Association between MDM2‑SNP309 and p53R72P polymorphisms and the risk of bladder cancer in the Mongolian population
- Authors:
- Published online on: July 13, 2017 https://doi.org/10.3892/mco.2017.1317
- Pages: 412-420
Abstract
Introduction
The incidence of bladder cancer is increasing and it is the most common urinary malignant tumor worldwide (1). It is the second most common urological malignancy in the Mongolian population (2). A histological study examining 108 Mongolian patients with urinary bladder cancer revealed that 2, 12 and 86% of the patients were diagnosed with papilloma, papillary urothelial neoplasm of low malignant potential and transitional cell carcinoma, respectively (3). Various environmental and genetic risk factors such as smoking, occupational exposure to aromatic amines, male sex, older age and bladder infections are associated with bladder cancer development (4,5). Furthermore, certain genetic alterations in bladder cancer including p53 tumor suppressor gene and its regulating gene, human homolog of murine double minute (MDM2) are considered to be associated with cancer susceptibility (6). Whether genetic polymorphisms in these genes also has a role in the risk of bladder cancer remains to be addressed.
P53 genomic sequence mutation is typically concentrated in the region of exons 4–9 when it is detected in human cancer (7,8). P53 codon 72 G to C polymorphism (p53R72P) is found in exon 4 and is a well-known single nucleotide polymorphism (SNP) that alters arginine to proline (9). The two forms are morphologically wild-type, but such variants of p53 may target varying transcriptional elements (10). Previous meta-analyses have reported that p53R72P is associated with the risk of bladder cancer in Asian but not Caucasian patients (11,12). However, these studies did not include Mongolian populations, and the risk association in this population remains to be investigated.
A functional SNP was also identified at position 309 of the first intron at the P2 promoter region of human MDM2, termed MDM2-SNP309 (rs2279744) (13). The conversion from the T to G allele in this SNP increases the binding of the general transcription activator Sp1 to the promoter and enhances the expression of the MDM2 gene, which in turn promotes the degradation of p53 and leads to a higher risk of carcinogenesis (14,15). For bladder cancer, the homozygous variant of MDM2-SNP309 is not associated with overall risk of bladder cancer but may influence the invasive growth of bladder cancer in German populations (16). A study examining Turkish populations reported that the MDM2 T309G polymorphism is a potential genetic susceptibility factor for bladder cancer (17). Another previous study, conducted in the USA, revealed that SNP309 is associated with earlier onset of superficial tumors, poorer clinical outcomes and TP53 mutation status in invasive bladder cancer (18). A study conducted in Japan revealed no significant associations between each genotype of the MDM2 SNP309 and p53 Arg72Pro polymorphisms and bladder cancer risk, but the polymorphisms may influence the clinical outcome of bladder cancer (19). However, investigations have been inconsistent, and the hypothesized association remains controversial. These inconclusive and conflicting results may be associated with the different patient subgroups and ethnicities studied.
In the present study, the risk association between p53R72P and MDM2-SNP309 and bladder cancer in Mongolian patients was analyzed using the logistic regression method. The age onset of patients with bladder cancer was also compared among the different genotypes of these two genetic polymorphisms.
Patients and methods
Patients
A total of 79 cancer-free healthy controls and 63 patients with histologically confirmed bladder cancer who were diagnosed in the First Central Hospital of Mongolia (Ulaanbaatar, Mongolia) were enrolled in the study. Patients with bladder cancer were not selected according to their age, sex or tumor stage. Age- and gender-matched controls were enrolled in this study. All patients provided informed consent and completed structured questionnaires. The questionnaire included information about risk factors for bladder cancer such as age, sex, body mass index, socioeconomic status, occupational exposure, dietary factors, smoking, alcohol drinking, drug usage, history of chronic urinary tract diseases and exercising. This study was approved by the Ethics Committee of the Ministry of Health of Mongolia (No. 4), and the institutional review board of National Yang-Ming University, Taiwan (YM102005). The approval documents will be provided upon request. All participants were native Mongolians and aged >18 years.
Genotyping
In total, 3 ml whole blood was collected from cancer-free healthy controls and patients with bladder cancer for genotyping of p53R72P and MDM2-SNP309 after written informed consent was obtained. The procedures of MDM2-SNP309 and p53R72P genotyping was performed as described previously (18). In brief, DNA was extracted from 200 µl of blood using the Qiagen mini blood DNA extraction kit (Qiagen, Inc., Valencia, CA, USA) and MDM2 SNP309 was amplified by polymerase chain reaction (PCR) using the following primers: Forward, 5′-CGGGAGTTCAGGGTAAAGGT-3′; and reverse, 5′-AGCAAGTCGGTGCTTACCTG-3′. The PCR reactions consisted of 100 ng of genomic DNA, 0.2 µM primer, 200 µM dNTP, 1.5 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl and 1 U of Platinum Taq DNA polymerase (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA). The thermal cycling conditions were 1 min at 94°C; 40 cycles of denaturing at 94°C, annealing at 58°C, and elongation at 72°C for 30 sec each, followed by one cycle at 72°C for 10 min. For restriction fragment length polymorphism analysis, 10–20 µl of the amplified 352-bp fragment was digested with 1 U of MspA1I restriction enzyme (New England Biolabs, Inc., Ipswich, MA, USA) at 37°C in a water bath for 30 min to 1 h. The T/T, T/G and G/G genotypes were distinguished by bands with lengths of 233 and 88; 233, 187 and 88 bp; and 187 and 88 bp, respectively, following electrophoresis. The same procedure was used for genotyping p53 codon 72 polymorphism (20). The primers were as follows: Forward, 5′-TTTCACCCATCTACAGTCCC-3′; reverse, 5′-CGGTGTAGGAGCTGCTG-3′. The length of the PCR product was 166 bp, then it was digested with BstU1 at 60°C for 1 h. The digestion of the P/P variant yielded a 166-bp band, the R/R variant yielded 135- and 31-bp bands and the P/R heterozygous variant yielded 166, 135 and 31 bp bands.
Statistical analysis
The genotype and allele frequency were tested for Hardy-Weinberg equilibrium. Discrepancies between controls and cases were determined using 2×2 χ2 tables or two-sample t-tests. Multivariate logistical regression analysis with adjustment or stratification was used to determine odds ratios (OR) and 95% confidence intervals (CI) for evaluating the risk association of MDM2-SNP309 and p53R72P with the incidence of bladder cancer. The Kaplan-Meier method with the log-rank test was used to analyze the age at diagnosis of these genotypes. Statistical evaluation was performed using the demo version of Medcalc 9.5.1.0 software (MedCalc Software, Mariakerke, Belgium). P≤0.05 was considered to indicate a statistically significant difference.
Results
Characteristics of bladder cancer cases and controls
One hundred and twenty-nine cancer-free controls and 141 patients with bladder cancer were initially enrolled in the present study. However, characteristics of patients and controls were only recorded in 79 controls and 63 patients with bladder cancer, as they had provided the full information requested in the questionnaires. The demography of 142 Mongolian volunteers awas summarized in Table I. There were 3.5 times more male than female patients. Smoking, alcohol drinking and exercise, as well as the history of chronic urinary tract disease were significantly different between cases and controls (P<0.05). Thus, these characteristics were considered confounders that were adjusted for in the odds ratio in the multivariate logistic regression analysis.
Allelic frequency analysis of MDM2-SNP309 and p53R72P
The allelic frequencies of MDM2-SNP309 and p53R72P were next analyzed in these 142 Mongolians. For MDM2-SNP309, the number and percentage of wild-type (T/T), heterozygote (T/G) and homozygote (G/G) alleles was 18 (22.7%), 44 (55.7%) and 17 (21.6%) in the controls, respectively, whereas for bladder cancer cases, these values were 13 (20.6%), 30 (47.6%) and 20 (31.7%), respectively. The genotype frequency of MDM2-SNP309 in both controls and cases obeyed the Hardy-Weinberg equilibrium. The number and percentage of p53R72P genotypes were wild-type (R/R) 37 (46.8%), heterozygote (R/P) 23 (29.1%) and homozygote (P/P) 19 (24.1%) alleles in controls, whereas in bladder cancer cases the R/R, R/P and P/P alleles were 35 (55.6%), 20 (31.7%) and 8 (12.7%), respectively. Unlike MDM2-SNP309, the allelic frequency of p53R72P in controls (R, 61%; P, 39%) was different from that of bladder cancer cases (R, 71%; P, 29%). The genotype frequency of p53R72P in bladder cancer cases obeyed the Hardy-Weinberg equilibrium, but not in controls. The analysis of allelic frequencies of MDM2-SNP309 and p53R72P is summarized in Table II.
Association between MDM2-SNP309 and p53R72P and the risk of the bladder cancer
To investigate the association between MDM2-SNP309 and p53R72P polymorphisms and the risk of bladder cancer in Mongolian patients, a multivariate logistic regression model was used to calculate the ORs of heterozygotes and homozygotes relative to the wild-type genotype. For MDM2-SNP309, the crude OR of the homozygous genotype (G/G) was 1.629 (95% CI=0.622–4.2663) compared with the wild-type genotype (T/T). On the contrary, the OR of the heterozygous genotype (T/G) was 0.944 (95% CI=0.4031–2.2111). The confounders-adjusted ORs of the G/G and T/G genotypes were 1.306 (95% CI=0.3938–4.2204) and 0.993 (95% CI=0.3246–3.0403), respectively. Notably, the crude OR and adjusted OR for the homozygous genotype of p53R72P were 0.445 (95% CI=0.1727–1.147) and 0.491 (95% CI=0.1487–1.6199), respectively. However, the ORs of MDM2-SNP309 and p53R72P were not significant (Table III). Thus, the current analysis showed that the homozygous genotype (G/G) of MDM2-SNP309 tended to increase the risk of bladder cancer in Mongolian populations, but this effect was not observed for p53R72P.
 | Table III.Risk evaluation of the MDM2 SNP309 and p53R72P genotypes and the development of bladder cancer in the Mongolian population. |
Risk evaluation of MDM2-SNP309 and p53R72P genotypes and bladder cancer development
MDM2-SNP309 and p53R72P were examined by stratifying according to the following potential confounding factors: Smoking, alcohol drinking and history of chronic urinary tract diseases, as stated in Table I. The G/G genotype of MDM2-SNP309 had a higher risk in the smoking group (OR, 2.3704; 95% CI=0.4308–13.0436) than the non-smoker group (OR, 1; 95% CI=0.2077–4.8138). Furthermore, the G/G and T/G genotypes of MDM2-SNP309 were associated with the risk of bladder cancer following stratification by history of chronic urinary tract diseases. For alcohol drinking, the G/G genotype of MDM2-SNP309 had a protective effect on the risk of bladder cancer (OR, 0.6944; 95% CI=0.1296–3.7203), whereas the T/G genotype increased the risk of bladder cancer (OR, 1.5625; 95% CI, 0.3612–6.7587). The P/R genotype of the p53R72P was also associated with increased risk (OR, 1.875; 95% CI=0.4415–7.9631) of bladder cancer following stratification by alcohol drinking, but not the P/P genotype (OR, 0.5; 95% CI=0.108–2.3142). Although the ORs were altered after stratification, the results of the statistical analysis were not significant (Table IV). Therefore, the adjusted ORs of MDM2-SNP309 and p53R72P genotypes revealed no risk association between these SNPs and bladder cancer in Mongolian patients.
| Table IV.Stratification of smoking, history of CUTD and alcohol drinking to evaluate the risk of the MDM2 SNP309 and p53R72P genotypes regarding the incidence of bladder cancer in the Mongolian population. |
Evaluation of the effect of MDM2-SNP309 on the risk of bladder cancer after stratification by p53R72P
To investigate whether MDM2-SNP309 and p53R72P interact to influence the risk of bladder cancer, the genotypes of p53R72P were stratified by MDM2-SNP309 genotype. Based on this dataset, the results revealed no significant effects regarding the homozygous genotype (G/G) of MDM2-SNP309 and the risk of bladder cancer in Mongolian patients in the RR group of the p53R72P SNP (OR, 3.3554; 95% CI=0.3914–28.7657). For heterozygous (R/P) genotypes of p53R72P, both genotypes of MDM2-SNP309 showed no risk association with bladder cancer. All of these results are summarized in Table V.
Comparison of different genotypes of MDM2-SNP309 and p53R72P and the age at diagnosis of bladder cancer
The median age at diagnosis of bladder cancer in Mongolian patients was compared among different genotypes of MDM2-SNP309 and p53R72P. The results were obtained from 84 patients with bladder cancer with registered age at diagnosis. For MDM2-SNP309, the median ages of G/G (60 years) and T/G (59 years) genotypes were older than that of wild-type (T/T) genotype (50.5 years) (Table VI). However, there was no significant difference among these three genotypes of MDM2-SNP309 (Fig. 1A). For p53R72P, the median patients age at diagnosis for the R/P genotype (52 years) was lower than for the homozygous (RR, 58; PP, 61) genotypes (Table VII); however, among the p53R72P genotypes no significant differences were identified (Fig. 1B).
| Table VI.Comparison of age at diagnosis of bladder cancer in Mongolian patients according to MDM2-SNP309 genotype. |
| Table VII.Comparison of age at diagnosis bladder cancer in Mongolian patients according to p53R72P genotype. |
Discussion
The current study investigated the association between the MDM2 SNP309 and p53R72P polymorphisms and bladder cancer risk in Mongolian populations. According to the results, the homozygous (G/G) genotype of MDM2 SNP309 is associated with increased risk of bladder cancer in Mongolian populations, but not significantly so. This result is similar to the result of a previous Turkish study, which indicated that the G/G genotype was a potential genetic susceptibility factor for bladder cancer (OR=2.68; 95% CI=1.34–5.40) (17). Furthermore, it is partially consistent with previous studies reporting that the G allele of MDM2-SNP309 is associated with the development of an aggressive tumor phenotype but not overall risk of bladder cancer in Caucasian Japanese populations (16,19).
In the present study, several risk factors for bladder cancer including smoking, drinking alcohol and history of urinary tract infections exhibited significant differences between cases and controls, as determined via structured questionnaires (21). Smoking and history of chronic urinary tract disease increased the risk association of the G/G genotype of MDM2-SNP309 with bladder cancer, whereas the T/G genotype of MDM2-SNP309 may increase the risk following stratification by history of urinary tract disease and drinking alcohol, but not smoking.
In the current study, for p53 Arg72Pro genotypes, each genotype individually was not associated with the risk of bladder cancer in Mongolian populations. These results were similar to the results of studies conducted among Japanese and north Indian populations (19,22). Notably, according to a meta-analysis of 15 publications, p53R72P is associated with the risk of bladder cancer in Asian but not Caucasian patients (9). However, the north Indian population was categorized as Caucasian in this previous meta-analysis (11). Furthermore, patients with the P/P genotype of p53R72P increased the risk (OR=3.02; 95% CI=1.42–6.40) of the development of bladder cancer in Bangladeshi populations (23). Another meta-analysis reported that, in Caucasians, the wild-type (R/R) genotype of p53R72P increases the risk (OR=1.64; 95% CI=1.18–2.28), but the heterozygous (R/P) genotype reduces the risk (OR=0.62; 95% CI=0.44–0.99) of bladder cancer (24).
In the current study, when the genotypes of p53R72P were stratified by the confounding factors, only the R/P genotype increased the risk of bladder cancer in patients drinking alcohol. Furthermore, the combination of the R/R genotype of p53R72P with the G/G genotype of MDM2-SNP309 was associated with increased risk of bladder cancer in the present study (Table V). It is speculated that the G/G genotype of MDM2-SNP309 may suppress the level of wild-type (R/R) p53 and increase the risk of cancer development accordingly. The present results found no association between these two SNPs and early age onset of bladder cancer. However, the sample size was too small to determine the interaction between these two SNPs.
The current study has several limitations that may have influenced the results. A major potential limitation of the study was the small patient sample size; the study involved 79 controls and 63 patients with bladder cancer who participated fully, although 129 controls and 141 patients were initially enrolled in the research. This may be due to the population density, large geographical area and lack of specialized cancer registry system. However, the number of volunteers in the present study may be standard compared with similar studies in regards to population (16,17,19,22). For Mongolians, lifestyle and diet (primarily red meat and dairy) are not the critical risk factors for bladder cancer development in the present study. Regarding sex, males were 3.5 times more affected by bladder cancer than females in the current study. This result is similar to a previous study in that the incidence, staging and prognosis of bladder cancer was associated with sex (25).
In conclusion, the current data suggest no significant association between MDM2-SNP309 and p53R72P and the risk of bladder cancer in Mongolian populations. Patients who smoke or have urinary tract disease have an enhanced bladder cancer risk effect associated with MDM2-SNP309 but not p53R72P. The present study failed to demonstrate any association between early age at onset of MDM2-SNP309 and p53R72P and the development of bladder cancer. As these statistical results were based on small sample sizes of cases and controls, enlargement of the volunteer pool may be important to further elucidate the effect of MDM-SNP309 and p53R72P on the risk of Mongolian bladder cancer.
Acknowledgements
The present study was supported by The National Science Council (now Minister of Science and Technology), Taiwan (NSC 101-2923-B-010-001-MOST 105-2628-B-010-013-MY3) and The Mongolian Foundation for Science and Technology (NSC-MECS2012004). The authors would also like to thank Miss Odgerel, Miss B. Chuluuntsetseg, Miss D. Odgerel, Miss Chun-Yuan Chang and Miss Hsian-Wen Lins' for technical support.
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