Association between tumor necrosis factor-alpha gene rs1800629 ( - 308G/A) and rs361525 ( - 238G > A) polymorphisms and prostate cancer risk in an Iranian cohort

Abstract

BACKGROUND:

Prostate cancer (PCa) as the first men’s common cancer in the world and the third cancer in Iranian men is a heterogeneous disorder which sometimes several biopsies are needed for its diagnosis.

OBJECTIVES:

The aim of current study is finding new biomarkers in order to diagnose of PCa at the earliest possible stage. Hence, the relationship between rs1800629 and rs361525 polymorphisms of TNF- $\alpha$ gene with PCa was investigated.

MATERIALS AND METHODS:

Blood DNA samples were collected from 100 patients with PCa, 110 with BPH, and 110 controls. Collected samples were examined using PCR-RFLP and Tetra-ARMS-PCR techniques to detect the desired polymorphisms.

RESULTS:

The frequency of rs1800629 genotypes in smokers was significantly different from non-smokers with PCa ( $p=$ 0.001). Logistic regression analysis results showed that GA heterozygotes in comparison to GG homozygotes had higher risk of developing Benign Prostatic Hyperplasia (BPH) or prostate cancer. However, no significant correlation was considered between the risk of PCa and the TNF- $\alpha$ gene polymorphisms (rs1800629 and rs361525).

CONCLUSIONS:

Although, the achieved results of this investigation demonstrated that the two examined genetic variants do not seem to be suitable markers for early diagnosis of prostate cancer in this pilot study; however increased risk for the disease is shown in GA heterozygotes and smokers which is indicative of some epigenetic factors influence on prostate cancer etiology.

Keywords

Prostate cancer benign prostate hyperplasia TNF-α polymorphism

1. Introduction

The prostate is the largest accessory gland of the male reproductive system and the organ associated in Prostate cancer (PCa), Benign prostatic hyperplasia (BPH), and Prostatitis [1, 2]. The prostate cancer, is the second most common malignancies in men all over the world and the third common cancer among Iranian males [3]. It is a heterogeneous disorder as a normal age-related phenomenon [4] and rare amongst men younger than 40 years [5] which includes 7–9 percent of all cancers in Iran where most cases occur over 50 years of age [6]. Risk factors such as age, ethnicity/race, familial inheritance, and lifestyle may induce PCa growth in addition to genetic and epigenetic factors [7]. There are many clinical tests which have been used to diagnose the prostate cancer including the prostate-specific antigen (PSA) blood test [8], digital rectal exam (DRE) [9], magnetic resonance imaging (MRI) [10], sonography [11], and prostate biopsy (Gleason scoring) [12]. The above-mentioned clinical tests have their own disadvantages. For instance, information obtained from a single biopsy is not sufficient to diagnose and guide treatment decisions due to the heterogeneity of the prostate tissue [13]. So, the current study was carried out to determine new biomarkers for early diagnosis of PCa.

Genetic up to 42% and/or epigenetic alterations affect PCa through changing in the gene transcription and function in many malignancies [14]. Recent studies suggest that genetic polymorphisms in cytokines might be associated with increasing cytokine production and inflammation and eventually increasing prostate cancer risk [15].

Tumor necrosis factor alpha (TNF- $\alpha$ ), as a pro-inflammatory cytokine, which is secreted by various types of cells such as macrophages, T cells, and natural killer (NK) cells [16], plays significant roles in the pathogenesis of several diseases [17]. TNF- $\alpha$ activates oncogenic transcription factors such as NF-kB and AP-1 [18] which accelerate cell proliferation, inflammation reaction, and anti-apoptotic activity. The prolonged inflammation in prostate tissue and escape from immune response may increase the fortuity of initiation and progression of cancer [19, 20, 21]. The TNF- $\alpha$ protein induces the expression of adhesion molecules and invasion of metastatic tumor cells [22].

Moreover, TNF- $\alpha$ gene is located in the class III region of the major histocompatibility complex (MHC III) on chromosome 6p21.3 [23] with 4 exons [24] and has significant roles in the regulation of differentiation, death, innate, and adaptive immune responses. Any impairment in the gene structure may associate with a variety of human diseases like infectious, cancer, autoimmune, etc. [25]. Several single nucleotide polymorphisms (SNPs) of the gene have been associated with susceptibility to several cancers including the breast, stomach, lung, and prostate cancers [26, 27, 28, 29]. rs1800629 is one of the most common polymorphisms with a G to A transition at $-$ 308 (TNF- $\alpha$ $-$ 308G/A) nucleotide upstream from the transcription initiation site in the promoter region, that can affect the TNF- $\alpha$ levels [30]. The other common polymorphism which is located at $-$ 238 of the same promoter is rs361525 where a G to A substitution is happened (TNF- $\alpha$ -238G/A) and possibly affects the expression levels of TNF- $\alpha$ [31]. Previous data have shown that these polymorphisms are considered as significant modifiers of transcription activation and are associated with increased release of TNF- $\alpha$ . Therefore, it is assumed that TNF- $\alpha$ polymorphism may be positively related to BPH and prostate cancer risk [32].

Hence, the current case-control study was done to investigate any association of TNF-a gene variants (rs361525 and rs1800629) with increasing the risk of the disease in a group of Iranian population.

2. Materials and methods

2.1 Subjects

In this study, blood samples include 100 PCa patients, 110 BPH, and 110 healthy individuals which were collected from the Pathology Department, Shohada-e Tajrish hospital. All participants provided a personal questionnaire including body mass index (BMI), PSA level, smoking consumption, family history of cancer, and detailed medical history. All participants had over 45 years old [33] and were matched by age.

Patients were selected based on the pathological findings including high PSA level and tissue biopsy with or without a family history of the disease. BPH group was selected according to the digital rectal examination (DRE), prostate volume (more than 30 ml), and urologist’s opinion (based on symptoms) without any history of malignancy and cancer. Patients with prostate volume less than 25 cm ${}^{2}$ were not selected for BPH group. Healthy subjects were selected based on healthy prostate through clinical and paraclinical examinations such as DRE and PSA levels ( $<$ 4 ng/ml) and no personal or family history of PCa or BPH. The recipients of any hormone/chemotherapy, and/or radiotherapy were excluded from the study. All participants provided written informed consent.

Table 1
Primers sequences, PCR conditions, and fragment sizes for genotyping of the TNF- $\alpha$ gene polymorphisms

Method	SNP	Primers sequences	PCR	Fragment size
			conditions
PCR-RFLP	rs361525 ( $-$ 238G/A)	F: 5’-ATCTGGAGGAAGCGGTAGTG-3’ R: 5’-AGAAGACCCCCCTCGGAACC-3’	95 ${{{}^{\circ}}}$ –5 min 95 ${{{}^{\circ}}}$ –30 sec 59 ${{{}^{\circ}}}$ –30 sec 38 cycles 72 ${{{}^{\circ}}}$ –20 sec 72 ${{{}^{\circ}}}$ –7 min	G allele: 132 $+$ 20 bp A allele: 152 bp
T-ARMS-PCR	rs1800629 ( $-$ 308G/A)	${\text{F}}_{\text{Ou}}$ : 5’-AGGACTCAGCTTTCCGAAGCCCCTCCCA-3’ $\text{F}_{\text{in}}$ : 5’-GGAGGCAATAGGTTTTGAGGCGCAGGG-3’ $\text{R}_{\text{Ou}}$ : 5’-TTCTGTCTCGGTTTCTTCTCCATCGCGG-3’ $\text{R}_{\text{in}}$ : 5’-GTAGGACCCTGGAGGCTGAACCCCGTACT-3’	95 ${{{}^{\circ}}}$ –5 min 95 ${{{}^{\circ}}}$ –30 sec 71.5 ${{{}^{\circ}}}$ –25 sec 38 cycles 72 ${{{}^{\circ}}}$ –20 sec 72 ${{{}^{\circ}}}$ –7 min	G allele: 304 $+$ 197 bp A allele: 304 $+$ 162 bp

Figure 1.

The electrophoresis analysis of TNF- $\alpha$ promoter region amplified PCR product. A fragment of 152 bp indicates TNF- $\alpha$ rs361525 on 2% agarose gel M: 50 bp DNA marker, S1 and S2: two examined samples.

Figure 2.

Polyacrylamide gel electrophoresis analysis of rs361525 polymorphism genotypes of the TNF- $\alpha$ gene on 12% polyacrylamide gel (PAGE) after PCR-RFLP amplification of the DNA samples and digestion by MspI restriction enzyme. Column 1: 100 bp DNA ladder, Column 2: GA heterozygote (152, 132 and 20 bp), Column 3: GG homozygote (132 and 20 bp). ${}^{*}$ The 20 bp fragments are not visible due to the short length in both columns. However, the sequencing of the larger fragments confirmed the separation of the 20 bp genomic segment from it.

2.2 DNA extraction

Genomic DNA of peripheral blood samples was extracted using salting-out method. Nanodrop spectrophotometer and 1% agarose gel electrophoresis were used to measure the concentration, degree of purity, and protein contamination status of the DNA and evaluate its quality.

Figure 3.

The sequencing result of TNF- $\alpha$ rs361525 gene polymorphism PCR products. A: The homozygous GG genotype indicates the presence of the wild allele G. B: The heterozygous GA genotype contains two alleles A and G, respectively.

2.3 Genotyping of rs361525 polymorphism

TNF- $\alpha$ gene promoter $-$ 238G/A (rs361525) polymorphism was detected by PCR-RFLP according to Özhan et al. [34]. Primer sequences and PCR conditions for this SNP are listed in Table 1. PCR reactions were performed using 1 $\mu$ l (100 ng) of genomic DNA, 0.5 $\mu$ l (10 pMol) of each primer (Bioneer, South Korea), 8 $\mu$ l of Taq DNA Polymerase 2x Master Mix RED (Ampliqon, Denmark), and 8 $\mu$ l of ddH ${}_{2}$ O. Moreover, PCR amplification was accomplished by thermal cycler PCR (SimpliAmp Thermal Cycler, ABI, USA). In the next step, PCR products were analyzed by 2% of agarose gel electrophoresis containing 4 $\mu$ l of gel stain (SMOBIO, Taiwan) and was examined on gel documentation apparatus (UVIdoc HD5, UVItec Cambridge, UK). The PCR products of 152 bp fragments were considered alongside a 50 bp of DNA Ladder (SMOBIO, Taiwan), as shown in Fig. 1. Afterwards, the PCR products were digested by MspI restriction enzyme (Fermentas, Hunover, MD, USA) at 37 ${}^{\circ}$ C for 16 h and then separated by polyacrylamide gel electrophoresis (PAGE, 12%), beside a 100 bp marker (SMOBIO, Taiwan), and then was stained with silver nitrate and visualized under a UV Transilluminator (VILBER lourmat). According to the Fig. 2, three genotypes were identified after the digestion which are including two 152 bp fragments (attributed to the AA genotype), two fragments of 132 bp/20 bp (the GG genotype), and three different fragments of 152/132/20 bp bands (the GA genotype). Finally, some of the PCR products of each genotype were randomly sequenced to confirm PCR-RFLP results (Genomin Company, Iran), as shown in Fig. 3.

Figure 4.

Electrophoresis view of Tetra-ARMS-PCR generated fragments. Different TNF- $\alpha$ $-$ 308G $>$ A gene polymorphism genotypes of undergoing examination subjects are observed in columns 1 and 2. From left to right: M: DNA marker; 1: Heterozygous GA genotype (304,197 and 162 bp); 2: Homozygous GG genotype (304 and 197 bp).

Table 2

Demographic and clinical characteristics of the studied groups

	Control ( $n=$ 110)	BPH ( $n=$ 110)	PCa (100)	$P$ value
Characteristics
Age ${}^{*}$	66.28 $\pm$ 8.43	67.43 $\pm$ 8.6	67.78 $\pm$ 8.17	0.396
PSA ${}^{*}$ (ng/ml)	1.67 $\pm$ 0.87	5.81 $\pm$ 4.05	20.75 $\pm$ 29.19	$<$ 0.001
BMI ${}^{*}$	24.12 $\pm$ 4.14	25.07 $\pm$ 3.45	25.07 $\pm$ 3.67	0.104
Smoking status
Non smokers	71 (64.5%)	61 (55.5%)	56 (56%)	0.312
Current or former smokers	39 (35.5%)	49 (44.5%)	44 (44%)
Prostate volume ${}^{*}$	Not available	62.69 $\pm$ 23.48	50.01 $\pm$ 24.24	$<$ 0.001
Gleason score
$\leqslant$ 6	NA ${}^{**}$	NA ${}^{**}$	50 (50%)
$>$ 6	NA ${}^{**}$	NA ${}^{**}$	50 (50%)

Significant $p$ values are shown in bold. ${}^{*}$ Mean $\pm$ SD, ${}^{**}$ Not applicable.

Figure 5.

The frequency of genotypes in the understudy groups for the TNF- $\alpha$ polymorphisms. A: rs361525 and B: rs1800629.

2.4 Genotyping of rs1800629 polymorphism

TNF- $\alpha$ gene promoter $-$ 308G/A (rs1800629) polymorphism was investigated using Tetra-ARMS-PCR according to Solhjoo et al. [35]. The sequences of used primers and PCR conditions for this SNP are presented in Table 1. PCR reactions were prepared using 2 $\mu$ l (100 ng) of genomic DNA, 0.2 $\mu$ l of forward outer primer, 0.2 $\mu$ l of revers outer primer, 0.7 $\mu$ l of forward inner primer, 0.7 $\mu$ l of reverse inner primer (10 pMol each primer) (Bioneer, South Korea), 10 $\mu$ l of taq DNA Polymerase 2x Master Mix RED, and 10 $\mu$ l of ddH ${}_{2}$ O. PCR was carried out using FlexCycler (Analytic Jena, Germany). The PCR products were analyzed by 2% agarose gel containing 4 $\mu$ l Gel stain and 100 bp of DNA marker and were detected by a UV transilluminator. The PCR products including 304/162 bp fragments (homozygote AA genotype), those with bands of 304/197/162 bp (GA heterozygote genotype), and samples with 304/197 bp bands (homozygote GG genotype) were shown in Fig. 4.

2.5 Statistical analysis

We used Chi-square test ( $\chi^{2}$ ) to compare the frequency of genotypes and alleles in three studied groups to investigate the association between the two polymorphisms genotypes to analyze the demographic data and the Hardy-Weinberg equilibrium. $P$ -values less than 0.05 were considered as statistically significant. Multivariable logistic regression test was used to compute the odd ratio (OR) with 95% confidence interval (CI). All analyses were performed using SPSS v.25 software.

Table 3
Association between the genotypes of polymorphisms with demographic information

			rs1800629 $n$ (%)				$P$ -value	rs361525 $n$ (%)				$P$ -value
			GG		GA			GG		GA
Normal	Age	$<$ 70	67	(83.8)	13	(16.3)	0.070	75	(93.8)	5	(6.3)	0.161
		$>$ 70	29	(96.7)	1	(3.3)		30	(100)	0	(0)
BPH	Age	$<$ 70	60	(84.5)	11	(15.5)	0.704	67	(94.4)	4	(5.6)	0.672
		$>$ 70	34	(87.2)	5	(12.8)		36	(92.3)	3	(7.7)
Cancer	Age	$<$ 70	53	(81.5)	12	(18.5)	0.360	61	(93.8)	4	(6.2)	0.081
		$<$ 70	31	(88.6)	4	(11.4)		29	(82.9)	6	(17.1)
Normal	PSA	$<$ 4	96	(87.3)	14	(12.7)	–	105	(95.5)	5	(4.5)	–
BPH	PSA	$<$ 4	37	(82.2)	8	(17.8)	0.632	44	(97.8)	1	(2.2)	0.199
		4–10	45	(86.5)	7	(13.5)		48	(92.3)	4	(7.7)
		$>$ 10	12	(92.3)	1	(7.7)		11	(84.6)	2	(15.4)
Cancer	PSA	$<$ 4	6	(75.0)	2	(25.0)	0.304	7	(87.5)	1	(12.5)	0.577
		4–10	32	(91.4)	3	(8.6)		33	(94.3)	2	(5.7)
		$>$ 10	46	(80.7)	11	(19.3)		50	(87.7)	7	(12.3)
Normal	Smoke	Yes	35	(897)	4	(10.3)	0.564	37	(94.9)	2	(5.1)	0.828
		No	61	(859)	10	(14.1)		68	(95.8)	3	(4.2)
BPH	Smoke	Yes	42	(857)	7	(14.3)	0.945	45	(91.8)	4	(8.2)	0.488
		No	52	(852)	9	(14.8)		58	(95.1)	3	(4.9)
Cancer	Smoke	Yes	31	(705)	13	(29.5)	0.001	37	(84.1)	7	(15.9)	0.081
		No	53	(946)	3	(5.4)		53	(94.6)	3	(5.4)
BPH	Volume	$<$ 30	5	(71.4)	2	(28.6)	0.512	7	(100)	0	(0)	0767
		31–70	62	(87.3)	9	(12.7)		66	(93.0)	5	(70)
		$>$ 70	27	(84.4)	5	(15.6)		30	(93.8)	2	(63)
Cancer	Volume	$<$ 30	20	(83.3)	4	(16.7)	0.501	23	(95.8)	1	(42)	0387
		31–70	49	(81.7)	11	(18.3)		52	(86.7)	8	(133)
		$>$ 70	15	(93.8)	1	(6.3)		15	(93.8)	1	(63)

Significant $p$ values are shown in bold.

Table 4

Multivariable logistic regression in comparing BPH and PCa patients relative to the healthy group

Variable	BPH			PCa
	$P$ -value		OR (95% CI)	$P$ -value		OR (95% CI)
rs1800629 (GA vs. GG)		0.47	1.47 (0.5–4.29)		0.39	2.30 (0.33–15.88)
rs361525 (GA vs. GG)		0.94	1.06 (0.16–6.72)		0.19	4.30 (0.48–38.37)
Age		0.64	0.99 (0.94–1.03)		0.28	1.03 (0.96–1.11)
BMI		0.03	1.10 (1.0–1.21)		0.08	1.13 (0.98–1.31)
PSA	$<$	0.001	2.74 (2.03–3.69)	$<$	0.001	3.19 (2.17–4.68)
Smoke (Yes vs. No)		0.49	1.30 (0.61–2.76)		0.74	0.81 (0.24–2.74)

Significant $p$ values are shown in bold.

3. Results

3.1 Characteristics of subjects

The demographic and clinical characteristics of the studied subjects are summarized in Table 2. The mean age of control, PCa, and BPH group was 66.28, 67.43, and 67.78 years ( $P=$ 0.396), respectively. The mean of total serum PSA level was significantly higher in PCa (20.75 ng/ml) and BPH (5.81 ng/ml) than in healthy individuals (1.67 ng/ml) ( $P<$ 0.001). Furthermore, there was no significant difference for the BMI index ( $P=$ 0.104) and smoking status ( $P=$ 0.312) but the prostate volume was significantly higher in the BPH patients among three under study groups ( $P<$ 0.001).

3.2 Frequency analysis

The comparison between genotype frequencies of rs361525 and rs1800629 polymorphisms are presented in Fig. 5. Based on the Fig. 5, there was no significant difference in the distribution of genotypes among the three groups for each SNP. Besides, none of the samples had AA genotype of the polymorphisms. In addition, since none of the SNPs were in the equilibrium, we were not able to do a haplotype study.

3.3 Association between the SNPs genotypes and demographic information

Based on the Table 3, there is no correlation between age, PSA level, prostate volume, and rs1800629/ rs361525 genotypes in the studied groups ( $P>$ 0.05).

While the relative frequency of each rs1800629 genotype was not significantly different between smokers and non-smokers of the BPH and healthy groups ( $P>$ 0.05), this value was significantly different in all studied smokers and non-smokers ( $P=$ 0.001).

3.4 Multivariable logistic regression

To match the variables, all quantitative and qualitative changeable and the studied polymorphisms evaluated by the multivariable logistic regression modeling. A comparison between the odds ratio (OR) for all individuals is indicated in Table 4.

The higher BMI showed to be statistically related to 10% increased chance of having BPH [ $p=$ 0.03; OR $=$ 1.10; 95% CI (1.0–1.21)]. In addition, increased PSA was related to up to 2.74 times statistically higher chance of having BPH [ $P<$ 0.001; OR $=$ 2.74; 95% CI (2.03–3.69)] and the 3.9 times increased risk of cancer [ $P<$ 0.001; OR $=$ 3.19; 95% CI (2.17–4.68)].

4. Discussion

Prostate cancer is a multifactorial disease in which genetics and environmental factors are involved [36]. The etiology of PCa is unclear [37] but some polymorphisms of anti-inflammatory and pro-inflammatory cytokine genes regulatory regions affect the gene function [25]. PCa has tumor heterogeneity nature and numerous genes are revealed in its etiology. Therefore, looking for genomic variations as new potential biomarkers in the blood could enable us to detect the risk of disease before gene expressional changes in the early stage of the disease in a specific population.

TNF- $\alpha$ as a key mediator of inflammation has an important role in PCa development [38]. By considering the potency of TNF- $\alpha$ promoter rs1800629 and rs361525 SNPs and their gene expression effect, the SNPs were counted as possible functional variants and as new biomarkers for early detection of susceptibility to prostate cancer.

A meta-analysis results regret to count on TNF- $\alpha$ -238G/A polymorphism as a risk factor for prostate cancer, while accepts that TNF- $\alpha$ $-$ 308G/A may have a considerable role in the disease risk [15]. Many studies have also shown that the latest variant is associated with TNF- $\alpha$ expression level elevation [39]. Lopez and his colleagues studied the effect of the variant on prostate cancer susceptibility and found that the A allele of TNF-A-308 showed 60% increased risk of PCa in Spain [40]. However, Jang and colleagues had previously reported that the TNF-A-238 polymorphism can play a protective role against cancers [41]. Moreover, many studies have been focused on the association between TNF-A-238 and various types of cancers [42]. Bandil and colleagues showed that either TNF- $\alpha$ -238G $>$ A or a haplotype of GA-AA were associated with the development of PCa and BPH. They also found a higher risk for smokers of TNF- $\alpha$ SNP haplotypes [25].

The prostate cancer is an age-dependent phe- nomenon which increases with age [43]. Some researches indicate no difference in the distribution of genotype between younger and older control groups, while others have reported an age-related difference of the polymorphisms of specific genes [40]. In the present study, the mean age of the healthy group, BPH and prostate cancer patients did not show any significant difference which confirms that the age alone cannot be a cause of the prostate disease and its malignancies.

Kesarwani and colleagues showed that the PSA concentration was significantly different in the healthy group and PCa patients [44]. Other study showed that the PSA level was higher than normal (4 ng/ml) in cancer patients compared to patients with BPH [25]. Our study also showed that the PSA level increases 2.47 and 3.9 times in BPH and prostate cancer groups in comparison to the controls. Therefore, PSA while not enough but is still an important factor in determining the benign and malignant status from a healthy condition.

On the other hand, while our study showed a 10% gain between increasing BMI and the risk of the disease, some articles have reported a lack of correlation between the increase of BMI and prostate cancer [45]. Therefore, it seems that BMI parameters should be more considered regarding prostate diseases, especially in the context of the cancer.

Tobacco and alcohol consumption may play critical roles in the etiology of many cancers including PCa [46, 47].

Some studies have been reported that smoking and alcohol drinking suppresses TNF- $\alpha$ production which adversely affect the immune response in inflammatory and infectious diseases and contributes to the incidence of cancers [48, 49, 50]. There are other sources that do not report any significant association between TNF-a $-$ 308G/A polymorphism and PCa risk in smokers compared to non-smokers [42]. In the current study, it was shown that the GA genotype of rs1800629 polymorphism has a higher significant incidence in smoker PCa patients than the non-smokers ( $p=$ 0.001). So, it can be concluded that smoking in interaction with the GA genotype can increase the risk of prostate cancer.

In the actual study, the percentages of rs1800629 GA genotype in the three groups of healthy, BPH, and cancer were 12.7%, 14.5%, 16%, and 14.4% in total (46 out of 320 individuals), respectively. Moreover, the frequencies of rs361525 GA genotype were 4.5%, 6.4%, 10%, and in total 6.9% (22 out of 320 individuals), respectively. Analysis of the polymorphisms in the studied groups showed that although the frequencies of GG and GA genotypes had no significant differences, their relative frequencies were significantly different and GA showed a possible dominant model which could be a methylation-related site where the methylation level increases [51]. We had no AA genotype in this pilot study which indicates low frequency of A allele in the studied population. Investigation on a larger population may indicate the exact frequency of A allele of the studied TNF- $\alpha$ polymorphisms in the future and will overcome the understanding of the SNPs mechanism of actions in the etiology of the prostate cancer.

5. Conclusions

Our study indicates that there is no significant association between TNF- $\alpha$ gene (rs361525 or rs1800629) variant with the risk of BPH and PCa. However, we realized that GA genotype of the examined polymorphisms in comparison to the GG genotype increases the risk of BPH and PCa while the other variables do not show significant differences between the understudy groups ( $P>$ 0.05) but, still, a potential association is considered between them (OR $>$ 1) which could be more interpreted for their possible PCa risk factor role. Hence, studies with a larger number of samples should be done to reveal the existence of the probable associations.

Footnotes

Acknowledgments

Special thanks to Dr. Seyed Ali Ziai the research deputy of the faculty of medicine at Shahid Beheshti University of Medical Sciences for authorizing the project performance at the Central Laboratory. Our sincere thanks to Dr. Seyed Mostafa Hosseipour mashkani from UTS University, Australia, for reviewing the article and submitting his suggestions, and appreciate to those who generously presented their blood samples to allow us to exam the research goals. This study was funded by Shahid Beheshti university of Medical Sciences (Grant number: 15139).

Conflict of interest

Farkhondeh Pouresmaeili has received research grants from Shahid Beheshti university of Medical Sciences. The rest authors have no conflict of interest.

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Association between tumor necrosis factor-alpha gene rs1800629 ( - 308G/A) and rs361525 ( - 238G > A) polymorphisms and prostate cancer risk in an Iranian cohort

Abstract

BACKGROUND:

OBJECTIVES:

MATERIALS AND METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Subjects

Table 1 Primers sequences, PCR conditions, and fragment sizes for genotyping of the TNF- α gene polymorphisms

2.5 Statistical analysis

Table 3 Association between the genotypes of polymorphisms with demographic information

3.1 Characteristics of subjects

3.2 Frequency analysis

3.3 Association between the SNPs genotypes and demographic information

3.4 Multivariable logistic regression

4. Discussion

5. Conclusions

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Primers sequences, PCR conditions, and fragment sizes for genotyping of the TNF- $\alpha$ gene polymorphisms

Table 3
Association between the genotypes of polymorphisms with demographic information