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Author's Personal Copy

MDM2 SNP309T>G polymorphism and risk of hepatocellular

carcinoma: A case–control analysis in a Moroccan population

Sayeh Ezzikouri a,*, Abdellah Essaid El Feydi b, Rajae Afifi b, Latifa El Kihal b,Mustapha Benazzouz b, Mohammed Hassar a, Agnes Marchio c, Pascal Pineau c,

Soumaya Benjelloun a

a Laboratoire de Virologie, Institut Pasteur du Maroc, Casablanca, Moroccob Service de Medecine C, CHU Ibn-Sina, Rabat, Morocco

c Unite d’Organisation Nucleaire et Oncogenese, INSERM U579, Institut Pasteur, Paris, France

Accepted 21 January 2009

Abstract

Background: The Murine double minute 2 (MDM2) gene encodes a negative regulator of the p53 tumor suppressor protein. A single

nucleotide polymorphism (SNP) in the MDM2 promoter (a T to G exchange at nucleotide 309) has been reported to produce accelerated tumor

formation. The aim of this study was to investigate whether this functional SNP is associated with an enhanced risk of liver tumorigenesis in

Moroccan patients. Methods: The study consisted in the comparison of 96 hepatocellular carcinomas (HCC) cases and 222 controls without

HCC matched for age, gender and ethnicity. PCR–RFLP and sequencing methods were used to determine the genotype at the MDM2

SNP309T>G locus. Results: Overall, our results indicate that the GG genotype of SNP309 is significantly associated with an increased risk of

HCC (odds ratio, OR = 2.60, 95% CI, 1.08–6.28). Interestingly, despite a wide range of confidence interval, there is a trend associating the GG

genotype with a high risk of HCC in males (OR = 3.31; 95% CI, 0.93–11.82) and in HCV-infected patients (OR = 3.7; 95% CI, 0.82–16.45).

By contrast, no association between age at diagnosis and MDM2 SNP309 genotypes was observed in HCC patients (P = 0.610). Conclusion:

Our findings suggest that the MDM2 309T>G polymorphism is an important modulator of hepatocellular carcinoma development in

Moroccan patients.

# 2009 Elsevier Ltd. All rights reserved.

Keywords: Hepatocellular carcinoma; MDM2 polymorphism; Morocco

www.elsevier.com/locate/cdp

Cancer Detection and Prevention 32 (2009) 380–385

1. Introduction

Hepatocellular carcinoma (HCC) is the fifth most

common malignant tumor in the world and the third most

common cause of cancer-related mortality. Approximately

667,000 new cases are diagnosed each year and 550,000

deaths are recorded [1]. There are major differences in the

incidence rates according to geographical areas, such

heterogeneity being due the different prevalence of several

oncogenic factors such as hepatitis B and C viruses (HBV

* Corresponding author at: Laboratoire des Hepatites Virales, Institut

Pasteur du Maroc 1, Place Louis Pasteur, 20100 Casablanca, Morocco.

Tel.: +212 22434470; fax: +212 22260957.

E-mail address: sayeh.ezzikouri@pasteur.ma (S. Ezzikouri).

0361-090X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cdp.2009.01.003

and HCV), aflatoxin intake and alcohol abuse in different

populations [2].

The p53 gene is deleted or mutated, thus, inactive as a

transcription factor in �50% of all human tumors. Previous

studies reported that the p53 tumor suppressor gene is

involved in hepatocarcinogenesis, and that p53 mutation

spectrum differs according to tumor etiology [3,4]. The p53

pathway is to respond to a wide variety of stress signals.

The triggering of one or more of these signals is

associated with the biochemical modification of the p53

protein (phosphorylation, methylation, acetylation, neddy-

lation, ubiquitination, or sumoylation) and a dramatic

increase in the half-life of the p53 protein [5]. Rapid p53

turnover in normal cells is largely due to the Murine double

minute 2 (MDM2) oncoprotein, a pivotal p53 regulator.

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S. Ezzikouri et al. / Cancer Detection and Prevention 32 (2009) 380–385 381

MDM2 is an E3 ubiquitin ligase: its binds specifically to p53

and promotes the covalent conjugation of ubiquitin residues

to it, leading to subsequent p53 degradation by the 26S

proteasome [6]. In addition, the mere binding of MDM2

blocks p53 function, by preventing its interaction with

components of the transcription machinery. Remarkably,

p53 transactivates the MDM2 gene, thus establishing a

negative feedback loop that helps maintain p53 in check in

nonstressed cells [6]. The levels of the MDM2 protein in a

cell and/or organism seem to have widespread consequences

for p53 response and cancer susceptibility [7]. In mice that

produce reduced levels of the MDM2 protein, the offspring

are small, lymphopenic, and radiosensitive with increased

rates of apoptosis in both lymphocytes and epithelial cells

[8]. By contrast, a 4-fold overexpression of MDM2 in

transgenic mice results in a 100% tumor incidence [9].

Recently, Bond et al. identified a functionally significant

SNP in the promoter for MDM2; this polymorphism occurs

at nucleotide 309 of intron 1 of the MDM2 gene, and

changes a T to G, which they called SNP309 (rs 2279744)

[10]. Notably, this change is predicted to create a higher-

affinity binding site for the transcription factor Sp1, which is

an important controller of MDM2 RNA levels. The G allele

bound with 2–4-fold enhanced affinity to purified Sp1 both

in vitro and in vivo. In line with this finding, cell lines

homozygous for the G allele of SNP309 were shown to have

an attenuated p53 transcriptional and proapoptotic apoptotic

response, owing to a decreased ability of p53 to stabilize

following DNA damage [10].

The MDM2 309T>G polymorphism has been shown to

be associated with an increased risk of HCC in Japanese

patients [11]. The aim of the present case–control study was

to provide information about the risk associated with

MDM2 SNP309 in a rarely studied population of patients

with HCC.

2. Patients and methods

2.1. Patients and controls

We studied 96 HCC patients and 163 healthy controls

matching for age, sex and ethnicity. The controls did not

have a previous diagnosis of any type of cancer. Blood of 59

subjects with HCV infection and without HCC were added

to control group. Serum was used for the ELISA tests, and

EDTA sample collected for the preparation of leukocyte

DNA. The procedures and purposes of the study were

explained to all the subjects. After informed consent was

obtained, each participant was interviewed, and a structured

questionnaire on demographic data and selected risk factors

was given and facilitated by interviewers. The following risk

factors were investigated in the questionnaire: heavy alcohol

intake, smoking behaviour, non-insulin-dependent diabetes

mellitus. Cases and controls accepted to be enrolled in a

proportion of 98% and 80%. The major cause of refusal was

merely a lack of interest for research work. All subjects were

recruited in Western-Central Morocco (Rabat and Casa-

blanca) in a population of mixed berberic and arabic

ethnicity. Two groups of controls were recruited: (i) the first

group among individuals coming for blood testing unrelated

to liver pathology at the Pasteur Institute of Morocco; (ii) the

second group among HCV-infected patients with low grade

liver disease. The severity of liver disease was routinely

assessed by non-invasive methods. Controls were graded as

‘‘low’’ when abdominal ultrasonographic examination

confirmed the presence of a mild liver disease concomitant

to moderately elevated plasma liver enzymes. These

investigations were approved by the Ethics Committee of

the Faculte de Medicine of Casablanca.

2.2. Serological markers and genetics

Serological markers for hepatitis viruses were tested with

commercially available kits (Axsym, Abbott Diagnostics,

Germany) for HBsAg, Hepatitis B e antigen (HBeAg), anti-

HBe, anti-HBc IgG, anti-HBsAg and anti-hepatitis C virus

(anti-HCV) for HCC patients and HBsAg and anti-HCV for

control subjects. Genomic DNA was isolated from

peripheral leukocytes. The blood samples were submitted

to digestion in SDS/proteinase K buffer at 37 8C for 6–12 h,

followed by two phenol and one chloroform extraction.

DNA was then ethanol-precipitated and resuspended in TE

buffer.

MDM2 SNP309 polymorphism was detected using an

amplification followed by restriction analysis (PCR–RFLP)

method. A step-down amplification was performed at

annealing temperature of 55 8C for analysis of SNP309. The

polymerase chain reaction was performed with a set of

primers forward 50-CCCGGACGA TATTGAACA-30and

reverse 50-AGAAGCCCAGACGGAAAC-30) (Eurogentec,

Seraing, Belgium). The fragment was amplified in a

reaction volume 25 ml using 0.2 mM primers, 200 mM

dNTP and 1 U Taq polymerase. A 226-bp PCR product was

digested 16 h at 37 8C using 10 units of MspA1 I

(Fermentas-Euromedex, Souffelweyersheim, France).

Digestion products were loaded on a 4% STG agarose

gel stained with ethidium bromide (Eurobio, Les Ulys,

France). The wild-type (SNP309T) allele produces tow

fragments 117- and 109-bp. Presence of the T>G

polymorphism creates an additional restriction site cleaving

the 109-bp fragment in 63- and 46-bp products. About 60%

of the samples were randomly selected to be sequenced on

SNP309 in order to confirm the predicted pattern of

restriction. PCR products were purified by using the

Exonuclease I/Shrimp Alkaline Phosphatase and sequenced

using Big Dye Terminator version 3.1 kit (Applied

Biosystems, Foster City, CA) and run on an ABI automated

fluorescent 3130 DNA sequencer. Sequencing data were

analyzed using SeqScape v2.5 software (Applied Biosys-

tems). The results of sequencing were 100% concordant

with the results of RFLP.

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S. Ezzikouri et al. / Cancer Detection and Prevention 32 (2009) 380–385382

Table 1

Characteristics of HCC patients and control subjects.

Characteristics HCC patients

(n = 96)

Controls

(n = 222)

P-value

Age (mean � SD years) 59.3 � 14.1 56.4 � 10.1 0.053a

Sex ratio (male/female) 1.53 1.33 0.987b

HBV infection

HBsAg positive 12 (12.5%) 7 (3.1%) 0.005c

HBsAg negative 84 (87.5%) 215 (96.7%) –

HCV infection

Anti-HCV positive 55 (57.3%) (d) –

Anti-HCV negative 41 (42.7%) 161 (72.5%) –

Hepatitis B and C 5 (5.3%) 1 (0.4%) 0.018c

Alcoholic 3 (3.1%) 3 (1.3%) 0.534c

NIDDMe 8 (8.3%) 3 (1.3%) 0.007c

a Calculated using ANOVA.b Calculated using Wilcoxon.c Calculated using the Chi-square test.d Prevalence was not calculated because the control group included two

groups (healthy subjects and subjects with HCV infection).e Non-insulin-dependent diabetes mellitus.

Table 2

Distribution of MDM2 SNP309 genotypes in HCC cases and controls.

Genotype Controls

(n = 222), (%)

HCC cases

(n = 96), (%)

OR (95%CI)a P-value

MDM2 309T>G

TT 120 (54.04) 39 (40.62) 1.00

TG 89 (40.09) 46 (47.92) 1.59 (0.96–2.64) 0.071

GG 13 (5.85) 11 (11.46) 2.60 (1.08–6.28) 0.029

TG+GG 102 (45.94) 57 (59.37) 1.72 (1.06–2.79) 0.027

Allele

T 0.74 � 0.02 0.65 � 0.03 1.00

G 0.26 � 0.02 0.35 � 0.03 1.56 (1.09–2.26) 0.014

aMultivariate analysis adjusted for age, sex, HBV and HCV infection.

2.3. Statistical analysis

In a preliminary study on 100 control individuals, we

found 7 subjects with a GG homozygotes genotypes. The

sample size was therefore estimated according to the

following equation: n = Z2( pq)/d2; n is the number of

individual included in the study, Z is the value of the normal

law interval of confidence 95% ( p < 0.05); p = the

frequency of GG; q = 1 � p and d is the risk error. One-

way analysis of variance was conducted to compare two

means. Differences in sex, HBV infection and HCV

infection between HCC cases and controls were evaluated

using the Wilcoxon test and Chi-square test. Frequencies

were compared by Fisher’s exact test and Chi-square test.

Departures from Hardy–Weinberg equilibrium were deter-

mined by comparing the observed genotype frequencies

with expected genotype frequencies calculated using

observed allele frequencies by a Chi-square test. The

associations between the MDM2 309T>G polymorphism

and HCC risk were estimated by computing the ORs and

their 95% CIs from both univariate and multivariate logistic

regression analyses with adjustment for age, sex (male vs.

female), HBVand/or HCV infections (negative vs. positive).

Statistical analysis of the data was performed on SISA

(Simple Interactive Statistical Analysis). A P-value of <5%

was considered statistically significant.

3. Results

We investigated the relationship between MDM2

309T>G polymorphism in the promoter region of MDM2

gene and hepatocellular carcinoma in case–control study of

96 cases (mean age 59.3 � 14.1 years; range 26–81 years)

and 222 controls (mean age 56.4 � 10.1 years; range 26–85

years). Liver parenchyma was cirrhotic in more than 80% of

cases. Mean tumor size was 4.5 � 3.6 cm. Chronic hepatitis

C is the main risk factor in the population studied, HCV-

targeted antibodies are present in 57.3% of the cases. HBsAg

was present in only 12.5% of cases. HBV and HCV were

both present in 5.3% of the cases. One patient has auto-

immune hepatitis seropositive for liver kidney microsome 1

(LKM1) antibody. Eight patients (8.3%) had non-insulin-

dependent diabetes mellitus (NIDDM). Three patients

(3.1%) were alcoholic. The general characteristics of the

case patients and controls are shown in Table 1. There were

no significant differences between patients with HCC and

control subjects in terms of age and sex (P > 0.05; Table 1),

which suggest adequate matching based on these variables.

However, there was more HCV infection among cases than

in the first group control (healthy subjects). This difference

was controlled for in the multivariate analyses.

We analyzed the MDM2 309T>G polymorphism in 96

HCC cases and 222 controls using PCR–RFLP. The results

of PCR–RFLP were subsequently confirmed by direct

sequencing of 20 randomly chosen samples of each

genotype. A 100% concordance was found with RFLP-

predicted genotypes. The genotype and allele distribution of

the MDM2 309T>G polymorphism in the HCC cases and

controls are shown in Table 2. The genotype distributions of

the MDM2 309T>G polymorphism among the controls

were in Hardy–Weinberg equilibrium (Chi-square = 0.43,

P = 0.60). The genotype frequencies were 54.04% (TT),

40.09% (TG) and 5.85% (GG), respectively, among the

controls, which were significantly different from those of

HCC (40.62% TT, 47.92% TG and 11.46% GG). The GG

genotype was more frequent in the HCC cases than in

controls (11.46% vs. 5.85%, P = 0.029). Multivariate

logistic regression analyses revealed that compared with

the SNP 309 TT genotype, the ORs for HCC patients

carrying the TG, GG and TG+GG genotypes were 1.59 (95%

CI, 0.96–2.64; P = 0.071), 2.60 (95% CI, 1.08–6.28;

P = 0.029) and 1.72 (95% CI, 1.06–2.79; P = 0.027),

respectively. The MDM2 309T>G genotype distributions

in HCC cases and controls stratified by gender and HCV

infection status are shown in Tables 3 and 4. Subgroup

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S. Ezzikouri et al. / Cancer Detection and Prevention 32 (2009) 380–385 383

Table 3

Distribution of MDM2 SNP309 genotypes and associated odds ratio in

relation to sex.

Genotype Controls,

no. (%)

HCC cases,

no. (%)

OR (95%CI)a P-value

Male

TT 69 (54.33) 25 (43.10) 1.00

TG 53 (41.73) 27 (46.55) 1.41 (0.73–2.70) 0.304

GG 5 (3.94) 6 (10.34) 3.31 (0.93–11.82) 0.054

TG + GG 58 (45.67) 33 (56.89) 1.57 (0.84–2.94) 0.156

Female

TT 51 (53.68) 14 (36.84) 1.00

TG 36 (37.89) 19 (50) 1.92 (0.85–4.32) 0.111

GG 8 (8.42) 5 (13.16) 2.28 (0.64–8.06) 0.194

TG + GG 44 (46.32) 24 (63.16) 1.98 (0.92–4.30) 0.079

aAdjusted for age, HBV and HCV infection.

analysis revealed that the effect was higher among men.

Male patients, SNP309 GG carriers, had odds ratio of 3.31

(95% CI, 0.93–11.82) (Table 3).

Patients with HCV infection and GG genotype had 3.7-

fold (0.82–16.45) increased risk for HCC than controls

(Table 4). Age at diagnosis with hepatocellular carcinoma

(mean � standard deviation) was not significantly different

among MDM2 SNP309 genotypes (TT: 61.23 � 11.81, TG:

58.69 � 12.52 and GG: 62.33 � 14.68, P = 0.610).

4. Discussion

MDM2 functions as key negative regulator of p53. It

binds to the N-terminal transactivation domain of p53,

thereby inhibiting its transcriptional activity. MDM2 has

also been shown to promote tumorigenesis by interacting

with Rb and to inhibit its growth regulatory function [12–

14]. Recently a SNP309 in the promoter region of MDM2

has been shown to be associated with both hereditary and

sporadic cancers in humans [10]. Several groups have

reported an association between the MDM2 309 G variant

and increased risk for epithelial cancer, including gastric

cancer [15–17], breast [18], lung [19] and bladder cancers

[20]. In other studies, however, no association between

Table 4

Distribution of MDM2 SNP309 genotypes and associated odds ratio in HCV inf

HCV infection Genotype Controls, (%)

Negative TT 87 (54.04)

TG 64 (39.75)

GG 10 (6.21)

TG + GG 74 (45.96)

Positive TT 33 (54.10)

TG 25 (40.98)

GG 3 (4.92)

TG + GG 28 (34.41)

Allele T 0.75 � 0.03

G 0.25 � 0.03

aAdjusted for age, HBV and sex.

MDM2 309T>G polymorphism and cancer risk was found

[21–23]. It is possible that some of these studies did not find

a correlation between the polymorphism and cancer because

they did not control for ethnic differences in allele frequency

in unaffected individuals. It is also possible, however, that

relevant environmental factors attenuate or increase the

impact of MDM2 polymorphism on cancer development.

In this case–control study, we investigated, the associa-

tion between the promoter polymorphism of MDM2

309T>G and hepatocellular carcinoma. The analysis of

96 HCC patients and 222 matched controls demonstrates

that the functional polymorphism in the MDM2 promoter

had a significant impact on the risk of developing HCC in

Moroccan patients. We found that patients with MDM2 309

GG genotype had 2.6-fold increased risk when compared to

MDM2 309 TT genotype. This finding seems to be in line

with the well-documented functional relevance of this SNP.

Furthermore, Dharel et al. recently was found a high

prevalence of G alleles of the SNP309 in Japanese patients

with HCC specifically in patients with hepatitis C [11]. In

fact, previous study has been shown that the G alleles

increases the binding affinity of Sp1 to the promoter of

MDM2, resulting in increased MDM2 expression and

attenuated the p53 tumor suppressor pathway [10]. Studies

using MDM2 transgenic mice have shown that 100% of the

MDM2-overexpression mice developed spontaneous tumors

in a lifetime [9]. Given the role of MDM2 in cancer

formation, one might expect that individuals who carry the G

allele and thus have heightened expression of MDM2 over a

lifetime are at higher risk for developing HCC.

The frequency of the MDM2 309G allele among control

Moroccans was 0.260, which was significantly higher than

those reported in African-Americans (0.114) [22] but lower

than those in Koreans (0.534) [16], Han Chinese of northeast

China (0.456) and Caucasians (0.358–0.402) [19,22,24].

When controlled for gender, the analysis of HCC

patients revealed a modest but significant association with

male sex (see Table 3). Indeed, gender differences for age

at cancer onset have been already described for MDM2

SNP309 polymorphism. The G allele was more frequent in

younger women than young men, suggesting a synergic

ection.

HCC, (%) OR (95%CI)a P-value

21 (51.22) 1.00

15 (36.58) 0.97 (0.46–2.03) 0.937

5 (12.19) 2.07 (0.64–6.70) 0.217

20 (48.78) 1.12 (0.56–2.22) 0.747

18 (32.73) 1.00

31 (56.36) 2.27 (1.04–3.30) 0.037

6 (10.91) 3.7 (0.82–16.45) 0.076

37 (67.27) 2.4 (1.14–5.16) 0.020

0.61 � 0.04 1.00

0.39 � 0.04 1.88 (1.08–3.30) 0.025

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S. Ezzikouri et al. / Cancer Detection and Prevention 32 (2009) 380–385384

activity of estrogen receptor response element with Sp1

binding site at SNP309 [25]. MDM2 SNP309 accelerates

tumor formation in a gender-specific and hormone-

dependent manner [10,25]. The situation is, thus, quite

different in HCC with a mildly enhanced male suscept-

ibility. However, it has been shown in a recently published

mouse model as well as in tumor cells culture that MDM2

plays a critical role in the growth of hormone-dependent

prostate cancer [26]. Combined targeting of epidermal

growth factor receptor and MDM2 by gefitinib and

antisense MDM2 cooperatively inhibits hormone-inde-

pendent prostate cancer [26,27]. We can, therefore,

hypothesize that MDM2 SNP309 GG synergizes with

androgens during human liver carcinogenesis.

In Moroccan patients, more than 57% of hepatocellular

carcinoma are caused by the HCV infection alone.

Persistence of the viral infection in hepatic cell is strongly

associated with the hepatocarcinogenesis [28]. In our study,

stratification based on HCV infection status showed that

patients with HCV are more likely to be carriers of the GG

genotype (OR = 3.7, 95% CI, 0.82–16.45). However,

initially due to the small sample size, wide CI were defined

by our analysis. Our data warrant thus confirmation on a

larger series of individuals. This result is in agreement with

previous study and strong p53 expression suppresses

replication of the HCV in vitro, the viral replication is

significantly enhanced when p53 gene expression is

suppressed [11]. On the other hand, in HCV-positive

HCC patients, high prevalence of G alleles of the

SNP309 implies that the p53 functions could have been

indirectly suppressed by the heightened MDM2 levels,

making them more vulnerable to tumor development.

In previous studies, an association between SNP309 and

lower age at diagnosis of cancer was shown [10,24,29].

However, in Moroccan population, SNP309 was not

significantly associated with age at diagnosis (P = 0.61).

In conclusion, our results indicate an association between

the SNP309 GG genotype and risk of hepatocellular

carcinoma in Moroccan population. SNP309 GG was

especially more prevalent among males and HCV-infected

patients.

Conflict of interest

None.

Acknowledgements

The authors would like to acknowledge all patients for

their participation in this study. We thank the Direction des

Programmes Transversaux de Recherches of the Institut

Pasteur, Paris for their financial supports (PTR no. 130). We

are particularly grateful to Benoit Robert and Michele Joliy

for their advises and encouragements. We also thank

Nathalie Jolly and Christine Sadorge for their skillfull

expertise in Medical Research Protocol Elaboration.

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