40 (72.7)
|
30 (76.9)
|
|
250-500
|
21 (22.3)
|
12 (21.8)
|
9 (23.1)
|
0.333
|
≥500
|
3 (3.2)
|
3 (5.5)
|
0
|
|
Education2
|
|
|
|
|
Elementary school/Illiterate
|
44 (45.8)
|
23 (41.8)
|
21 (51.2)
|
0.341
|
High school
|
46 (47.9)
|
27 (49.1)
|
19 (46.3)
|
University
|
6 (6.3)
|
5 (9.1)
|
1 (2.4)
|
|
Smoker2
|
|
|
|
|
No
|
81 (84.4)
|
48 (87.3)
|
33 (80.5)
|
0.365
|
Yesa
|
15 (15.6)
|
7 (12.7)
|
8 (19.5)
|
Use of alcoholic beverage2
|
|
|
|
|
No
|
39 (40.6)
|
22 (40.0)
|
17 (41.5)
|
0.885
|
Yesa
|
57 (59.4)
|
33 (60.0)
|
24 (58.5)
|
Marital status2
|
|
|
|
|
Married/Fixed Partner
|
77 (80.2)
|
40 (72.7)
|
37 (90.2)
|
0.073
|
Single
|
9 (9.4)
|
8 (14.5)
|
1 (2.4)
|
Widow/Divorced
|
10 (10.4)
|
7 (12.7)
|
3 (7.3)
|
Age at first vaginal intercourse (years)1
|
|
|
|
|
<18
|
41 (43.6)
|
25 (45.5)
|
16 (41.0)
|
0.670
|
≥18
|
53 (56.4)
|
30 (54.5)
|
23 (59.0)
|
Lifetime sexual partners2
|
|
|
|
|
1
|
23 (23.9)
|
13 (23.6)
|
10 (24.4)
|
0.936
|
2
|
18 (18.8)
|
11 (20.0)
|
7 (17.1)
|
≥3
|
55 (57.3)
|
31 (56.4)
|
24 (58.5)
|
Use of hormonal contraceptive2
|
|
|
|
|
No
|
61 (63.5)
|
39 (70.9)
|
22 (53.7)
|
0.082
|
Yes
|
35 (36.5)
|
16 (29.1)
|
19 (46.3)
|
Pregnancies2
|
|
|
|
|
0
|
15 (15.6)
|
9 (16.4)
|
6 (14.6)
|
0.130
|
1
|
19 (19.8)
|
14 (25.5)
|
5 (12.2)
|
2
|
26 (27.1)
|
10 (18.2)
|
16 (39.0)
|
3
|
22 (22.9)
|
12 (21.8)
|
10 (24.4)
|
≥4
|
14 (14.6)
|
10 (18.2)
|
4 (9.8)
|
|
|
|
|
|
HPV infection
|
52 (49.1)
|
|
|
|
Negative
|
54 (50.9)
|
37 (61.7)
|
15 (32.6)
|
0.003
|
Positive
|
|
23 (38.3)
|
31 (67.4)
|
|
Participants excluded due to absence of information: 1Twelve; 2Ten. aAmount or frequency not determined. Remission: presence of pre-neoplastic lesion at T1, and normal cytology at T2; Persistence: pre-neoplastic lesion detected at T1 and T2.
Regarding the genetic polymorphisms evaluated, MTRR A66G genotypic frequencies were 10.4% (n=11), 76.4% (n=81), and 13.2% (n=14) of AA, AG, and GG, respectively. The genotypic frequency of TSER was 35.8% (n=38) of 2R/2R, 31.1% (n=33) of 2R/3R, and 33.0% (n=35) of 3R/3R. However, we could not find the distribution of genotypes of MTRR A66G and TSER of the Remission group under Hardy-Weinberg equilibrium (p=0.000). Thus, these polymorphisms were excluded from further analyses in this study.
MTHFR C677T genotypic frequencies were 50.0% (n=53) of CC, 45.3% (n=48) of CT, and 4.7% (n=5) of TT; the T allelic frequency was 27.4%. We detected the MS A2756G polymorphic genotype in 3.8% (n=4) of the samples, and G allele in 19.8%. On the other hand, a higher frequency of women presented polymorphic genotype for TS3’UTR genetic variation (16.0%, n=17), and the frequency of del allelic was 41.5% (Table 3).
TABLE 3: Frequencies of genetic polymorphisms according Remission or Persistence of pre-neoplastic cervical lesions.
Genetic
polymorphisms
|
|
Cytological abnormality
|
|
|
Total
|
Remission
(n=60)
|
Persistence
(n=46)
|
OR (IC95%)a
|
p
|
MTHFR C677T1
|
Genotype n (%)
|
|
|
|
|
|
CC
|
53 (50.0)
|
28 (46.7)
|
25 (54.3)
|
1.0
|
|
CT
|
48 (45.3)
|
28 (46.7)
|
20 (43.5)
|
0.93 (0.40 – 2.12)
|
0.856
|
TT
|
5 (2.2)
|
4 (6.6)
|
1 (2.2)
|
0.25 (0.02 – 2.48)
|
0.245
|
Allele %
|
|
|
|
|
|
C
|
72.6
|
70.0
|
76.1
|
1.0
|
|
T
|
27.4
|
30.0
|
23.9
|
0.71 (0.34 – 2.11)
|
0.842
|
MS
A2756G2
|
Genotype n (%)
|
|
|
|
|
|
AA
|
69 (65.4)
|
42 (70.0)
|
27 (58.7)
|
1.0
|
|
AG
|
33 (31.1)
|
17 (28.3)
|
16 (34.8)
|
1.20 (0.50 – 2.89)
|
0.690
|
GG
|
4 (3.8)
|
1 (1.7)
|
3 (6.5)
|
4.99 (0.46 – 54.57)
|
0.188
|
Allele %
|
|
|
|
|
|
A
|
80.2
|
83.3
|
76.1
|
1.0
|
|
G
|
19.8
|
16.7
|
23.9
|
1.63 (0.59 – 4.53)
|
0.349
|
TS3’UTR3
|
Genotype n (%)
|
|
|
|
|
|
ins/ins
|
34 (32.1)
|
26 (43.3)
|
8 (17.4)
|
1.0
|
|
ins/del
|
55 (51.9)
|
28 (46.7)
|
27 (58.7)
|
3.22 (1.19 – 8.69)
|
0.021
|
del/del
|
17 (16.0)
|
6 (10.0)
|
11 (23.9)
|
6.50 (1.71 – 24,70)
|
0.006
|
Allele %
|
|
|
|
|
|
ins
|
58.5
|
66.7
|
47.8
|
1.0
|
|
del
|
41.5
|
33.3
|
52.2
|
2.28 (1.00 – 5.22)
|
0.051
|
GRS n(%)
|
≤2
|
77 (72.6)
|
48 (80.0)
|
29 (63.0)
|
1.00
|
|
≥3
|
29 (27.4)
|
12 (20.0)
|
17 (37.0)
|
2.21 (0.89 – 5.48)
|
0.086
|
Hardy-Weinberg Equilibrium (HWE): 1p=0.389; 2p=0.625; 3p=0.699. aAdjusted for HPV infection. Remission: presence of pre-neoplastic lesion at T1, and normal cytology at T2; Persistence: pre-neoplastic lesion detected at T1 and T2.
To evaluate the association between the MTHFR C677T, MS A2756G, and TS3’UTR polymorphisms according to the course of cytological abnormalities, we compared the genotype distribution of the Remission and Persistence groups (Table 3).
There were no differences in the distribution of MTHFR C677T and MS A2756G and the course of cytological abnormalities (Table 3). On the other hand, women with persistent lesions had higher heterozygote and polymorphic genotypic frequencies of TS3’UTR than those from the Remission group. Furthermore, the ins/del and del/del genotypes increased the risk of persistence at least three times [OR (IC95%): 3.13 (1.21 – 8.12), p=0.019; OR (IC95%): 5.96 (1.67 – 21.25), p=0.006 - respectively] (Table 3).
To simultaneously evaluate the presence of MTHFR C677T, MS A2756G, and TS3’UTR, we determined the Genetic Risk Score (GRS) (13). A score of ≥3 meant a high number of genetic variants. GRS≥3 was more frequent in the Persistence group (n=17, 37.0%) than in the Remission group (n=12, 20.0%). Also, a high number of genetic variants presented higher risk of persistent lesions [OR (IC95%): 2.21 (0.89 – 5.48), p=0.086] (Table 3). However, when adjusted for TS3’UTR, the risk of persistence according GRS was modified [OR (IC 95%): 1.26 (0.44-3.61), p=0.669], showing that, between the three polymorphisms analyzed, only TS3’UTR was associated with the course of cytological abnormality.