Genetic polymorphisms of MDM2 and TP53 genes are associated with risk of nasopharyngeal carcinoma in a Chinese population

This study included 522 NPC patients and 712 healthy population controls. All subjects were ethnically homogenous Han Chinese. Patients with newly diagnosed NPC were consecutively recruited from March 2001 to May 2007, at the Sir Run Run Shaw Hospital, Zhejiang University (Hangzhou) and Zhejiang Cancer Hospital (Hangzhou). All eligible patients diagnosed at the hospital during the study period were recruited, with a response rate of 94%. Patients were from Hangzhou city and its surrounding regions and there were no age, stage, and histology restrictions. The tumor, node, metastasis (TNM) classification and tumor staging was evaluated according to the 2002 American Joint Committee on Cancer staging system [11]. The clinical features of the patients are summarized in Table 1. Population controls were cancer-free people living in Hangzhou region; they were selected from a nutritional survey conducted in the same period as the cases were collected. The control subjects were randomly selected from a database consisting of 2500 individuals based on a physical examination. The selection criteria included no history of cancer and frequency matched to cases on age and sex. Median age was 46 years (range 26-81) for case patients and 47 years (range 22-85) for control subjects (P = 0.78). At recruitment, informed consent was obtained from each subject. This study was approved by the Medical Ethics Committee of Sir Run Run Shaw Hospital and Zhejiang Cancer Hospital.

Genomic DNA was isolated from the peripheral blood lymphocytes of the study subjects. Genotypes were analyzed using PCR-based methods as described below. Genotyping was performed without knowledge of subjects' case/control status. A 30% masked, random sample of cases and controls was tested twice by different persons and the results were concordant for all masked duplicate sets.

The genotypes of TP53 Arg72Pro (rs1042522, G>C) were analyzed by PCR-RFLP method on the basis of that reported previously [12]. The primers used were TP53 F, 5'-TTG CCG TCC CAA GCA ATG GAT GA-3' and TP53 R, 5'-TCT GGG AAG GGA CAG AAG ATG AC-3', which produce a 199-bp fragment containing the G/C site. Amplification was accomplished with a 25 μl reaction mixture containing ~100 ng template DNA, 0.5 μM each primer, 0.2 mM each dNTP, 1.5 mM MgCl₂, and 1.2 units of Taq DNA polymerase with 1 × Reaction buffer (Promega, Madison, WI, USA). PCR profile consisted of an initial melting step of 2 min at 94°C, followed by 35 cycles of 30 s at 94°C, 30 s at 56°C, and 30 s at 72°C, and a final elongation step of 7 min at 72°C. The 199-bp PCR products were then subject to the digestion with BstUI (New England Biolabs, UK) and separated on a 3.0% agarose gel. The genotypes identified by BstUI digestion were confirmed by DNA sequencing (Figure 1).

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MDM2 SNP309 (rs2279744, T>G) genotypes were analyzed using the tetra-primer amplification refractory mutation system (ARMS)-PCR method [13]. The primers for ARMS-PCR amplification of DNA fragment containing the MDM2 SNP309 T allele were MDM2 F1: 5'-GGG GGC CGG GGG CTG CGG GGC CGT TT-3' and MDM2 R1: 5'-TGC CCA CTG AAC CGG CCC AAT CCC GCC CAG-3'; For the MDM2 SNP309 G allele they were MDM2 F2: 5'-GGC AGT CGC CGC CAG GGA GGA GGG CGG-3' and MDM2 R2: 5'-ACC TGC GAT CAT CCG GAC CTC CCG CGC TGC-3'. The amplification was accomplished with a 10-μl reaction mixture containing 10 ng of template DNA, 0.8 μM of primers MDM2 F1 and MDM2 R1, 4.8 μM of primers MDM2 F2 and MDM2 R2, 0.2 mM of dNTPs, 1.5 mM of MgCl2, and 0.4 U of HotStar TaqTM with 1 × buffer and 1 × Q-solution (Qiagen, Chatsworth, CA). The reaction was carried out with an initial melting step of 15 min at 95°C, followed by 35 cycles of 45 sec at 95°C, 45 sec at 64°C, and 1 min at 72°C, and a final elongation step of 7 min at 72°C. The amplified DNA was visualized on agarose gel containing ethidium bromide. The MDM2 SNP309 T allele generated a 122-bp band, and the G allele generated a 158-bp band. They had a common 224-bp band, which was amplified by primers MDM2 F2 and MDM2 R1. The genotypes identified by PCR-RFLP and ARMS-PCR digestion were confirmed by DNA sequencing (Figure 2).

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Total RNA was isolated from Seventy-one NPC tissues using the Trizol reagent (Molecular Research Center, Inc., Cincinnati, OH) and converted to cDNA using an oligo (dT)₁₅ primer and Superscript II (Invitrogen, Carlsbad, CA). Relative gene expression quantitation for MDM2, with β-actin as an internal reference gene, was carried out using ABI Prism 7300 sequence detection system (Applied Biosystem, Foster City, CA) in triplicates, based on the SYBR-Green method [8]. The primers used for MDM2 were 5'-TGT AAG TGA ACA TTC AGG TG-3' and 5'-TTC CAA TAG TCA GCT AAG GA-3'; and for β-actin were 5'-GGC GGC ACC ACC ATG TAC CCT-3' and 5'-AGG GGC CGG ACT CGT CAT ACT-3'. The PCR reaction mixture consisted of 0.1 μmol/L of each primer, 1 × SYBR Premix EX Taq (Perfect Real Time) premix reagent (TaKaRa, Dalian, China), and 50 ng cDNA to a final volume of 20 μL. Cycling conditions were 95°C for 10 minutes, followed by 40 cycles at 95°C of 15 seconds and 62°C for 1 minute. PCR specificity was confirmed by dissociation curve analysis and gel electrophoresis. All analysis were done in a blinded fashion with the laboratory persons unaware of genotyping data. The expression of individual MDM2 measurements was calculated relative to expression of β-actin using a modification of the method described by Lehmann et al [14].

χ² tests were used to examine the differences in the distributions of genotypes between cases and controls. The association between the TP53 and MDM2 polymorphisms and risk of NPC were estimated by ORs and their 95% CIs, which were calculated by unconditional logistic regression models. We tested the null hypotheses of multiplicative gene-gene interactions by evaluated departures from multiplicative joint effect models by including main effect variables and their product terms in the logistic regression model [15]. A more-than-additive interaction was suggested when OR₁₁ > OR₁₀ + OR₀₁ -1, for which OR₁₁ = OR when both factors were present, OR₁₀ = OR when only factor 1 was present and OR₀₁ = OR when only factor 2 was present. A more-than-multiplicative interaction was suggested when OR₁₁ >OR₁₀ ×OR₀₁. The correlation of genotypes and clinical parameters was analyzed via the Fisher's exact test or χ² test as appropriate. The normalized expression values of MDM2 were compared by Kruskal-Wallis one way ANOVA. All P-values were two-sided with a P-value < 0.05 considered to be statistically significant. All analysis was carried out with Statistical Analysis System software (Version 9.0; SAS Institute, Cary, NC, USA).

The genotype results are shown in Table 2. The allele frequencies for MDM2 G and TP53 Pro were 0.423 and 0.426 in controls, and 0.555 and 0.518 in cases respectively. The observed genotype frequencies of MDM2 and TP53 polymorphisms in both controls and cases did not deviated from those expected from the Hardy-Weinberg equilibrium. Distributions of these MDM2 and TP53 genotype were then compared among cases and controls. The frequencies of MDM2 TT, TG and GG genotypes among patients were significantly different compared to controls (P _trend < 0.001), with the GG homozygotes being significantly overrepresented among patients compared to controls (P < 0.001). Moreover, Logistic regression analysis showed that subjects with TP53 Pro allele significant increased risk of NPC compared with subjects carrying the Arg allele (OR for the Arg/Pro genotype, 1.43; 95%CI, 1.22-2.13; OR for the Pro/Pro genotype, 2.22; 95%CI, 1.58-3.10; P _trend < 0.001), suggesting that the Pro allele is the high-risk allele.

The effects of the TP53 and MDM2 polymorphisms were additionally examined with stratification by age, tumor size, metastatic status and Epstein-Barr virus (EBV) infection status. However, no significant association was observed between age and the TNM stage at the time of NPC diagnosis and the polymorphism of this gene, and no interaction was detected between the polymorphism and status of EB virus infection (data not shown).

We examined whether there was a statistical interaction between the MDM2 and TP53 polymorphisms (Table 3). The data showed that patients who carried the MDM2 GG genotype were also more likely to carry the TP53 Pro/Pro genotype than the controls (10.3% vs. 2.8%, P < 0.001). The presence of one MDM2 GG genotype, but not one TP53 Pro/Pro genotype, were associated with an increased risk of NPC (OR = 2.83, 95% CI = 1.33-5.90), compared to the lack of such a genotype. However, the presence of both MDM2 GG and TP53 Pro/Pro genotypes was associated with an even higher risk for NPC increase (OR = 7.75, 95% CI = 3.53-17.58; P < 0.05, test for homogeneity) compared to those who lacked both genotypes. These results clearly indicate a more than multiplicative interaction [15] between the MDM2 GG and TP53 Pro/Pro genotype in the risk of developing NPC.

To examine the effect of the MDM2 309T>G polymorphism on MDM2 expression in the target tissues, the levels of MDM2 mRNA in individual NPC tissues were quantified by real-time PCR (Figure 3). The results showed that the MDM2 GG genotype carriers (n = 19) had significantly higher MDM2 mRNA level than the MDM2 TT genotype carriers [0.050 ± 0.033 (n = 19) versus 0.014 ± 0.009 (n = 18), P < 0.001]. However, the MDM2 TG genotype carriers (n = 34) had a MDM2 mRNA level that was very similar to that of the TT genotype carriers (0.015 ± 0.013 versus 0.014 ± 0.009, P = 0.610).

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