A case-control study on the SNP309T → G and 40-bp Del1518 of the MDM2 gene and a systematic review for MDM2 polymorphisms in the patients with breast cancer.

OBJECTIVE: The current research was conducted to study the association between the SNP309 and del1518 polymorphisms with the breast cancer in the patients with the Kurdish ethnic background from western Iran. Also, a systematic review of the relevant case-control studies on the MDM2 polymorphisms in the patients with breast cancer was performed.
METHODOLOGY: Two mL of peripheral blood was taken from 100 patients with breast cancer and 100 healthy individuals. The frequencies of MDM2 SNP309 and del1518 genotypes and alleles were determined using the PCR-RFLP and PCR methods, respectively.
RESULTS: The frequency of the TT, TG, and GG of MDM2-SNP309 genotypes in the patients was obtained as 23%, 52%, and 25%, and they were equal to 22%, 40%, and 38% in the control group, respectively. Also, considering the MDM2-del1518 polymorphism, the frequencies of ins/ins, ins/del, and del/del genotypes were equal to 52%, 41%, and 7% in the breast cancer group and they were equal to 62, 30, and 8% in the control group, respectively. Analysis of the results indicated that the GG genotype plays a protective role for the breast cancer in the recessive model (GG vs TT + TG) of SNP309 (χ2  = 3.916, P = .048, and OR = 0.54).
CONCLUSION: Our findings revealed that the GG genotype of MDM2-SNP309 can play a protective role in the breast cancer disease. Also, our systematic review indicated that the SNP309, SNP285, and del1518 of MDM2 gene in different populations mostly did not have a significant association with the risk of breast cancer.

INTRODUCTION

Breast cancer (BC), as the most common type of cancer in the women, is a multi‐factorial and heterogeneous malignancy in the world. , , The crude incidence rate of BC in the Iranian women has raised from 24 cases in 2004 to 33.8 cases per 100 000 in 2018. , , This cancer is caused by the interaction effects of the genetic, hormonal, and environmental factors. , , Exposure to the carcinogen agents, ionizing radiation, diet, physical activity or exercise, gender, woman's hormonal history, age, and obesity are the main effective risk factors for the breast cancer. , Single nucleotide polymorphisms (SNPs) in the functional genes could influence the incidence of breast cancer and its progression. , , Results of a recent meta‐analysis and research studies have confirmed a correlation between the SNPs of candidate genes with the risk of breast cancer.

Main cellular regulatory mechanisms for the arrest of cell cycle and apoptosis in the stressed or damaged cells are involved in the cancer susceptibility. The p53 is a primary regulatory factor that can be expressed in response to the increased expression of oncogenic proteins in the stressed conditions of the cells. , , Under normal conditions, it is important to keep the p53 at the low level, so human homologue of mouse double minute 2 (MDM2) is a negative regulator of the p53 reducing the p53 level in the cell. The MDM2 controls the expression level of the p53 using two mechanisms: Firstly, it prevents the transcriptional activity of p53 through direct binding as a physical blocker. Secondly, it can be degraded by the proteasome through the ubiquitination of the p53. , , , , Mono‐ubiquitination causes the p53 to transfer from the nucleus to the cytoplasm and ultimately to be degraded by the proteasome, while poly‐ubiquitination targets p53 cause the degradation with the proteasome in the nucleus.

There are controversial findings regarding the association of the SNP309 and del1518 of the MDM2 gene with the breast cancer in different populations. On the other hand, there are big ethnicities in the Iran's population. Also, the frequency and association of these two main polymorphisms of the MDM2 gene together have not been investigated in the patients with breast cancer among the Kurdish population so far. Therefore, the current study was conducted for the first time to investigate the SNP309T>G and del1518 of the MDM2 gene in the patients with breast cancer and healthy individuals with the Kurdish ethnic background from western Iran. Also, a systematic review was designed and the case‐control studies on evaluating the effect of all the investigated MDM2 variations on the breast cancer risk were perused for the first time.

PATIENTS AND METHODOLOGY

Patients

Two mL of the blood samples in tubes was obtained from every 100 patients with breast cancer (mean age ± SD of 48 ± 11 years old, with the age range of 24‐71 years old) and 100 age‐matched (≤5 years) healthy individuals as the control group, with no history of cancer. The sampling was carried out from September 2017 to May 2018. Patients with a history of other types of cancer and fibroadenoma were excluded from the study. Also, all the cases and controls had the Kurdish ethnicity and were from the western Iran. Information on the age onset of the disease, first‐degree family history of breast cancer, and a first‐degree family of other types of cancer, estrogen receptor (ER), HER's‐2 expression, progesterone receptor (PgR), P53, Ki‐67, lymph node metastasis, and tumor grade was obtained from the patients̕ medical history. Tumor grading was carried out according to the pathologists̕ report. HER‐2 was tested by the immunohistochemistry (IHC) method, and the samples with score of 2+ were then checked by the FISH protocol. Scores of 3+ and 2+ with the positive FISH report were recorded as the HER‐2 positive. The sample size of the study was defined based on the MDM2 genotype frequency data presented in the studies by Akisik et al (2011) and Alshatwi et al (2012) using the following formula:

A sample size of ~80 was calculated for each study group considering the alpha value of 0.01 and beta value of 0.05, but to ensure regarding the sample adequacy, a sample size of 100 was studied in each group.

Selection of the SNPs and bioinformatics analysis

The SNPs were selected based on the reported studies on the main polymorphisms in the promoter region of the MDM2 gene in the different populations of breast cancer worldwide. Also, the selected SNPs were checked in some web servers and online prediction tools. SNP309T>G (rs2279744) and del1518 (rs3730485) were checked by the Variant Effect Predictor (Ensembl Tools) (https://www.ensembl.org/Homo_sapiens/Tools/VEP), rSNPBase 3.1 (http://rsnp3.psych.ac.cn/), SNP Function Prediction (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html), TFBIND (http://tfbind.hgc.jp/), and MethPrimer 2.0 (http://www.urogene.org/methprimer2/).

DNA extraction

DNA was extracted from the blood samples using the DNA extraction kit based on the manufacturer's instructions (Zagros Bioidea Co., Iran). Concentration and purity of the extracted DNA were evaluated using a NanoDrop (Thermo‐2000/2000c, USA).

Genotype analysis

The PCR‐RFLP and PCR techniques were carried out to investigate the distribution of MDM2 SNP309 and del1518 genotypes, respectively. The final volume was equal to 25 μL for each PCR process, containing 12 μL of Master Mix PCR (SinaClon Co., Iran), 100‐500 ng of the extracted DNA and 400 nmol/L of each specific primer. , The sequences of the primers were checked and confirmed using the UCSC In‐Silico PCR (http://genome.ucsc.edu/). Amplification conditions of the SNP309 were as follows: first denaturation cycle at 95°C and 5 minutes, 40 cycles: 35 seconds at 94°C, 35 seconds at 59°C and 30 seconds at 72°C, followed by a final extension (72°C for 10 minutes). Also, PCR parameters for del1518 were as follows: initial denaturation cycle for 5 minutes at 94°C, then 35 cycles: 30 seconds at 94°C, 30 seconds at 58°C, 30 seconds at 72°C, followed by a final extension cycle for 10 minutes at 72°C. The amplified fragment of MDM2‐SNP309 with 352 bps was digested by two units of restriction enzyme, MspA1I (New England Biolabs, Ipswich, MA) for 15 hour of incubation period at 37°C. Then, the obtained fragments of MDM2‐SNP309 and amplified products of MDM2‐del1518 were electrophoresed on the 2% and 2.5% agarose gels and were stained by the GelRed, respectively. An amount of 5% of MDM2‐SNP309 and MDM2‐del1518 genotypes in the patients was randomly selected and identified by the DNA sequencer (BiONEER, South Korea).

Statistical analysis

Frequencies of the genotypes in the studied SNPs were analyzed using the online Hardy‐Weinberg equilibrium calculator. The genotype frequencies were compared using the Pearson's chi‐squared test in the IBM SPSS software (version 16). Analysis of the odds ratios and 95% confidence intervals were done by the online MedCalc statistical software (MedCalc, Ostend, Belgium). Haplotype analysis was carried out using a web‐based application. The analysis of the age onset of the disease was done by the one‐way analysis of variance (ANOVA).

Search strategy, inclusion and exclusion criteria, and data extraction

The relevant papers were selected from the PubMed and Scopus databases. A time limit was set in the searches up to August 7, 2019 (last updated search). The terms of (MDM2 OR "mouse double minute 2" OR "murine double minute 2" OR HDM2) and (breast) and (polymorphism OR SNP OR variant) were used to search in the Scopus database, and the used terms in the PubMed database were (MDM2 OR mouse double minute 2 homolog OR human homolog of mouse double minute 2 OR HDM2 OR murine double minute 2) AND (breast cancer OR breast carcinoma OR breast neoplasm) AND (single nucleotide polymorphism OR SNP OR polymorphism OR variant).

Inclusion criteria in this systematic review were as follows: (a) original studies investigated the MDM2 gene variations by the experimental methods for genotyping of the patients with breast cancer, (b) using the blood or tissue of the human samples, and (c) evaluating both case and control groups. Exclusion criteria were as follows: (a) cell line surveys; (b) studies that did not report the association of SNP status at least with one of the BC risk, IHC/outcome, or onset age variables; (c) review and meta‐analysis papers; (d) lack of reporting the frequency of each genotype; (e) papers that had the full text in a non‐English language; (f) the conference, letter, note, and editorial papers; and (g) history of cancer in the individuals of control groups (Figure 1). Full text of the eligible papers was checked. Information on the studied variation, country, geographic area of the population, ethnicity, sample size, main case background, matching in case‐control studies, source of samples, and the associated risk with BC, IHC/outcome and onset age was obtained from the selected papers. In some included studies, the corresponding author or co‐authors were contacted (email) to complete the study information.

Figure 1

PRISMA flow diagram of article selection for systematic review on the MDM2 variations in breast cancer patients

RESULTS

Population specifications

Among the patients, there were 98 females and 2 males. A family history (breast cancer) was detected among 7.4% of the patients. Among 94 patients with the recorded breast cancer history in the first‐degree relative, 80.4% of them were positive. Also, the frequency of IHC markers including ER+, PgR+, and P53+ was equal to 69.4%, 62.4%, and 48.6%, respectively. Patients were classified into four groups of Luminal A, Luminal B, HER2‐enriched, and Basal‐like based on the tumor markers with the values of 14.6%, 54.9%, 24.4%, and 6.1%, respectively. These molecular subclasses were classified according to the presentations provided in the IMPAKT Breast Cancer Conference in Brussels, Belgium, on May 2012.

Bioinformatics analysis

In the rSNPBase database, SNP309 and del1518 polymorphisms were introduced as rSNPs, and the presence of TF binding region as the related regulatory element was confirmed. The performed analysis by the VEP tool indicated the rs2279744 as a risk factor in the ClinVar database, and a high score was reported for rs3730485 in the transcript support level. SNP Function Prediction and TFBIND tools confirmed a new site region for the SNP309G allele and del allele in del1518. The CpG island predictions of two polymorphisms were checked in the MethPrimer 2.0 tool, and the results indicated that the rs2279744 is located in the CpG island region.

SNP309 and Del1518 frequencies in the control and breast cancer groups

Figure 2 shows the electrophoresis pattern of the RFLP fragments of MDM2‐SNP309 and amplified products of MDM2‐del1518. The frequencies of SNP309 genotypes were as follows: TT (23%), TG (52%), and GG (25%) among the patients, and they were obtained as follows: TT (23%), TG (52%), and GG (25%) in the controls (P‐value = .118). Also, the rate of del1518 genotypes in the patient group was as follows: ins/ins (52%), ins/del (41%), and del/del (7%), and they were obtained as follows: ins/ins (62%), ins/del (30%), and del/del (8%) in the control group (P‐value = .266). The frequencies of SNP309 and del1518 genotypes were evaluated among all the individuals in the two study groups using the Hardy‐Weinberg equilibrium by the available software, and the results showed no significant deviation (P‐values > .05). Frequency distribution of MDM2 SNP309 and del1518 genotypes was analyzed in the patients and control subjects, and the results indicated no positive difference in the genotype frequencies of the co‐dominant, dominant, recessive, and over‐dominant models between the two study groups. Also, no significant difference was found for MDM2 SNP309 and del1518 allele frequencies between the patients and controls. However, in SNP309 polymorphism, the GG genotype in the recessive model was positively different among the patients and controls (P = .048, OR = 0.54, 95% CI = 0.30‐1.00) indicating its protective role against the breast cancer. Then, genotyping results were confirmed by the DNA sequencing. Figure 3 shows the results obtained from each genotype for SNP309 and homozygous genotypes for del1518. Haplotype study of MDM2 gene polymorphisms showed a strong linkage disequilibrium between the SNP309 and del1518 variants (D' = 0.9995) in our studied population.

Figure 2

Gel (agarose) electrophoresis of PCR products for del1518 and PCR‐RFLP for SNP309 of the MDM2 gene. (A) Lane 1 shows del/del genotype; lanes 2, 4, 5, and 6 show ins/ins genotypes; and lane 3 shows ins/del genotype. (B) Lanes 1 and 2 show TG genotypes, and lanes 3 and 4 demonstrate GG and TT genotypes, respectively. Lane 7 in (A) and lane 5 in (B) show 100 bp DNA ladder. The map showed the 100‐bp upstream and downstream of the SNP309

Figure 3

Sequencing results of SNP309 and del1518 in case patients. 1. SNP309 positions were marked with the arrows for TT (1.A), TG (1.B), and GG (1.C) genotypes. 2. The sequence of 40‐bp del1518 was showed in the box in ins/ins genotype (2.A). The Figure 2.B shows the del/del genotype of 40‐bp del1518. Sequencing results were viewed in Chromas (Version 2.6.6)

Polymorphisms, clinicopathological features and demographic factors

The possible association between the clinicopathological features of the patients with SNP309 and del1518 genotypes was analyzed in the co‐dominant, dominant, recessive, and over‐dominant models. Our results indicated a significant association between the MDM2‐SNP309 genotypes in an over‐dominant model with HER2 and p53 status and also in a recessive model with the family history of other types of cancer (Table 1). Also, the association of the SNP309 and del1518 genotypes of the MDM2 gene with the age onset of the disease was evaluated in the patients with breast cancer. Our results demonstrated no significant association between the genotypes of the SNP309 and del1518 and onset age of disease (Data not shown).

Table 1

Association of SNP309 with HER2, P53, and family history of other cancers’ characteristics in breast cancer patients

Variable	SNP309			SNP309
	TG	TT + GG	P‐value* (χ2)	GG	TT + TG	P‐value* (χ2)
Family history of other types of cancer cancer
No	43	31	.142 (2.155)	13	61	.049 (3.869)
Yes	7	11		7	11
P53
Positive	23	12	.018 (5.595)	7	28	.152 (2.054)
Negative	14	23		13	24
HER2
Positive	25	14	.031 (4.677)	8	31	.268 (1.226)
Negative	15	23		12	26

P‐values were calculated with the chi‐square test.

DISCUSSION

The MDM2 gene is located on the chromosome 12 q14.3‐q15.1. The MDM2 gene (HDM2) is transcribed through two promoters: basic promoter (p1) and alternative promoter (p2). , , , Some functional and regulatory sequences in the promoter regions of the MDM2 (rSNP) can change the gene expression level. , It has been indicated that the rSNPs of the genes associated with DNA damage and apoptosis mechanisms can influence the individual's sensitivity to the cancer progression. Several types of research have studied the SNPs in the MDM2 gene and their association with the risk of breast cancer in different populations. Common studied polymorphisms are located in the first intron of the MDM2 gene as P2 promoter. The SNP309T>G (rs2279744) is located at position 309 (IVS1 + 309) in the P2 region so that T to G transversion enhances the binding affinity of specificity protein 1 (Sp1) to a specific sequence in the promoter and, thus, increases the transcriptional level of the MDM2 gene. , , Rs3730485 (GRCh38.p7 assembly), also known as del1518 polymorphism (merged into rs150550023 in GRCh38.p12 assembly), is located in the P1 region of the MDM2 gene having a putative TATA motif. In the del1518 polymorphism, del‐allele can increase the binding of some transcription factors, such as Myocyte Enhancer Factor 2A (MEF2A), RAR‐Related Orphan Receptor A (RORA), and MBD2‐Interacting Zinc Finger protein (MIZF) to the regulatory sequence in the promoter regions. The del1518 has high linkage disequilibrium (LD) with SNP309 locus. , High‐level expression of MDM2 can influence the p53 signaling pathway allowing the damaged cells to escape from the control point of the cell cycle leading to the increase in the carcinogenesis. , , ,

Risk of BC is influenced by the environmental, hormonal, and genetic factors. , , The MDM2 protein is a main negative regulator in controlling the p53 expression. It has been found that the MDM2 protein binds to the p53 and induces the ubiquitination and therefore degradation of the p53 by proteasome. Therefore, high‐level expression of the MDM2 gene contributes to the reduced p53 activity and results in escaping from the checkpoints in the cell cycle. , , The P53, as a tumor suppressor protein, activates the cellular processes, such as halt of the cell cycle, autophagy, and apoptosis process in response to the genotoxic stresses and damages. , , Studies have shown that the genotypes of SNP309GG and del/del in 40‐bp ins/del polymorphisms in the MDM2 gene could influence its expression and may play a significant role in the cancer susceptibility. The association of MDM2‐SNP309 and del1518 with different types of cancer has been investigated in relation to the breast, , esophageal, , uterine leiomyomas, endometrial, bladder, lung, and colorectal cancers ; however, the reported results were contradictory. There are no available reports related to the T309G and del1518 of the MDM2 gene among the Iranian population in the west of Iran; therefore, this study was conducted to investigate the relationship between these MDM2 polymorphisms with the breast cancer for the first time.

Our results showed a negative association between the MDM2‐del1518 polymorphism and development of the breast cancer. Limited studies have been done with the contradictory results on the correlation of MDM2‐del1518 polymorphism and BC. , , , Consistent with our results, Ma et al found no correlation between the MDM2‐del1518 polymorphism and BC in the Chinese population. Gansmo et al (2016) in a meta‐analysis concluded that the MDM2‐del1518 polymorphism was not associated with the BC. Also, Hua et al (2017) performed a meta‐analysis study to investigate the role of MDM2 del1518 polymorphism associated with the cancer susceptibility. Their results provided a significant support for the lack of association between the MDM2 del1518 polymorphism and cancer risk. In other studies conducted in the Chinese population, no association has been reported between the MDM2‐del1518 polymorphism with esophageal squamous cell carcinoma and uterine leiomyomas. , However, inconsistent with the results of our study, Hashemi et al (2014) revealed an association between the MDM2‐del1518 polymorphism with the risk of BC in the Zahedan population, Sistan and Baluchestan province (southeast of Iran). Also, a significant correlation has been observed between the MDM2‐del1518 polymorphism and the BC in the Mexican population. Contradictory results in the reported studies can be due to the differences in the sample size and ethnicity of the populations. It is suggested that the 40‐bp ins/del in the promoter of the MDM2 might not play a major role in the risk of BC.

Herein, no positive association was found between the frequencies of MDM2‐SNP309 alleles with the risk of BC. Consistent with our results, Hosein Pour et al and Tavakkol Afshari et al reported no positive association between the SNP309 with the BC across populations of the northwest and northeast of Iran. Different studies in the various populations failed to confirm a significant association between the MDM2‐SNP309 and an increased risk of BC. , , , , , , , Also, Zhao et al (2012) in a meta‐analysis reported no significant correlation between the MDM2‐SNP309 and the incidence of BC in the European populations. A negative association has been confirmed between the MDM2‐SNP309 and other types of cancer, such as endometrial, bladder, esophageal squamous cell, Kaposi's sarcoma, and uterine leiomyomas. However, in several studies, an association has been reported between the SNP309 polymorphism and the incidence of BC worldwide. , , , , , , ,

Our results indicated a protective role of the GG genotype in SNP309 against the BC (P = .048, OR = 0.54, 95%CI = 0.30‐1.00). Consistent with our findings, some studies have revealed the protective role of GG genotype of MDM2‐SNP309 in different cancers including the lung, colorectal, and esophageal cancers. Protective role of GG genotype in MDM2‐SNP309 may be due to the formation of haplotypes with other unknown effective rSNPs. It can reduce the positive effect of SNP309 and highly influences the expression level of the MDM2 gene and decreases it.

The strong linkage disequilibrium was observed between the SNP309 and del1518 variants in the studied population. In fact, when the alleles are in the linkage disequilibrium condition, haplotypes do not occur with the expected frequencies. It can be used to improve the power of the cancer‐genetic association studies, and it helps to detect the true associations in the case‐control studies. So, the LD results suggested the homogeneity of these polymorphisms in the Kurdish population.

The contradictory results for the function of MDM2‐SNP309 in different types of cancer may be due to several reasons: (a) The effect of this variant is related to the changes in the binding of tissue specific‐transcription factors (TF) in the promoter ; (b) malignancies in various tissues can have different molecular mechanisms, and even in one type of cancer because of heterogeneity across different individuals ; (c) the different sample size of the studied populations ; (d) differences in the ethnicity and lifestyle‐related factors in various studied populations may influence the effect of MDM2‐SNP309 on the incidence of BC , ; (e) function and expression level of the MDM2 gene or TFs‐associated genes may be related to the DNA methylation as a main epigenetic mechanism so that the methylation status of the regulatory sequence in the promoter is different in various populations based on the specific environmental and ethnic conditions , ; (f) polymorphism effect can be related to the interaction with the haplotypes and other SNPs in the MDM2 gene ; and also (g) variable of minor allele frequency (MAF) can show up in the demographic layers of the populations, and as a result, the risk of BC may change with respect to the ethnicity because of their allele frequencies.

Some diseases, such as cancer, are complex, meaning that they are caused by the multiple genes and environmental factors. Studying a few genetic polymorphisms in such small populations is like only a piece of the large puzzle presenting their ability to influence the cancer risk in different populations, especially ethnicities. Also, it can provide an overall perspective for the subsequent studies on the genes involved in the desired pathway in the ethnic groups.

In the next step, our research was expanded with a systematic review of all the variations of the studied MDM2 gene in the patients with breast cancer for the first time. There were several case‐control studies on assessment of the functional variations of the MDM2 and the susceptibility to BC. As indicated in Table 2, seven single nucleotide variations (SNVs) and an ins/del were investigated in the eligible reviewed studies. Commonly studied polymorphisms are located in the first intron (Intron 1‐2) as P2 promoter of MDM2 gene.

Table 2

Properties of included studied polymorphisms in the systematic review

RsID	Name	Polymorphism	Risk allele * , a	Position b	Consequence in MDM2	Main TF‐related SNP	Frequency of minor allele c
Rs150550023 d	Del1518	40‐bp ins/del	Deletion	chr12:68806996‐68807065	2KB Upstream	RORA, MEF2A, MIZF	0.37 e
Rs2870820	SNP55	C → T	T	chr12:68808546	Intron 1‐2	Sp1	0.23
Rs117039649	SNP285	G → C	C	chr12:68808776	Intron 1‐2	Sp1	0.01
Rs2279744	SNP309, G2580T	T → G	G	chr12:68808800	Intron 1‐2	Sp1	0.36
Rs1196333	SNP344	T → A	A	chr12:68808835	Intron 1‐2	TFAP2A	0.04
ND	SNP443	G → T	T	chr12:68808934	Intron 1‐2	NR	NR
Rs769412	SNP354	A → G	G	chr12:68839435	Exon 11 f	NR	0.07
Rs937283	G2164A	A → G	G	chr12:68808384	5 Prime UTR	NR	0.30

Minor alleles were considered as the risk allele.

Based on GRCh38.p12 assembly.

1000 genome.

Rs3730485 based on GRCh38.p7 assembly.

Based on the frequency of GenomAD.

Synonymous mutation.

Totally, 46 studies on the SNVs and one ins/del reported in 36 papers and the current research had the eligibility to be included in the systematic review (Figure 4). The status of eight variants in the patients with BC was included in the populations from 23 countries in the systematic review. Table 3 shows the characteristics of the eligible studies.

Figure 4

The positions of the all assessed MDM2 variations in the current systematic review on breast cancer patients

Table 3

All investigated MDM2 variations in the case‐control studies on the BC patients

rs# (variation name)	Country (population1)	Ethnicity	Sample source	No. of case/control	Patients		Control group		Genotyping method(s)	Association with			References
					Type	Gender	Match with BC	Properties		Risk2	IHC & outcome3	Age of onset4
rs3730485 (del1518)	China (Nanjing And Southeast)	Chinese, Unrelated Ethnic Han	Blood	366/605	BC	Female	Age, gender	Cancer‐free	PCR	Not significant	NR	Negative	1
	Iran (Kermanshah Province, West)	Kurdish	Blood	100/100	BC	98 Females, 2 Males	Age, Area Of Residence	Healthy, Cancer‐Free	PCR	Not Significant	Negative	Negative	Present Study
	Iran (Southeast)	NR	Blood	236/203	BC	NR	Age	Cancer‐Free	PCR	Higher Risk	Negative	Negative	72
	Mexico (Guadalajara City)	NR	Blood	742/345	BC	Female	Gender, Area Of Residence	Healthy	PCR	Higher Risk	NR	NR	73
	Norway (CONOR) Study	NR	Blood	1717/1872	BC	Female	Age, Gender, Area Of Residence, Same Cohort	Healthy, Cancer‐Free	PCR	Not Significant	NR	NR	74
rs2870820 (SNP55)	Norway (CONOR) Study	NR	Blood	1707/1858	BC	Female	Age, Gender, Same Cohort	Healthy	LightSNiP	Not Significant	NR	Negative	75
rs117039649 (SNP285)	Austria	Austrian	Blood	406/254	BC	Female	Gender, Geography	benign gynecological lesion/Healthy	Real‐Time PCR	Not Significant	Negative	Negative	76
	Mixed (Norway And Netherlands)	Mixed (Norwegian And Dutch)	Blood & Cancerious Breast Tissue	1973/2518	1. BC 2. BC, ER+	NR	Same Countries	Healthy	Sequencing	NR	NR	1. NR 2. Negative	77
	Norway (CONOR) Study	NR	Blood	1717/1872	BC	Female	Age, Gender, Area Of Residence, Same Cohort	Healthy	LightSnip	Not Significant	NR	NR	78
	Poland (Wielkopolska)	Caucasian	Blood	468/550	BC	Female	Gender, Geography, Ethnicity	Healthy	Sequencing	Lower Risk	Negative	NR	79
	Scotland	Scottish Caucasian	Blood	299/275	BC	Female	Gender, Geography, Ethnicity	Cancer‐Free	Sequencing	NR	NR	Negative	80
rs2279744 (SNP309)	Austria	Austrian	Blood	406/254	BC	Female	Gender, Geography	Benign gynecological lesion/Healthy	Real‐Time PCR	Not Significant	P53, Ki67	Positive	76
	Brazil	NR	Blood/ Non‐Tumoral Tissue	39/186	BC, R337H Mutation Carriers	NR	NR	Cancer‐Free, No Family History Of Cancer, Without The R337H Mutation	Real‐Time PCR	NR	Negative	NR	81
	Canada	Caucasian	Blood	38/379	BC, Pre‐Menopasual	Female	NR	NR	Sequencing	Not Significant	NR	NR	82
	China (Shanghai)	NR	Blood	1. 402/84 2. 402/605	BC	Female	1. Gender, Geography 2. Gender	Healthy, Cancer‐Free	Sequencing	1. Higher Risk 2. Higher Risk	Negative	Positive	83
	China (Nanjing And Southeast)	Chinese, Unrelated Ethnic Han	Blood	366/605	BC	Female	Age, Gender	Cancer‐Free	PIRA‐PCR, Sequencing	Not Significant	NR	Negative	71
	Czech Republic	NR	Tissue/ Blood	158/149	BC	NR	NR	Mixed Of Healthy And Ischemic Disease (Cancer‐Free)	PCR‐RFLP	Not Significant	Negative	Negative	84
	England (WCGS)	NR	NR	59/102	BC/ With BRCA1 Mutations	Female	NR	NR	Pyrosequencing	NR	NR	Negative	85
	England (Anglo‐Saxon Population)	British	NR	351/258	BC	Female	Gender, Geography	Cancer‐Free	Allele Specific PCR	Not Significant	NR	Negative	86
	German	NR	Blood	549/1065	Familial BC, Lacking BRCA1&2 Mutations	Female	Ethnicity	Healthy	Real‐Time PCR, Sequencing	Not Significant	NR	Negative	87
	India (North)	NR	Blood	100/100	BC, IDC	NR	Age	Healthy, Cancer‐Free	Allele Specific PCR	Not Significant	HER2, Distant Metastasis	NR	88
	India (Lucknow, North)	NR	Blood	104/105	BC	Female	Gender, Geography, Ethnicity	Tumor/Cancer‐Free	ARMS‐PCR	Not Significant	NR	NR	89
	Iran (Kermanshah Province, West)	Kurdish	Blood	100/100	BC	98 Females, 2 Males	Age, Area of residence	Healthy, Cancer‐Free	PCR	Lower Risk	HER2, P53, Family history of cancer	Negative	Present Study
	Iran (Mashhad City, Southeast)	NR	Blood	128/126	BC	Female	Age, Gender	Healthy	ARMS‐PCR	Not Significant5	Negative	Positive	90
	Israel	Ashkenazi–Jewish (AJ) Origin	NR	187/138	BC, BRCA1/2 Mutation Non‐Carrier	Female	Gender	Cancer‐Free	MALDI‐TOF	Not Significant	NR	Negative	91
	KSA	Arabian	Blood	100/100	BC	Female	Gender, Ethnicity	Healthy	Real‐Time PCR	Higher Risk	Negative	Negative	92
	Kyrgyzstan	Kyrgyz	Blood	117/102	BC	Female	Age, Gender	BC‐Free	PCR‐RFLP	Not Significant	NR	NR	93
	Mixed (Norway And Netherlands)	Mixed (Norwegian And Dutch)	Blood & Cancerious Breast Tissue	1973/2518	1. BC 2. BC, ER+	NR	Same Countries	Healthy	Sequencing	Not Significant	NR	1. NR 2. Negative	77
	Mixed (UK)	Scottish Caucasian	Blood	299/182	BC	Female	Gender, Geography, Ethnicity	Cancer‐Free	Sequencing	Not Significant	Tumour Grade, Lymph Node, NPI	Negative	80
	Netherlands (South–West)	NR	Blood	343/126	Familial BC	340 Females, 3 Males	Geography	Heterozygous carriers of cystic fibrosis gene mutations	Sequencing	Not Significant	NR	Positive	94
	Norway (CONOR) Study	NR	Blood	1717/1872	BC	Female	Age, Gender, Area Of Residence, Same Cohort	Healthy	Lightsnip	Not Significant	NR	NR	78
	Poland (Wielkopolska)	Caucasian	Blood	468/550	BC	Female	Gender, Geographically, Ethnicity	Healthy	PCR‐RFLP, Sequencing	Not Significant	Negative	NR	79
	Sweden (South‐East Sweden Health Care Region)	NR	Blood/ Normal Lymph Node Tissues	123/146	BC, Young Women	Female	Gender, Geography	Healthy	Pyrosequencing	Not Significant	Negative	NR	95
	Taiwan	Asian Taiwanese, Not Immigrants From America Or Europe.	Blood	255/324	BC	254 Females, 1 Male	‐	Healthy, Cancer‐Free	PCR‐RFLP, Sequencing	Higher Risk	NR	NR	96
	Taiwan	Taiwanes	Blood	124/97	Sporadic BC	Female	Gender, Ethnicity	Healthy, Cancer‐Free	PCR‐RFLP, Sequencing	Higher Risk	NR	Positive	97
	Turkey	NR	Blood	110/138	BC	Female	Age, Gender	Healthy	PCR‐RFLP	Higher Risk	Negative	NR	98
	Turkey	NR	Blood	147/120	Familial BC	Female	Gender	Healthy	PCR‐RFLP	Higher Risk	NR	NR	99
	Turkey	Turkish	Blood	223/149	BC (Ductal Carcinoma)	Female	Age, Gender, Ethnicity	Healthy, Cancer‐Free	PCR‐RFLP	Not Significant	NR	NR	100
	US (NHS Study)	NR	Blood	1519/2271	BC	Female	Age, Menopausal Status, Recent Postmenopausal Hormone (PMH) use	Healthy	PCR‐RFLP	Not Significant	NR	NR	101
	US (Baltimore)	1. African American Descent 2. Caucasian White Descent (Not Hispanic White)	Blood & Cancerious & Noncancerious Breast Tissue	1. 165/178 2. 125/136	BC	Female	Age, Gender, Geography, Race	Cancer‐Free	Real‐Time PCR	Not Significant	Tumor P53 Expression	Negative	102
	US (Carolina Breast Cancer Study)	1. African‐Americans 2. Whites	Blood	1. 767/680 2. 1270/1133	BC	Female	NR	NR	Real‐Time Pcr	Not Significant	ER, PgR	Negative	103
rs1196333 (SNP344)	Mixed (Norway And Netherlands)	Mixed (Norwegian And Dutch	Blood	1271/2954	BC	Female	NR	Healthy	Sequencing	Not Significant	NR	Negative	104
	Scotland	Scottish Caucasian	Blood	299/275	BC	Female	Geography, Ethnicity	Cancer‐Free	Sequencing	Not Significant	NR	Negative	80
rs769412 (SNP354)	US (Baltimore City)	1. African American Descent 2. Caucasian White Descent (Not Hispanic White)	Blood & Cancerious & Noncancerious Tissue	1. 166/176 2. 127/134	BC	Female	Age, Gender, Race	BC‐Free	Real‐Time PCR	1. Higher Risk 2. Not Significant	NR	Negative	102
(SNP443)	Scotland	Scottish Caucasian	Blood	299/275	BC	Female	Geography, Ethnicity	Cancer‐Free	Sequencing	Not Significant	NR	Negative	80
rs937283	China (Central Chinese Population)	NR	Blood	480/500	BC	Female	Age, Gender, Smoking Status, Drinking Status	Healthy, Cancer‐Free	PCR‐RFLP, Sequencing	Higher Risk	NR	NR	105

1. Region of the hospital‐based studies was not reported. 2. Combined effect with other SNPs and classified groups (based on demographic characteristics and clinical features) were not reported. 3. Association between IHC reports and clinical/outcome features with distribution of alleles and genotypes in BC patients. 4. Association with age of incidence in breast cancer patients was considered for each variation alone, and it was not reported in cases of haplotypes or in the subgroup of other variants. Also, age similarity was not included. 5. According to the stated result in the article, no significant association is reported (P = .048).

Abbreviations: ARMS, Amplification Refractory Mutation System; BC, Breast Cancer; CONOR, Cohort of Norway; ER, Estrogen Receptor; IDC, Invasive Ductal Carcinoma; IHC, Immunohistochemistry; MALDI‐TOF, Matrix Assisted Laser Desorption Ionization‐Time of Flight; NPI, Nottingham Prognostic Index; NR, Not Reported; PgR, Progesterone Receptor; PIRA, Primer‐Introduced Restriction Analysis; RFLP, Restriction Fragment Length Polymorphism; WCGS, Wessex Clinical Genetics Service.

As demonstrated in Table 3, the main sample sources in the reviewed studies were taken from the blood. Two studies , have used the samples including the cancerous tissue for evaluation of two variations in MDM2 gene that may change the genotype frequency of the target polymorphisms and influence the association of SNP with the studied outcome and, therefore, intervening in the interpretation of the results. Most studies have been conducted on the female patients with BC. In most studies, the control groups were matched with the patients in terms of age, sex, and geographical location parameters. BC‐free and healthy individuals were mostly considered as the control group. Also, rs2279744 known as SNP309 and G2580T was the well‐studied SNP in the MDM2 gene. This polymorphism occurs as a result of T to G transversion that has a higher affinity to the transcription factor of Sp1 in its mutant allele. Our review revealed that there are contradictory results for this SNP. Six studies conducted in Asia have confirmed the positive result of this polymorphism in increasing the BC incidence. In overall, in accordance with our case‐control research, studies conducted worldwide until 2019 have shown that this SNP did not increase the BC risk. Also, a recent meta‐analysis performed in 2018 showed that this polymorphism could not have a significant intervention in the carcinogenesis of BC. Results of some studies on the correlation between the SNP309 with onset age of disease indicated no positive association in this regard. , ,

SNP285(G>C) is located in intron1‐2 and the upstream of the SNP309 (Figure 4). Some studies have demonstrated that the C allele plays a role as an antagonist factor for SNP309 and reduces the Sp1 affinity for binding to the gene promoter and decreases the transcription activity. , In the literature review, reported results did not confirm the role of SNP285 as a risk factor for BC susceptibility.

Rs3730485 (GRCh38.p7 genome assembly), known as del1518, is located in the promoter region of the 2kb upstream. Deletion allele can make a higher potential binding site for the RORA, MEF2A,and MIZF TFs. This 40‐bp ins/del is located near the 40‐bp ins/del (rs150550023) so that, in the GRCh38.p12 genome assembly, these polymorphisms are merged into rs150550023 with three alleles: 40‐bp double insertion, 40‐bp insertion, and 40‐bp deletion. The current review revealed controversial results regarding the lack of significant association and positive correlation for an increasing role of del1518 on the BC susceptibility in different populations.

Limited number of studies have been conducted on the relationship between the SNP55, SNP344, , SNP354, SNP443, and rs937285 with the risk of BC. SNP443 does not have the RefSeq ID and is not validated as a variation in the National Center for Biotechnology Information (NCBI) and Ensemble databases. SNP55 and Rs1196333 have a higher affinity to bind to the Sp1 and TFAP2A TFs in the mutant allele, respectively, leading to the increase in the expression level of MDM2 in the cell.

In conclusion, our results indicated that the GG genotype of SNP309‐MDM2 plays a protective role in the BC in our studied population. However, our findings indicated no positive association between the MDM2 del1518 polymorphism and the risk of BC in the Kurdish population from western Iran. Also, our systematic review indicated that the SNP309, SNP285, and del1518 polymorphisms of MDM2 gene in different populations mostly were not associated with the BC risk. Thus, there is a need to evaluate other novel rSNPs in the MDM2 gene in the future researches as well as the expression of the MDM2 gene and its associated transcription factors, such as RORA, Sp1, MEF2A, and MIZF.

There were some limitations in the present study including a small number of included studies with the contradictory results for 40‐bp del1518 polymorphism to calculate the sample size, obtaining the patients̕ consent to participate in the study, achieving a maximum number of the included new cases, time constraint, and obtaining a maximum data from the patients' medical history in new cases. Therefore, more time should be taken to collect the sample of new cases, increase the sample size, and obtain the complete IHC characteristics of the patients for the future studies.

AUTHOR CONTRIBUTIONS

AJ collected the samples, performed experiments, extracted the systematic review data, and wrote the first draft of the manuscript. KY and MA designed the case‐control and systematic review studies and analyzed the data. ZR and IS edited the final version of the manuscript. PM draw the schematic figure. All authors reviewed the final version of the manuscript and approved it for publication.

ETHICAL APPROVAL

Ethics Committee in Kermanshah University of Medical Sciences, Iran, approves the current study. All control and patient's individuals agreed to participant in the study and signed the form of informed consent (Helsinki II declaration).