|Year : 2018 | Volume
| Issue : 2 | Page : 179-183
Impact of gene polymorphism of TNF-α rs 1800629 and TNF-β rs 909253 on plasma levels of South Indian breast cancer patients
Karuvaje Thriveni1, Anisha Raju1, Girija Ramaswamy1, S Krishnamurthy2
1 Department of Biochemistry, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India
2 Department of Surgical Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India
|Date of Web Publication||31-Dec-2018|
Dr. Karuvaje Thriveni
Department of Biochemistry, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
AIM: Inflammation plays a lead role in the tumor microenvironment and promotes metastasis. Single-nucleotide polymorphism (SNP) in the tumor necrosis factor (TNF) gene locus may alter the expression of genes and proteins. The objective of the study is to find the distribution of genetic polymorphism in the sites of TNF-α −308G>A and TNF- β +252A>G in breast cancer and evaluate polymorphism effects on plasma levels. MATERIALS AND METHODS: The study group consisted of 109 invasive ductal primary breast cancer patients and 75 age-matched healthy female controls. Plasma cytokine concentrations were measured by the MILLIPLEX® MAP Human Cytokine/Chemokine Panel magnetic bead kits. The genotyping procedure for SNP included allele-specific polymerase chain reaction for TNFα and restriction fragment length polymorphism for TNFβ. RESULTS: Odds ratio with 95% confidence interval showed that these polymorphisms were not a causative risk factor, and both polymorphisms were consistent with Hardy–Weinberg equilibrium. Plasma TNFα and TNFβ median concentrations were significantly higher in cases when compared to controls (P < 0.01). When plasma TNFα levels were grouped under polymorphic subtypes, patients with mutant TNF- α −308A allele showed significantly higher values (P < 0.001). In addition, plasma TNFα values were significantly elevated in mutant TNF-β +252G allele (P < 0.01). CONCLUSION: This study demonstrated that there is no significant association between SNPs and breast cancer susceptibility in South Indian population. However, plasma TNFα level is significantly elevated with mutant-recessive TNF-α −308 A and TNF-β +252 G alleles of patients.
Keywords: Breast cancer, inflammation, polymorphism, tumor necrosis factor
|How to cite this article:|
Thriveni K, Raju A, Ramaswamy G, Krishnamurthy S. Impact of gene polymorphism of TNF-α rs 1800629 and TNF-β rs 909253 on plasma levels of South Indian breast cancer patients. Indian J Cancer 2018;55:179-83
|How to cite this URL:|
Thriveni K, Raju A, Ramaswamy G, Krishnamurthy S. Impact of gene polymorphism of TNF-α rs 1800629 and TNF-β rs 909253 on plasma levels of South Indian breast cancer patients. Indian J Cancer [serial online] 2018 [cited 2019 Jan 22];55:179-83. Available from: http://www.indianjcancer.com/text.asp?2018/55/2/179/249212
| » Introduction|| |
Breast cancer is highly heterogeneous, and several factors contribute to its development and progression. Chronic inflammation, the seventh hallmark of cancer, is a critical component responsible for increased risk of developing various cancers by recruiting inflammatory cells to precancerous lesions. As tumor necrosis factor (TNF) receptor is present in both epithelial cells and stromal cells, circulating TNF in the microenvironment can directly foster tumor proliferation and survival of neoplastic cells. TNFα is involved in all the steps of tumorigenesis as it induces tumor initiation and promotion. Proinflammatory cytokine TNF plays an important role in inflammation, cell differentiation, proliferation, and immune response. TNFα and TNFβ genes are located closely and tandem arranged within highly polymorphic and the largest (approximately 4 Mb) Class III major histocompatibility complex separated at a distance of 7 kb on chromosome 6p21.3. TNFα is synthesized in activated macrophages and has a role in immunoregulatory pathways, causing neoplastic cachexia., The presence of single-nucleotide polymorphism (SNP) in the promoter, regulatory, or intronic region may influence the transcription of TNF gene and affect the circulating level of TNF.
TNF production is regulated at the transcriptional level and involved in acute-phase reaction. Lymphocytes produce TNFβ, also known as lymphotoxin alpha, as a cytotoxic factor. Systemic release of TNFα and TNFβ from macrophages and lymphocytes may influence the disease prognosis. Among the cytokines, TNFα elicits a wide spectrum of activities by interacting with other cytokine networks. TNFα expression level correlates with HLA DR alleles and polymorphic sequences of TNFβ locus, namely, NcoI restriction sites of first intron region of TNFβ gene.
Polymorphism exists in synonymous and nonsynonymous forms. The promoter region of TNF- α–308G>A reference sequence (rs) 1800629 is a synonymous polymorphism and impacts the transcriptional activity of TNFα production. TNF- β+252A>G rs 909253 polymorphism is in the first intronic region. A functional polymorphism in the intronic region may have a great impact on gene and protein expression. It is known that cytokines play a pivotal role in inflammation, immune mediation, and regulation of cancer development. This study aimed to investigate whether the SNP TNF- α –308G>A (rs 1800629) and TNF- β+252A>G (rs 909253) are associated with the plasma levels of TNFα and TNFβ and breast cancer risk.
| » Materials and Methods|| |
The scientific review board and Medical Ethics Committee of the institute approved this study. Each individual signed the informed consent and agreed to participate in the study. The study cohort included 109 histopathologically confirmed breast cancer cases with invasive ductal carcinoma registered from June 2014 to December 2014. A healthy control group included 75 age-matched females who did not suffer from fever or any other inflammatory diseases in the study. We collected 5-ml blood samples from controls and cases prior to treatment. Blood samples were centrifuged at 3,000 rpm, and plasma was stored at −80°C till the analysis. From the leukocytes, genomic DNA (gDNA) was extracted by the modified conventional phenol-chloroform method and stored at −20°C. The concentration of gDNA was measured by Eppendorf Biospectrometer.
We measured plasma cytokine concentration by the MILLIPLEX® MAP Human Cytokine/Chemokine Panel magnetic bead kits. Genotyping analysis was carried out by allele-specific polymerase chain reaction (AS-PCR) for TNFα and restriction fragment length polymorphism (RFLP) for TNFβ polymorphism by a modified method.
AS-PCR was carried out with a forward primer (F) 5'-TCTCGGTTTCTTCTCCATCG-3' for the polymorphism of −308G>A 184 base pair fragments of TNF gene. We used a reverse primer (R1) 5'-ATAGGTTTTGAGGGGCATGG-3' as pair F-R1 for G allele and F-R2 (5'-ATAGGTTTTGAGGGGCATGA-3') for A allele. gDNA of each sample was tested with F-R1 and F-R2 primer pairs. For a heterozygous trait, amplification was found with both sets of primers. An internal control primer pair F-R3 (5'-GAGTCTCCGGGTCAGAATGA-3') was used to amplify 531 bp of TNF gene fragment, which improved the specificity of AS-PCR. The PCR was performed in a 30-μl reaction mixture with 50–100 ng template DNA, 1 U of Taq polymerase enzyme (SRL) and 1X buffer provided along with enzyme. Respective primers (0.3 μM) (Sigma), 0.5 mM deoxyribonucleotide triphosphate (dNTP) mix, and 1.5–2 mM MgCl2 constituted the PCR mix. After initial denaturation at 95°C for 10 min, PCR reactions were carried out for 31 cycles, which consisted heat denaturation at 95°C for 1 min 30 s, annealing for 150 s at 62°C for primer pair F-R1 and 60°C for primer pair F-R2, and extension at 72°C for 1 min. The PCR amplification processes were carried out in the thermal cycler S1000 Bio Rad Laboratories, USA.
Genetic polymorphism +252A>G was detected by the PCR-RFLP method. The PCR reactions were carried out in a 30-μl reaction mixture containing 1 μM mixture of forward 5'-CTCCTGCACCTGCTGCCTGGATC-3' and reverse primers 5'-GAAGAGACGTTCAGGTGGTGTCAT-3'. PCR master mix contained 200 μM of dNTP, Taq DNA polymerase 1X buffer with 1.5 mM MgCl2, and 2.5 units Taq polymerase. To this, we added 200 ng/μL of gDNA as template. After initial denaturation at 95°C for 10 min, 31 cycles of PCR reactions were carried out as heat denaturation at 96°C for 1 min, annealing at 65°C for 1 min, and extension at 72°C for 2 min. This was followed by final extension at 72°C for 5 min. The 368 bp PCR amplicon was then subjected to restriction digestion.
A transition substitution at the nucleotide position +252 of TNFβ gene from A to G creates Nco I restriction enzyme-cleaving sites. The amplicons were digested by 2U of restriction enzyme Nco I (New England Biolabs, Ipswich, Massachusetts) at 37°C overnight followed by 2% agarose gel electrophoresis. PCR amplicon 368 bp of TNF- β +252AA wild-type allele was intact even after restriction digestion. The amplicon of homozygous mutant with GG allele was cleaved into two 133 bp and 235 bp fragments. The presence of all the three base pairs, 368 bp, 235 bp, and 133 bp, in a sample indicated the presence of heterozygous GA allele.
R-statistics software version 3.2.2 (R Foundation for Statistical Computing, Vienna, Austria) was used to perform all the statistical analyses. Two-way contingency table was used to calculate the odds ratio (OR) with 95% confidence interval (CI). Chi-square test was used to find whether polymorphism was consistent with Hardy–Weinberg equilibrium. The plasma values did not fall into normal distribution as checked by Kolmogorov–Smirnov and Shapiro–Wilk normality tests. Hence, nonparametric Mann–Whitney U-test was used to calculate the median values and interquartile range for continuous variables with tests of significance in different polymorphic subtypes. Kruskal–Wallis test measured median values of three groups of genotypes and calculated the t value. Type I error rate was 5% throughout the analysis.
| » Results|| |
Distribution of polymorphism
Mean age (with a standard deviation) of patients was 49 ± 12 years with a range of 25–80 years. Samples from age-matched healthy controls (±4 years) were collected, and the parameters were analyzed. [Table 1] shows the distribution of dominant G allele of TNF- α–308G>A polymorphism in controls and cases as 78% and 77%, respectively. The allele frequencies of dominant A allele in TNF- β+252 were 79.3% and 75% in controls and cases, respectively. OR with 95% CI indicated that cases with TNF- α –308 A recessive allele had 1.05 value and no risk of developing breast cancer. However, in cases with TNF- β+252G, recessive allele increased the risk to 1.27 (0.66–2.46) times but was not found to be associated with cancer (P > 0.05).
|Table 1: Distribution of tumor necrosis factor-α-308 and tumor necrosis factor-β +252 polymorphic alleles in case and control groups|
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Chi-square (χ2) with P value was calculated by Court lab calculator. The distribution of genotype frequencies did not deviate from Hardy–Weinberg equilibrium with χ2 value as 0.47 and 2.8, respectively, for both polymorphisms of cases (P > 0.05). The minor allele frequencies were 0.21 for TNFα and 0.25 for TNFβ polymorphism.
Plasma levels of tumor necrosis factor
Plasma TNFα levels in cases and controls according to the distribution of genotypes were calculated. The detailed values for the concentration of TNFs are summarized in [Table 2]. In overall cases, median plasma values were 10.0 pg/ml and 7.6 pg/ml for TNFα and TNFβ, respectively, which were significantly raised when compared to the control group. Cases with wild-type homozygote reference genotype GG (60%) showed decreased plasma TNFα values (≤12.6 pg/ml). Similarly, lower plasma TNFα values (≤16.2 pg/ml) were observed in wild-type TNFβ AA genotype (60%). Median plasma values of TNFα were increased significantly in TNF α−308 A variant allele (up to 36.75 pg/ml) and in + 252 TNFβ G variant allele (up to 35.6 pg/ml). The control group did not show significant plasma cytokine elevation in these alleles. Plasma TNFβ values were not altered in any of the TNF- α –308 and TNF- β+252 alleles, indicating a lesser impact of polymorphism on TNFβ expression.
|Table 2: Comparison of the plasma levels of tumor necrosis factor-α and tumor necrosis factor-β in tumor necrosis factor-α -308 and tumor necrosis factor-β +252 alleles|
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[Table 3] shows detailed median plasma TNFα case values in all the three genotypes of TNF- α–308 polymorphism. We considered plasma levels of wild-type versus heterozygous genotype (GG:GA) and wild-type versus variant genotype (GG:AA). The compared values were highly significant showing t value of 3.26 and 3.01, respectively. This table shows that breast cancer cases with heterozygous TNF- α–308 GA and recessive variant AA genotypes had significantly elevated plasma TNFα median values of 15.95 pg/ml and 27.23 pg/ml, respectively. Plasma TNFα values were also significantly elevated in genotype TNFβ GA (t = 2.52, P = 0.03). Plasma TNFβ values were not evaluated further because the values were not significant, as shown in [Table 2].
|Table 3: Plasma values of tumor necrosis factor-α in tumor necrosis factor-α-308 genotypes of cases by Kruskal-Wallis test|
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| » Discussion|| |
Breast cancer is the most prevalent cancer in India and is associated with a high degree of mortality and morbidity. Inflammation is prominent in the early stages, and proinflammatory TNFs are released as an early event. The link between inflammation and cancer is confirmed by TNFα. The epithelial-to-mesenchymal transition (EMT) process plays a convergence point between inflammation and tumor progression to metastasis. By inhibiting TNFα, a marked reduction in tumor onset and tumor burden has been found. TNFα elicits nuclear factor-κB (NF-κB) activation, which, in turn, inhibits programmed cell death. NF-κB influences prosurvival activity through antiapoptotic complex proteins.
In this study, we described TNF gene polymorphism, circulating TNF level, and risk of breast cancer. In the pathogenesis of breast cancer, inflammation is common and causes metastasis. Tumor aggressiveness is higher due to genetic alterations. When a genetic variation occurs in >1% population in a nucleotide of DNA sequence, it is considered as SNP. Most of the SNPs occur in the intronic region of genes; however, a few SNPs exist in exons and are commonly associated with chronic diseases including cancer. Till date, around 16 polymorphisms are known to be present in and around the TNF gene, of which −308 G/A and −238 G/A promoter polymorphisms are the most studied. These polymorphisms affect the promoter region with a consequent increase in the TNFα protein as evidenced by the studies carried out on non-Hodgkin's lymphoma and myeloma.
Polymorphism at TNF- α −308G>A promoter region has the highest impact on transcriptional activity and secretion levels of the cytokine and showed that the functional polymorphism with −308A allele is associated with increased TNFα production. TNF- α −308A allele has been associated with increased plasma levels of TNFα in comparison with TNF- α −308G allele. Transition substitutions from G to A allele may induce transcriptional factors to bind with higher affinity at this polymorphic sites, leading to the activation of TNFα gene. However, various studies have reported diverse distribution of these alleles. Previous studies on TNF- α −308G/A polymorphism in various other cancers such as pancreatic cancer, colorectal cancer, and non-small cell lung carcinoma reported an elevated serum, and plasma TNFα level in patients compared to the control group.
Circulating TNFα levels were significantly elevated in asthmatic patients who carry A allele (genotypes AA and GA) of polymorphism TNF- α −308G>A. Thus, SNP at promoter region showed a significantly raised plasma level of TNFα. A metaanalysis by Jin et al. showed that patients with TNF- α −308A allele had higher plasma levels of TNFα and vulnerability to disease progression, metastasis, and immune-related breast neoplasm. In contrast, Chae et al. showed that TNFα level is not altered due to polymorphism. Messer et al. reported that TNF- β+252A allele reduced the level of TNFβ cytokine secretion. Waters et al. showed that TNFα as an inflammatory marker in the tumor microenvironment promotes metastasis by triggering EMT pathways. Thus, transcription factors activated by TNFα are prime key to invasion and metastasis that rely on activation of NF-κB signaling pathways. Posttranslationally, TNFα can regulate the expression of transcription factor Snail and induce EMT through NF-κB signaling pathway and promote migration of tumor cells. Ubiquitination is the most widely used protein modification to regulate cellular signaling and homeostasis. The inflammatory cytokine TNFα-induced NF-κB pathway is capable of inducing proliferation or apoptosis depending on the state of K63-linked poly-ubiquitination.
The current study does not show an association between TNF- β+252A>G polymorphism and plasma levels of TNFβ and risk of breast cancer. The results of study by Pierce et al. suggested that circulating inflammatory markers might provide early information about the risk of disease recurrence in patients with no evidence of metastatic disease and no current indication of cancer. SNP as a confounding factor helps in the evaluation of cancer susceptibility mechanisms and the complexity of the genetic heritage of risk allele. Few studies showed that these polymorphisms were associated with breast cancer risk. Varying lifestyle and the presence of different carcinogens in the atmosphere are the diverse factors, which affect the regional variation patterns of cancer. One of the limitations of the study was the lesser number of samples. A better statistical outcome may be achieved with a larger number of cases. Studying more than two polymorphisms may help to find the linkage disequilibrium. It should be noted that this study performed concurrent analysis of plasma TNF value with the SNP.
| » Conclusion|| |
In general, the effect of polymorphism on plasma levels of cytokine is not clear. In our study, plasma TNFα was significantly elevated in TNF- α −308A and TNF- β+252G variant alleles. However, these polymorphisms were not a causative risk factor for breast cancer. The polymorphism in the promoter region is associated with excess production of TNFα, a candidate biomarker for tumor metastasis. Stratification of the patients and clinical pertinence may help in targeting therapy. Further, studies with large sample size may help to find the possible association of polymorphism with the circulating levels of TNF. Haplotype analysis with more number of SNPs in the nearby gene showing linkage disequilibrium will help in finding the association between polymorphism and breast cancer risk.
The Institutional Ethical Committee approved this study, which was performed with the standard laid down in the 1964 Declaration of Helsinki. Each individual gave their signed informed consent prior to blood sample collection.
The authors acknowledge the Science and Engineering Research Board, New Delhi, for funding this project. We thank all the women who participated in the study by giving blood samples.
Funding for this study was provided by the Science and Engineering Research Board, DST, New Delhi.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]