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  Volume 95
Numero 6
novembre-dicembre 2009 		I documenti sono in formato PDF, consultabili utilizzando Acrobat Reader
 
Role of glutathione S-transferase omega gene polymorphisms in breast-cancer risk

Sunanta Chariyalertsak, Wichai Purisa, Suleeporn Sangrajrang
Research Division, National Cancer Institute, Bangkok, Thailand

Key words: breast cancer, gene polymorphism, glutathione S-transferase.

abstract

Background/aims. Genetically influenced variations in the levels of activity and/or expression of some members of the glutathione S-transferase (GST) family have been identified as risk factors for cancer. One, GST omega (GSTO), has been found in a very limited number of studies. The aim of the present study was to investigate the influence of GSTO1 and GSTO2 polymorphisms on breast cancer risk.
Methods. DNA isolated from the blood of 101 patients with breast cancer and 151 healthy controls was investigated for GSTO1 and GSTO2 polymorphisms by polymerase chain reaction-restriction-fragment length polymorphism.
Results. Univariate and multivariate analyses showed no association between GSTO1 and GSTO2 genotypes and the risk of breast cancer. A higher prevalence of wild-type GSTO1 (A140/A140) was significantly correlated with advanced-stage breast cancer (OR = 0.1, 95% CI, 0.01-0.77), but the presence of the genotype did not correlate with patient age at diagnosis, menopausal status, tumor size, lymph node metastasis, or estrogen-receptor status. No association was found between GSTO2 genotype and clinicopathological features.
Conclusions. The results of the study suggest that GSTO1 and GSTO2 variants are not associated with breast cancer risk, but that wild-type GSTO1 (A140/A140) is likely among cases at an advanced stage.

Introduction
Breast cancer is the second most common cancer of women in Thailand, accounting for about 15% of all female cancers. Its incidence has been increasing1. The etiology of breast cancer is still poorly understood, although several risk factors are well established, including high estrogen exposure, life-style risk factors (e.g., alcohol and diet), and family history2. Since human breast cancer results from genetic-environmental interactions, genetic factors need to be identified for a more accurate evaluation of overall breast cancer risk. Polymorphisms in breast cancer susceptibility genes with low penetrance have a greater contribution to breast tumorigenesis when combined with environmental exposure3.
Glutathione S-transferases (GST), a family of phase-II detoxification enzymes, play important roles in protecting cells against environmental carcinogens. Eight classes of cytosolic, soluble GST have been identified in humans: alpha, mu, pi, theta, zeta, sigma, and omega4,5, whereas GST kappa is located in the mitochondria6 and peroxisomes7. In contrast, unlike other human GST, GST omega (GSTO) mediates glutathione-dependent thioltransferase and dehydroascorbate reductase activities, which are similar to those catalyzed by the glutaredoxins. In addition, GSTO catalyzes the reduction of monomethylarsonic acid, the rate-limiting reaction in the biotransformation of inorganic arsenic8, a notorious environmental carcinogen.
In humans, two expressed genes (GSTO1 and GSTO2) and a pseudogene GSTO3p have been identified in the omega class GST. The GSTO1 and GSTO2 genes contain six exons, spanning 12.5 and 24.5 kb, respectively, and lie 7.5 kb apart on chromosome 10q24.3. Two GSTO gene polymorphisms, GSTO1*A140D and GSTO2*N142D, have been identified in population studies9. Both polymorphisms were investigated in a single, small-scale (30 cases, 33 controls) study, which found the GSTO1 polymorphism alone related to breast cancer10.
These findings are of epidemiologic importance, as it has been suggested that carriers of the GSTO1 and GSTO2 variant alleles account for 8-34% and 23-86%, respectively, of the general population9-13. In addition, only a few GSTO polymorphisms have been reported in the literature. The current study was conducted to evaluate any association between genetic GSTO1 and GSTO2 polymorphisms with breast cancer risk among Thai women. Since a number of key factors are involved in the etiology of breast cancer, multivariate analysis was used to elicit and adjust for the confounding effects of such factors. An association between gene polymorphisms and patient clinicopathological characteristics was also observed.

Patients and methods
Patients
In this case-control study, 101 cases of histologically diagnosed breast cancer were recruited from the National Cancer Institute, Bangkok, Thailand. A control group of 151 healthy individuals was selected from women who came to the same hospital for an annual health checkup. Informed consent was obtained from all individuals: demographic and anthropometric data, reproductive and medical history, physical activity, occupation, and dietary data were obtained by an interview. The study was reviewed and approved by the Ethics Committee of the National Cancer Institute, Bangkok. Clinical stage was classified according to the American Joint Committee on Cancer TNM staging system.
DNA isolation
A blood sample of approximately 7 ml was collected from each patient and control. Genomic DNA was isolated from buffy coats using a QIAmp DNA extract kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA was quantitated by a spectrophotometer and stored at -80 °C.
Genotype analysis
Genotyping was performed by polymerase chain reaction-restriction-fragment length polymorphism. GSTO1 and GSTO2 genotypes were determined as previously described10. All analyses were blinded to case or control status. Histopathological features (i.e., tumor size, lymph node metastasis, staging and estrogen receptor status assessed using immunohistochemistry) were analyzed at the Pathological Division of the National Cancer Institute in Bangkok.
Statistical analysis
The observed genotype frequencies were compared with those calculated from Hardy-Weinberg equilibrium (p2+2pq+q2; where p is the frequency of the variant allele and q = 1-p) using the chi-square of goodness-of-fit with one degree of freedom, with respect to the distribution of the allele groups under study. Chi-squared and Fisher’s exact tests were used to evaluate the differences between cases and controls, for age, menopausal status, pregnancy, breast feeding, oral contraceptive use, body mass index (BMI), tobacco smoking, alcohol consumption and education. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by unconditional logistic regression to evaluate associations between GSTO1 and GSTO2 genotypes and breast cancer risk. The OR were adjusted for the potential confounders comprising age, menopausal status, pregnancy, BMI, and education. Associations between the genotypes and clinicopathological parameters were analyzed using OR and 95% CI estimated by the logistic regression model.

Results
The characteristics of the case and control cohorts are shown in Table 1. There were no significant differences between cases and controls regarding breast feeding, oral contraceptive use, smoking, or drinking alcohol. However, age ≥40 years, postmenopause, pregnancy, BMI ≥25 kg/m2, and low educational level were more prevalent among cases than controls. The allelic frequencies for GSTO1 and GSTO2 polymorphisms were determined for both study populations. For GSTO1, the frequencies of the wild-type A140 alleles were 0.89 and 0.88 for cases and controls, respectively. For GSTO2, the frequencies of the N142 alleles were 0.77 for both cases and controls. The genotypic frequencies of the different GSTO1 and GSTO2 polymorphisms in cancer and control populations agreed with the Hardy-Weinberg equilibrium (P >0.1).
Univariate and multivariate logistic regression analysis of the overall effect of the different GSTO1 and GSTO2 polymorphisms did not support an association between the presence of one polymorphism genotype and individual susceptibility to breast cancer, since the OR of GSTO1 and GSTO2 genotypes did not reach the level of statistical significance (Table 2).

Table 1 - General characteristics for the breast cancer cases (n = 101) and control population (n = 151)

Characteristics

Cases

Controls

P

 

No. (%)

No. (%)

 

 

 

 

 

Age

 

 

0.000

   <40

12 (11.9)

79 (52.3)

 

   40-60

77 (76.2)

65 (43.0)

 

   >60

12 (11.9)

7 (4.6)

 

Menopause

 

 

0.002

   Pre

52 (51.5)

107 (70.9)

 

   Post

49 (48.5)

44 (29.1)

 

Pregnancy

 

 

0.001

   No

27 (26.7)

73 (48.3)

 

   Yes

74 (73.3)

78 (51.7)

 

Breast feeding

 

 

0.076

   No

12 (17.9)

6 (8.0)

 

   Yes

55 (82.1)

69 (92.0)

 

Oral contraceptive use

 

 

0.685

   No

63 (63.0)

100 (66.2)

 

   Yes

37 (37.0)

51 (33.8)

 

Body mass index (kg/m2)

 

 

0.014

   <25

64 (63.4)

118 (78.1)

 

   ≥25

37 (36.6)

33 (21.9)

 

Tobacco smoking

 

 

0.359

   No

98 (97.0)

149 (98.7)

 

   Yes

3 (3.0)

2 (1.3)

 

Alcohol consumption

 

 

0.661

   No

92 (91.1)

135 (89.4)

 

   Yes

9 (8.9)

16 (10.6)

 

Education

 

 

0.000

   ≤9 years

60 (59.4)

43 (28.5)

 

   >9 years

41 (40.6)

108 (71.5)

 


Table 2 - OR and 95% CI for GSTO1 and GSTO2 genotypes and breast cancer association

Genotype

Cases

Controls

OR crude

P

OR adjusted

P

 

 

 

 

 

 

 

GSTO1

 

 

 

 

 

 

A140/A140

80

117

1.00 (Reference)

 

1.00 (Reference)

 

A140/D140

20

 33

0.85 (0.46-1.59)

0.62

0.93 (0.46-1.88)

0.83

D140/D140

 1

  1

1.45 (0.09-23.52)

0.79

0.97 (0.06-17.29)

0.99

A140/D140 + D140/D140

 

 

0.87 (0.47-1.60)

0.66

0.93 (0.46-1.86)

0.83

A140 allele frequency

0.89

0.88

 

 

 

 

D140 allele frequency

0.11

0.12

 

 

 

 

GSTO2

 

 

 

 

 

 

N142/N142

59

86

1.00 (Reference)

 

1.00 (Reference)

 

N142/D142

38

60

0.95 (0.56-1.60)

0.84

0.85 (0.47-1.55)

0.60

D142/D142

 4

 5

0.88 (0.20-3.80)

0.86

1.32 (0.24-7.14)

0.75

N142/D142 + D142/D142

 

 

0.94 (0.57-1.57)

0.82

0.88 (0.49-1.58)

0.67

N142 allele frequency

0.77

0.77

 

 

 

 

D142 allele frequency

0.23

0.23

 

 

 

 

 

 

 

 

 

 

 


The relationships of clinicopathological parameters with GSTO1 and GSTO2 genotypes are shown in Tables 3 and 4, respectively. A significant association was found between higher prevalence of wild-type GSTO1 (A140/A140) and advanced stage breast cancer (OR = 0.1, 95% CI, 0.01-0.77). No other clinicopathological parameter was significantly associated with GSTO1 genotype. No significant association was found between GSTO2 polymorphism and age of patient at diagnosis, menopausal status, tumor size, lymph node metastasis, stage, or estrogen receptor status.

Table 3 - Association of GSTO1 polymorphism with clinicopathological parameters

Parameter

No.

A140/A140

A140/D140 + D140/D140

OR (95% CI)

P

 

 

 

 

 

 

Age

 

 

 

 

 

<40

12

 8

 4

 

 

40-60

77

62

15

0.48 (0.13-1.82)

0.28

>60

12

10

 2

0.40 (0.06-2.77)

0.35

Menopausal status

 

 

 

 

 

Pre

52

41

11

 

 

Post

49

39

10

0.96 (0.37-2.50)

0.10

Tumor size (cm)

 

 

 

 

 

≤2

34

25

 9

 

 

>2

67

55

12

0.61 (0.23-1.62)

0.32

Lymph node metastasis

 

 

 

 

 

Negative

40

34

 6

 

 

Positive

61

46

15

1.85 (0.65-5.25)

0.25

Stage

 

 

 

 

 

I + II

73

53

20

 

 

III + IV

28

27

 1

0.10 (0.01-0.77)

0.03

ER

 

 

 

 

 

Positive

45

36

 9

 

 

Negative

38

30

 8

1.01 (0.37-3.11)

0.91

 

 

 

 

 

 

OR, Odds ratios.


Table 4 - Association of GSTO2 polymorphism with clinicopathological parameters

Parameter

N

N142/N142

N142/D142 + D142/D142

OR(95% CI)

P

 

 

 

 

 

 

Age

 

 

 

 

 

<40

12

 7

 5

 

 

40-60

77

43

34

1.11 (0.32-3.80)

0.87

>60

12

 9

 3

0.47 (0.08-2.66)

0.39

Menopausal status

 

 

 

 

 

Pre

52

27

25

 

 

Post

49

32

17

0.57 (0.26-1.28)

0.17

Tumor size (cm)

 

 

 

 

 

≤2

34

20

14

 

 

>2

67

39

28

1.03 (0.44-2.37)

0.95

LN metastasis

 

 

 

 

 

Negative

40

25

15

 

 

Positive

61

34

27

1.32 (0.59-2.99)

0.50

Stage

 

 

 

 

 

I + II

73

40

33

 

 

III + IV

28

19

 9

0.57 (0.23-1.44)

0.24

ER

 

 

 

 

 

Positive

45

28

17

 

 

Negative

38

20

18

1.48 (0.62-3.56)

0.38

 

 

 

 

 

 

OR, Odds ratios.



Discussion
Differences between populations in the genotypic distribution of both GSTO1 and GSTO2 polymorphisms have been shown. For GSTO1, the frequency of the D140 allele was high in European Australians (f = 0.34) and low in Chinese (f = 0.17), Brazilians (f = 0.16), Mexicans (f = 0.12), Japanese (f = 0.12), and Africans (f = 0.08). In GSTO2, the D142 allele occurred most commonly in Africans (f = 0.86), with Australians (f = 0.31) and Chinese (f = 0.27) being similar9-13. In the present study, the frequencies for these alleles (D140, f = 0.12; D142, f = 0.23) among the controls were consistent with those for Asian populations.
Common low-penetrance cancer-susceptibility genes, acting together with endogenous and life-style risk factors, are likely to account for most sporadic breast cancers, which comprise the majority of all breast cancers14. Such genes can be identified by studying the biochemical or physiological pathways that are postulated to be involved in breast tumorigenesis. Polymorphisms in breast cancer susceptibility genes with low penetrance have a greater contribution to breast carcinogenesis in combination with exogenous and endogenous exposures3. Candidate polymorphic genes including GSTO, which reduce enzyme activity and lower capacity to biotransform inorganic arsenic, could play a role in breast carcinogenesis.
Arsenic is an obvious environmental carcinogen, and contamination of drinking water with inorganic arsenic is a worldwide health problem. Methylation is the main detoxification pathway for inorganic arsenic. Marked variations in methylation capacity have been found, and arsenic susceptibility has been noted among some individuals. GSTO genetic polymorphisms may contribute to such a variation between individuals15.
Only two previously published studies have demonstrated an association between GSTO polymorphism and breast cancer risk. One found a significantly higher risk of breast cancer among heterozygous or homozygous carriers of GSTO1*D140, but not carriers of GSTO2*D142. However, the study was conducted in small groups of Thai women10. The second study, of Danish women, found that homozygous carriage of GSTO1*D140 was significantly associated with higher risk, especially for estrogen-receptor-positive breast cancer16.
In contrast, our findings did not show a significant role for the GSTO gene, or its polymorphisms, in breast cancer risk. The main differences between the two previous studies and the present one were the sample size and the menopausal status of the study populations. In the first study10, 30 cases and 33 controls were recruited. In the second16, all participants were postmenopausal women. In contrast, in the present study, 101 patients and 151 healthy controls, either premenopausal or postmenopausal women, were enrolled. Thus, although no association of GSTO1 genotype with breast cancer risk was found in our study, such an association cannot currently be excluded for postmenopausal women, especially for estrogen-receptor-positive breast cancer.
To our knowledge, no association between GSTO1 polymorphism and the clinicopathological features of patients with breast cancer has been reported. Therefore, in the present study, we investigated the association of GSTO1 and GSTO2 genotypes with a number of clinical parameters in the total case group. It was found that wild-type GSTO1 (A140/A140) correlated with advanced stage breast cancer. Recently, a study demonstrated that mouse GSTO1 overexpressed in a lymphoma cell line with resistance to radiation and chemotherapeutics17. Since GSTO1 appears to be involved in drug and xenobiotic metabolism, it would be of great interest to investigate further whether resistance to radiation or chemotherapy occurs in carriers of wild-type GSTO1, particularly those with advanced stage breast cancer. Elucidating this relationship should lead to the improved clinical management of these patients.
In conclusion, no clear association between GSTO1 or GSTO2 polymorphism and breast cancer risk was found in the present study. However, we found wild-type GSTO1 (A140/A140) more frequently among patients with advanced stage breast cancer.

References
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