Association of TNF-308 gene polymorphism with Cervix Cancer susceptibility among women of Chhattisgarh

 

Tulsi Rani Thakre¹*, Abha Singh², Mitashree Mitra¹

¹School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur (C.G.)

²Department of Gynaecology, Dr. B.R. Ambedkar Memorial Hospital, Raipur (C.G.)

 

ABSTRACT:

Tumour Necrosis Factor (TNF), being an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumourigenesis. TNF has been shown to mediate carcinogenesis through induction of proliferation, invasion, and metastasis of tumour cells. Polymorphisms within TNF genes can result in pathogenesis and promoting malignant progression of cervix cancer. In the present hospital based case-control study, 230 cervix cancer patients (cases) and 230 controls were studied to determine the association of TNF-308 gene polymorphism with cervical cancer. TNF-308 null genotype showed significance distribution among cases and control (χ²=18.759, df =2, p = 0.00008). Women carrying the heterozygous A allele had a two-fold increased risk of developing cervix cancer (OR=1.775; 95% CI [1.178-2.674]) while the risk of cervix cancer raises to three-fold when A allele is preset in homozygous condition (OR=3.186; 95% CI [1.775-5.719]). These findings indicate that TNF-308 polymorphisms play crucial role in the development of cervix cancer.

 

 

 

INTRODUCTION:

Cervix cancer begins with abnormal changes in the cervical tissue. Cervix cancer is the second most common cancer in women world-wide with an annual 493,243 cases and 273,505 deaths¹. The incidence of cervical cancer is higher in developing countries (≈ 80%) and less in developed countries with an effective cervical screening program. Latin America, the Caribbean, sub-Saharan Africa and South and South-East Asia have the highest incidence rate^2-3.

 

Tumor necrosis factor-alpha (TNF-α) is an important inflammation regulator cytokine associated with the B-cell-mediated immune response⁴. It is secreted by macrophages and functions as a pro-inflammatory molecular in response to immune injury and infection^5-6.

 

 

Currently, little is known about the physiological role of this cytokine in various human cancers. It is reported that lower expression of TNF-α induces invasion and transformation of cancer cells⁷. In addition, cancer type and serum level in tissues are key factors to determine how TNF-α acts in these cancers⁸. It maps to chromosome 6p21.3, spans about 3 kb and contains 4 exons. The last exon codes for more than 80% of the secreted protein⁹. To date, there have been 16 single nucleotide polymorphisms identified in the TNF-α gene. A promoter polymorphism (rs1800629) which results in G to A transition at nucleotide position -308 of the transcriptional start site of the gene is directly and positively related to specific regulation of TNF-α synthesis at the transcriptional level¹⁰. Genotype-phenotype studies of the TNF-α rs1800629 polymorphism showed the G allele conferred two-fold lower effects on the transcription level when compared with the A allele¹¹. But the transcription activity affected by the TNF-α rs1800629 polymorphism may vary due to different cell types^10-12. Several previous meta-analyses have examined the association of TNF-α rs1800629 polymorphism with cervical cancer risk and showed similar results^13-16. In light of the outcomes of the studies worldwide, the present study was carried out to find association of TNF-308 polymorphisms with the risk of cervix cancer.

 

MATERIAL AND METHODS:

A case-control research design was used for the study. The study was based on diagnosed patients of cervix cancer attending the Indira Gandhi Regional Cancer Institute, Raipur (C.G.) and Gynecology Department of Dr. B. R. Ambedkar Memorial Hospital, Raipur (C.G.). The Department of Pathology from the same hospital had performed histo-pathological diagnosis of the lesions. The data were collected between 2011-2014 from 460 individuals. Of these, 230 were diagnosed patients of cervix cancer. Age-matched unrelated healthy peoples belonging to various districts of Chhattisgarh were chosen as controls.

 

Polymerase Chain Reaction (PCR) method along with primers forward 5’-GAGGCAATAGGTTTTGAGGGCCAT-3’ and reverse 5’-GGGACACACAAGCATCAAG-3’ were used to determine TNF-308 polymorphism in the isolated DNA. The PCR was performed using a programmable thermocycler in 10µl reaction buffer containing 200 µM dNTPs, 2 mM MgCl₂, 10 pmol of each primer, about 1µg DNA and 2 units of thermostable Taq DNA polymerase. PCR condition was 94^oC (5 min); [94^oC (30 sec), 60^oC (30 sec), 72^oC (10 sec)] X 40 Cycles; 72^oC (5 min). The amplification products were analyzed by gel electrophoresis (1.5% Agarose) and visualized under UV light in Gel Doc 200 Imaging System. The resultant band sizes were: +/+ = 126 bp, 21 bp; +/– = 147 bp, 126 bp, 21 bp; and –/– = 147 bp.

 

Genotype distributions were compared between groups using the χ2 test. The group attributed risk was estimated using standard methods (Odds Ratio). Statistical analyses were performed using SPSS 16.0 version and Microsoft Excel (Microsoft Office 2007). The protocol of study was approved by Institutional Ethical Committee for Human Research.

 

RESULTS:

Table -1 and figure no. 1 shows distribution of the genotype frequency of TNF-α-308 allele polymorphism among cases and controls. It can be seen that the frequency of GA genotype is higher among cases (36.52%) followed by controls with 29.13%. The frequency of AA genotype was higher among patients (19.57%) followed by controls 8.70%. The frequency of GG genotype was higher in controls (62.17%) than in cases (43.91%). TNF-308 null genotype showed significance distribution among cases and controls (χ²=18.759, df =2, p = 0.00008).

 

Table 1 - Distribution of genotype frequencies of TNF-308 gene among cases and controls and risk of Cervix Cancer

                   

 

Figure 1: Distribution of TNF-308 genotype among cases and controls

                   

Table 2: TNF-308 polymorphism in different stages of cervix cancer

Genotypes	Cases				Control
	Stage-I	Stage-II	Stage-III	Stage-IV
GG	13	49	28	11	101
GA	9	34	23	18	84
AA	6	16	11	12	45
Total	28	99	62	41	230

 

Figure 2: Distribution of TNF-308 polymorphism in different stage among cases and controls

 

Figure 3: Band pattern of TNF -308 (G/A) marker in cervix cancer cases

 

 

 

Table and figure no. 2 shows TNF- polymorphism frequencies at position – 308 for women with normal cervix cytology as compared to women with cervical diseases. At various stages it can be seen that all stages of cervix cancer are associated with TNF-α-308 phenotype GG compared to women with normal cervical cytology. 45.16% of stage III Cases (28/62) are GG with p-value is 0.948. Thus null genotype is significantly associated with stage III cervix cancer.

 

According to results (figure-3), women carrying the heterozygous A allele have a two-fold increased risk of developing cervix cancer [OR=1.775; 95% CI (1.178-2.674)] while the risk of cervix cancer raises to three-fold when A allele is present in homozygous condition [OR=3.186; 95% CI (1.775-5.719)]. Thus it is shown that the presence of the high producer allele -308 A in the TNF-alpha gene appears to be associated with an increased risk for the development of cervix cancer.

 

 

DISCUSSION:

In recent decades, the associations between gene polymorphisms and cancer susceptibility have been extensively investigated, suggesting that genetic polymorphisms of host factors could contribute to individual differences of susceptibility to different cancers. Inflammation developing through the action of various inflammatory mediators is considered as a cofactor in carcinogenesis¹⁷. The two most commonly studied polymorphisms were G to A substitutions in the promoter region at positions -308 and -238 which have been shown to influence the TNF-α expression^11,18.

 

In our study, GG genotype (62.17%) was more frequent among controls than in cases for –308 loci of TNF-α promoter. Women carrying A-alleles showed low increased risk of developing cervix cancer which is not in agreement with the studies in Portuguese population which shows that –308 A allele increased two times the risk of development of cervix cancer¹⁹. Our findings are in agreement with Korean population, which shows that though there was a tendency for more individuals to carry the TNF–308 A allele in the cervix cancer group but it could not attain statistical significance²⁰.

 

The present study revealed that –308 A carrier genotype dominant model: GA+GG vs. AA had a 2.5 -fold increased risk rate for the disease whereas the risk of cervix cancer rises to three-fold when A allele is preset in homozygous condition. Interestingly, the distribution for –308 A allele in Indian population is reported to be variable among different ethnic groups^21-22. Present study reveals a significant association of cervix cancer with TNF- 308 polymorphism. Therefore, it is suggested that TNF –308 G/A polymorphism could serve as an important biomarker in Indian population for their susceptibility to cervix cancer because it plays a role in alteration of TNF-α production and the inflammatory responses during the course of the disease.

 

CONCLUSION:

Our findings show that the TNF-308 gene polymorphism is an important co-factor for cervix cancer. The combination of different genotypes had high susceptibility risk for cervix cancer in Chhattisgarh. In conclusion, our result is consistent with earlier research findings.

 

ACKNOWLEDGEMENTS:

Our sincere thanks to Department of Science and Technology (DST), New Delhi for providing DST-FIST grant to School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur (CG). We are grateful for the cooperation received from all the patients and controls who voluntarily participated in the present study. We are also thankful to the doctors and staff of Department of Gynaecology, Dr. B.R. Ambedkar Memorial Hospital, Raipur (CG) and Indira Gandhi Regional Cancer Centre, Raipur (CG) for their support. Technical help provided by the laboratory technicians of the same department is duly acknowledged.

 

REFERENCES:

1.       Castellsague XS, de Aguado S, Louie D et al. HPV and cervical cancer in the world: 2007 report. Vaccine. 2007; 25(3):C1-C230.

2.       Parkin DM. Global cancer statistics in the year 2000. Lancet Oncology. 2001; 2:533-543. 

3.       Parkin DM, Bray FI and Devesa SS. Cancer burden in the year 2000: The global picture. European Journal of Cancer. 2001; 37(8):64-66.

4.       Bidwell J, Keen L, Gallagher G et al. Cytokine gene polymorphism in human disease: on-line databases. Genes Immunology. 1999; 1:3-19.

5.       Tracey KJ and Cerami A. Tumor necrosis factor, other cytokines and disease. Annual Review of Cell Biology. 1993; 9:317-343.

6.       Mosaffa F, Kalalinia F, Lage H et al. Pro-inflammatory cytokines interleukin-1 beta, interleukin 6, and tumor necrosis factor-α alter the expression and function of ABCG2 in cervix and gastric cancer cells. Molecular and Cell Biochemistry. 2012; 363:385-393.

7.       Polunovsky VA, Wendt CH, Ingbar DH et al. Induction of endothelial cell apoptosis by TNF-α: modulation by inhibitors of protein synthesis. Exp Cell Research. 1994; 214:584-594.

8.       Kroeger KM, Steer JH, Joyce DA et al. Effects of stimulus and cell type on the expression of the -308 tumour necrosis factor promoter polymorphism. Cytokine. 2000; 12:110-119.

9.       Nedwin GE, Naylor SL, Sakaguchi AY et al. Human lymphotoxin and tumour necrosis factor genes: structure, homology and chromosomal localization. Nucleic Acids Research. 1985; 13(17):6361-6373.

10.     Wilson AG, Symons JA, McDowell TL et al. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proceedings of National Academy of Sciences, USA. 1997; 94:3195-3199.

11.     Kroeger KM, Carville KS and Abraham LJ. The -308 tumor necrosis factor-alpha promoter polymorphism effects transcription. Molecular Immunology. 1997; 34:391-399.

12.     Wu WS and McClain KL. DNA polymorphisms and mutations of the tumor necrosis factor-alpha (TNF-alpha) promoter in Langerhans cell histiocytosis (LCH). Journal of Interferon and Cytokine Research. 1997; 17:631-635.

13.     Pan F, Tian J, Ji CS et al. Association of TNF-α-308 and -238 polymorphisms with risk of cervical cancer: a meta-analysis. Asian Pacific Journal of Cancer Prevention. 2012; 13:5777-5783.

14.     Liu L, Yang X, Chen X et al. Association between TNF-α polymorphisms and cervical cancer risk: a meta-analysis. Molecular Biology Reports. 2012; 39:2683-2688.

15.     Li M, Han Y, Wu TT et al. Tumor necrosis factor α -rs1800629 polymorphism and risk of cervical lesions: a meta-analysis. PLoS One. 2013; 8:e69201.

16.     Zhang HL and Zhang YJ. A systemic assessment of the association between tumor necrosis factor alpha 308 G/A polymorphism and risk of cervical cancer. Tumour Biology. 2013; 34:1659-1665.

17.     Coussens IM and Werb Z. Inflammation and cancer. Nature. 2002; 420(6917):860-867.

18.     Fong CL, Siddiqui AH and Mark DF. Identification and characterization of a novel repressor site in the human tumour necrosis factor alpha gene. Nucleic Acids Research. 1994; 22:1108-1114.

19.     Duarte I, Santos A, Sousa H et al. G-308 A TNF-α polymorphism is associated with an increased risk of invasive cervical cancer. Biochemistry and Biophysics Research Communication. 2005; 334:588-592.

20.     Jang WH, Yang YI, Yea SS et al. The 238 tumor necrosis factor-α promoter polymorphism is associated with decreased susceptibility to cancers. Cancer Letter. 2001; 166:41-46.

21.     Sengupta S, Farheen S, Mukherjee N et al. DNA sequence variation and haplotype structure of the ICAM 1 and TNF genes in 12 ethnic groups of India reveal patterns of importance in designing association studies. Annals of Human Genetics. 2004; 68:574-587.

22.     Gupta V and Sehajpal PK. Rapid detection of single nucleotide (-308) polymorphism in TNF-α promoter using ARMS-PCR. Current Science. 85; 2003: 1521-1523.

 

 

 

 

 

 

Received on 30.10.2018          Modified on 23.11.2018

Accepted on 21.12.2018        © RJPT All right reserved