Investigating the Association between rs712829 Polymorphism of EGFR Gene with Lung Cancer as a Predictive Marker in Iranian Population

Background and Objectives Lung cancer is the leading cause of cancer death globally. The epidermal growth factor receptor (EGFR) plays an important role in cell proliferation and signaling. In this study, we examined the association between EGFR (rs712829) gene polymorphism and lung cancer risk among the Iranian population. Subjects and Methods A total of 100 patients with primary lung cancer and 100 matched healthy controls were recruited into this study. EGFR rs712829 single nucleotide polymorphism (SNP) were genotyped by PCR-RFLP techniques for this association with lung cancer risk. Results A significant association was observed between the GG genotype (P= 0.039, OR= 5.500, CI=95%; 1.710-11.921) and also G (P= 0.001, OR= 2.967, CI=95%; 1.557-5.691) allele of rs712829 SNP with lung cancer risk, this was while the TT genotype and T allele of this polymorphism showed a protective role against risk of lung cancer. Conclusion In conclusion, EGFR rs712829 was associated with a risk of lung cancer among Iranians. More studies in other Iranian populations are required to confirm the present findings.


Extended Abstract
Introduction ung cancer (LC) is the cancer with the highest incidence and mortality rate in the world.In 2018, 2.09 million people were diagnosed with LC, and 1.76 million people died due to this disease.LC is the third most common type of cancer in Iran, and its prevalence is increasing rapidly.LC is classified into two major categories: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC).Approximately 85-87% of lung cancers are of the NSCLC type.Despite its unclear etiology, the development of LC is known to be multifactorial.Genetic and environmental factors such as addiction to tobacco, radiation therapy, other lung diseases, race, and family history of LC play an important role in disease susceptibility.
Recent studies indicate that genetic factors such as single nucleotide polymorphisms (SNPs) may play a role in the risk of LC.Several studies on different populations have been published, suggesting that SNPs of the EGFR (-216G/T) are associated with LC risk.Epidermal growth factor receptor (EGFR), a trans-membrane glycoprotein with tyrosine kinase activity encoded by a gene located on 7p12, is a main regulator of different signaling pathways.EGFR is frequently overexpressed in many cancers including LC.Among variants of EGFR gene, rs712829 is a functional polymorphism in the EGFR promoter.The replacement of G by T at position -216 increases promoter activity by 30%, thereby resulting in a higher EGFR expression level and causes carcinogenesis.Several reports have investigated the role of single nucleotide polymorphism of EGFR (rs712829) genes in lung cancer risk in different ethnic populations.The aim of the present study was to evaluate the association between the rs712829 (EGFR) polymorphisms with the risk of lung cancer in a population of Markazi province.

Methods
This study was designed to assess the association between the EGFR -216G/T gene polymorphisms and the risk of lung cancer in the Iranian population.A total of 100 patients with lung cancer and 100 age-and gender-matched healthy controls were recruited for the study.The participants were selected from Ayatollah Khansari Hospital, Arak, Iran, from January 2020 to May 2022.Lung cancer was diagnosed by an oncologist and was confirmed by histopathology examination.The exclusion criterion of the control group was previous malignancy or genetic diseases.Clinical data were collected from the patients' medical records, and demographic information was collected using a questionnaire.The study was approved by the Research Ethics Committees of Arak University of Medical Sciences.DNA extraction of blood samples was done using DNG-plus kit (Cinna Gen, Iran) according to the manufacturer's instructions, and the samples were stored at -20°C until molecular analysis.The SNPs of the EGFR (rs712829) gene were analyzed using PCR-RFLP method.The PCR products were studied after digestion with BseRI restriction enzyme (Fermentaz, USA) for -216G/T of EGFR gene (cut from 240 bp G type into 180 + 60 bp T type).
Digestion reaction conditions were performed following manufacturer recommendations, and the fragments obtained from enzymatic digestion were recorded with gel documentation system.Statistical analysis was performed using SPSS version 16.The P <0.05 was considered statistically significant.The odd ratios (OR) with their corresponding 95% confidence intervals (CI) were calculated by binary logistic regression to study the associations of the SNPs studied with the risk of lung cancer.

Results
The PCR-RFLP results of the SNP studied are shown in Figure 1.The age range of patients and healthy people was 44-88 (62.45±11.47)and 33-81 (61.5±10.70),respectively (P=0.385).As far as smoking status in the two groups was concerned, about 42% of the patients smoked cigarettes during their life, and this rate was 21% in the control group, indicating a significant difference between the two groups.In other words, smoking increased the risk of lung cancer (P=0.004).
According to Table 1, the frequency of TT genotype in the patient and control groups was 15% and 55%, respectively.Statistical analysis showed that the TT genotype has a protective role against the risk of lung cancer.In contrast, the frequency of GG genotype in patients was higher than that in the control group, and it had a significant relationship with the risk of lung cancer (P=0.039,OR=5.500,CI=95%; 1.710-11.921).Also, the frequency of GT genotype in patients was 2.2 times that of the control group and showed a significant relationship with the risk of lung cancer (P=0.003,OR=6.682,CI=95%; 12.324-1.798).The frequency of G allele in patient and control groups was 57.5% and 31%, respectively.Statistical analysis of allele frequency in both patient and control groups showed that the G allele of EGFR gene polymorphism rs712829 had a significant relationship with the risk of lung cancer (P=0.001,OR=2.967,CI=95%; 1.557-5.691),and unlike the T allele, this polymorphism acts as a protective factor.The relationship of rs712829 polymorphic genotypes of EGFR gene with tumor grade (T), lymph node involvement status (N) and metastasis status (M) was investigated.There was no significant relationship between the studied polymorphism genotypes and tumor grade (T1+T2 vs. T3+T4), lymph node status (involvement vs. non-involvement), and metastasis status (presence vs. absence).

Conclusion
In the present study, the relationship between rs712829 EGFR gene polymorphism and lung cancer risk in an Iranian population was investigated using PCR-RFLP technique.The results showed that the rs712829 polymorphism of the EGFR gene has a significant relationship with the risk of lung cancer.Comparing the frequencies of genotypes and alleles of rs712829 polymorphism in both control and patient groups showed that the TT genotype and the mentioned polymorphic T allele have a protective role against the risk of lung cancer and against the presence of the 2.9 G allele, which exposes a person to a higher risk of lung cancer compared to the T allele.Also, people with the GG genotype are about 5.5 times more likely to develop lung cancer than people who carry the TT genotype.The results of investigating the relationship between rs712829 EGFR gene polymorphism and the risk of lung cancer in our study are consistent with those of other studies conducted on different populations.This study identified that there is a significant association between rs712829 of EGFR gene polymorphisms and the risk of susceptibility to lung cancer.Therefore, this polymorphism can be used as a biomarker in personalized medicine for clinical prediction of the risk of lung cancer.To confirm the role of studied polymorphisms in the risk of lung cancer, more studies with larger sample sizes and different ethnic groups are required.

Figure 1 .
Figure 1.Results of PCR-RFLP analysis of the EGFR rs712829 polymorphism with BseRI restriction enzyme at 37°C/3 hr.

Table 1 .
The frequency of the alleles and genotypes for the SNP studied in patient and control groups.