The Correlation between Phospholipase C Epsilon (PLCE1) Gene Polymorphisms and Risk of Gastric Adenocarcinoma in Iranian Population.

Background: Phospholipase C epsilon 1 (PLCE1) gene harbors different single nucleotide polymorphisms (SNPs), which can be correlated with the risk of different types of cancers. In this case-control study, the relationship between rs2274223 (A>G), a single nucleotide polymorphism in phospholipase C epsilon gene, (PLCE1) and gastric cancer was evaluated among Iranian patients. Materials and Methods: The PLCE1 rs2274223 polymorphism was genotyped in 60 patients with gastric cancer and 69 control subjects using polymerase chain reaction and restriction fragment length polymorphisms (PCR-RFLP) methods. Clinical and pathologic parameters such as tumor characteristics and disease stage were also recorded. Results: There were 48 (80%) male patients and 45 (65.5%) healthy male individuals (p=0.077). About 34 (56.6%) patients were smokers. A family history of gastric cancer was found in 21 (35%) cases. GG genotype was observed among 15% of patients and 8.7% of normals, respectively. There was no significant difference between the AA and AG genotypes. Also, there were no significant correlations between AA, AG or GG genotypes and the risk of gastric cancer, gender, tumor size, tumor stage, grade, as well as tumor location and metastasis. Conclusion: The PLCE1 rs2274223 polymorphism was not correlated with gastric cancer in Iranian population. However, a further comprehensive study with larger sample sizes is needed.


INTRODUCTION
Gastric cancer is now considered as one of the most common cancers and leading cause of cancer death (more than 700,000 deaths each year) throughout the world 1,2 . Therefore, there are still urgent needs for early diagnosis and prevention of it. Adenocarcinoma gastric cancer is a multifactorial disease and various factors may play role in it, International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir including positive family history, Helicobacter pylori infection, nutrition, smoking, and alcoholic consumption 3 . Genetic background is considered as one of the significant causes of gastric cancer 4 . Recent evidence has shown that genetic variants, especially single nucleotide polymorphisms (SNPs), can mediate the effect of environmental risk factors through modifying functions of various signaling pathways involved in gastric carcinogenesis 5 . Genome Wide Association Studies (GWAS) provided good opportunity to identify potential candidates for single nucleotide polymorphisms. Recent GWAS have revealed that Phospholipase C epsilon 1 gene, PLCE1, which is located on chromosome 10q23, participates in cell growth, differentiation and gene expression, as well as induces small GTPases such as Ras, Rap and Rho families. Different SNPs in PLCE1 gene are shown to have correlation with the risk of different type of cancers including esophageal and gastric [6][7][8] . Rs2274223 (A>G), located in exon 26 of the PLCE1 gene, can cause the amino acid change, histidine (His) to arginine (Arg) at the codon position of 1927 of PLCE1protein. The PLCE1 gene encodes a phospholipase C that hydrolyses of Polyphatidylinositol to inositol 1,4,5-trisphosphate and 4,5-diacylglycerol 8 . This phospholipase also regulates a variety of proteins such as the protein kinase C (PKC) isoenzymes and the proto-oncogene Ras 9 . Recent studies have identified a new susceptibility for single nucleotide polymorphism (SNP) (rs2274223: A5780G), located in exon 26 of PLCE1, which may correlate with the risk of esophageal and gastric cancers 10,11 . For instance, Wang et al. reported a relatively high correlation between esophageal squamous cell carcinoma and rs2274223 A>G polymorphism in Chinese population 12 . In another study, Abnet et al. demonstrated a close relationship between rs2274223 polymorphism and increased risk of gastric adenocarcinoma and esophageal squamous cell carcinoma in Chinese population 13 . However, inconsistent results are also reported and several studies did not report any correlation between this polymorphism and gastric cancer 10,11 . In this study, we attempted to evaluate the correlation between the PLCE1 (rs2274223 A>G) polymorphism and the risk of gastric adenocarcinoma in northern Iran. Furthermore, it should be mentioned that this is the first study to date to assay the correlation between this polymorphism and gastric adenocarcinoma in Iran.

DNA extraction
Five to ten ml blood samples were collected from the antecubitalvein of patients and healthy donors using EDTA containing tube. Genomic DNA was extracted from 3 ml of blood sample using the salting out method 16 . Quantity and quality of extracted DNAs were determined using a NanoDropND-2000 spectrophotometer (Thermo Sci., Newington, NH). Isolated DNAs were stored at 20 o C until further analysis.

SNP Selection
The functional rs2274223 SNP was selected in PLCE1 gene for genotyping based on previous published data and the NCBI dbSNP database (https://www.ncbi.nlm.nih.gov/snp/?term=rs22742 23).

Genotyping using PCR-RFLP
The 397 bp PCR product was digested using AciIrestriction enzyme (0.3µl) (Thermo Fisher Scientific, US) overnight at 37° C. The digested products were then separated on 2% agarose gel electrophoresis, which was stained by safe stain (Sinaclone, Iran) and visualized with UV illumination. The A allele has a restriction site and produces two bands, 285 bp and 94 bp, while replacement of A nucleotide with G creates a new AciI restriction site in 94-bp fragment and produces a 60-bp and a 34-bp fragments. Therefore, AA genotype produces 94-bp and 285-bp; GG genotype produces 34-bp, 60-bp and 285-bp and AG genotype produces 34-bp, 60-bp, 94-bp, and 285-bp after enzyme digestion, respectively ( Figure 1).

Statistical analysis
Demographic and clinical characteristics of all patients were reported as means±SD. An independent student t-test was also considered to compare the mean of age between the patients and control groups. Descriptive statistics as well as Pearson's Chi-Square test were used to compare the frequency of each genotype between different groups. Data were analyzed using SPSS software (version 19) and a p<0.05 was considered as significant.

RESULTS
The basic demographic characteristics of all patients are summarized in Table 1. In total, 60 patients (48 males and 12 females) and 69 healthy individuals (45 males and 24 females) were entered into the study. The mean of age in the patient and control group was 64.4±11.5 and 56.9±9.5 years, respectively. More than half of the patients (56.66%) were active smokers and 35% of patients had a family history of gastric cancer (Table 1).  The frequency of AA, AG and GG genotypes among the control and patient groups is shown in Table 3.
There was no significant difference in the frequency of these genotypes between the patient and control group (p=0.36). While AA was the most common genotype in the patients (60%) and controls (71%), GG genotype was the least common in both groups (Table 3).
International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir The Correlation between the frequency of GG, AG as well as AA genotypes and tumor grading is depicted in Figure 2. There was no significant difference in the frequency of each genotype between patients with poorly or moderately differentiated tumor grading. Nevertheless, the frequency of AG genotype among moderately differentiated group (31%) was approximately greater than that in poorly differentiated group (19.4%). Additionally, the frequency of GG genotype in poorly differentiated group (22.6%) was partly greater than moderately differentiated group (6.9%). Also, allele frequency was calculated using genotype frequencies results. Allele A was 73% among patients and 81% among controls, respectively. Allele G was 27% among patients and 19% among controls, respectively (Table 3). There was no significant difference in the allele frequency between the patient and control group (p=0.81).
According to the data presented in Table 3, there was deviation from Hardy-weinberg equilibrium for genotypes in controls (P <0.017). This deviation from the HW equilibrium was probably due to the small sample size. The relationship between the frequency of GG, AG and AA genotypes and disease stages is depicted in Figure 3 and Table 4. There was no significant difference in the frequency of GG, AG and GG genotypes among different stages (p=0.089). However, the AA genotype was the most frequent genotype in patients with disease stage II (18.33%), III (13.33%), and IV (26.66%) compared to the other genotypes ( Figure 3 and Table 4). The correlation between lymph node involvement, metastasis and tumor location among all three different genotypes is presented in Table 5. We could not find a significant difference in the percentage of lymph node involvement (p=0.77), metastasis (p=0.42) and tumor place (p=0.77) among individuals with GG, AG, and AA genotypes. The mean of tumor size among patients with three different genotypes is presented in Figure 4. We could not find a significant difference between the tumor size and these three genotypes (p=0.073). Nevertheless, the mean of tumor size in GG genotype (4.33±1.25) was greater than that in the AA (4.06±0.89) and AG (3.53±0.61) groups.

DISCUSSION
In this case-control study, we considered the correlation between a potentially functional SNP of PLCE1 (rs2274223) and the risk of gastric adenocarcinoma in Iranian population. Although a large number of studies found a significant relationship between rs2274223 A>G and disease stage and tumor characteristics in different types of cancers, our findings revealed that there is no relationship between different genotypes of AA, AG and GG with disease stage. We also could not found a significant correlation between these genotypes with age, gender, tumor characteristics such as tumor size, metastasis, grading, tumor location, and other factors. Numerous studies have investigated several potentially functional SNPs of PLCE1 in various cancers such as gastric, esophagus and colorectal cancers 17 . For example, Abnetet al. 13 investigated the correlation between the rs2274223 A>G and the risk of esophagus cancer in 2,240 patients with gastric cancer. They found a close relationship between this SNP and the risk of esophagus cancer (OR=1.31). In another study, Wang et al. considered the correlation between the PLCE1 rs2274223 and the risk of gastric cancer in 1059 patients with pathologically confirmed gastric adenocarcinoma and 1240 frequency-matched healthy controls. They observed a significant correlation between this SNP and the risk of gastric cancer (OR = 1.26) 5 . In another similar study, Wu et al. 18 investigated the relationship between the rs2274223 with esophagus cancer and its tumorigenesis in 2,031 Chinese patients with gastric cancer and 2,044 controls. They identified a significant correlation between the SNP and esophagus cancer and its tumorigenesis. Ezgiet al. also reported a significant correlation between rs2274223 and the risk of colorectal cancer in Turkish population (OR = 2.018) 19 . Some studies have reported AG, GG or AG+GG genotypes are more susceptible to different cancers, particularly for esophageal/gastric cancer or esophageal squamous cell carcinoma compared to AA genotype 20,21 . In a meta-analysis study on 13676 patients with gastric cancer, it has been concluded that there is a significant relationship between PLCE1 rs2274223 polymorphisms and the incidence and increased risk of gastric cancer 22 . Cui et al. investigated the relationship between four functional SNPs in PLCE1 gene (including rs12263737, rs2274223, rs11187842 and rs753724) and the risk of esophagus cancer in 222 Chinese cases and 326 controls 23 . The results demonstrated that these polymorphisms are closely associated with higher risk of esophageal cancer. For these reasons, many researchers consider PLCE1 gene polymorphisms as one of the significant biomarker involved in esophagus cancer among Chinese populations. In other research, genetic variation in PLCE1 and the risk of upper gastrointestinal cancers and esophageal adenocarcinoma was evaluated in Caucasian populations 10 . The results showed that rs4072037 SNP in PLCE1 is associated with higher risk of upper gastrointestinal cancer, while rs2274223 and rs4072037 were correlated with increased risk of esophageal adenocarcinoma.  14 . Although G2274223, T3765524, and T7922612 haplotypes were significantly correlated with higher risk of gastric cancer, these polymorphisms were not independently correlated with gastric cancer and several associated factors such as smoking and family history may be involved 14 . In the present study, about 57% of cases were smokers and the family history of gastric cancer was 35%. Therefore, it can be suggested that these factors may also increase the risk of gastric cancer along with the suspected polymorphism genotypes. In a meta-analysis study, Zhang et al. analyzed the results of 13188 cases with gastric cancer and 14666 controls 25 . They demonstrated that there is a relationship between rs2274223 A>G polymorphism (all genotypes of AA, AG and GG) and disease stage, which was most popular among Asian populations. In another investigation, the correlation between rs2274223 polymorphism was considered in 380 esophageal cancer patients and 380 healthy controls. The results revealed that the rs2274223 is significantly associated with increased risk of the cancer. Additionally, AG genotype in women was greater than that in men. For these reasons, some studies considered rs2274223 SNP as a sensitive biomarker for gastric cancer 17 . Although various studies found a significant correlation between SNPs in PLCE1 and the risk of cancers, there are several studies that did not find any significant correlation. For example, Ma et al. investigated the relation between rs2274223G, rs3203713G and rs11599672T polymorphisms in patients with head and neck cancers, but did not find any significant correlation 26 .

CONCLUSION
In summary, in this case-control study, we investigated the correlation between the functional rs2274223 SNP in PLCE1 gene and increased risk of gastric cancer for the first time in Iranian population. Although previous studies reported a close relationship between this SNP and enhanced risk of different cancers, we could not find a significant correlation between rs2274223 SNP in PLCE1 gene and the risk of gastric cancer, tumor characteristics, and disease stage. Other factors such as smoking and family history may independently affect the risk of gastric cancer. However, additional larger studies among Iranian populations are needed to validate our findings.