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Egyptian Journal of Medical Human Genetics
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Investigating the importance of EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with risk of lung cancer as potential diagnostic biomarker in Iranian population

Egyptian Journal of Medical Human Genetics volume 24, Article number: 55 (2023) Cite this article

Abstract

Background

In Iran, lung cancer is the third most common type of cancer and its prevalence is increasing rapidly. Identification informative genetic polymorphisms in cancer causing genes including epidermal growth factor receptor (EGFR) as key gene in control of cellular proliferation via intrinsic tyrosine/kinase activity, exonuclease 1 (EXO1) as one of the upregulated gene in different human malignancies and leptin (LEP) participate in carcinogenesis in lung cancer appears to be used as potential genetic markers for predicting lung cancer risk. There is no study about investigate association of the EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with risk of lung cancer in Iranian population. The aim of this study was investigating the association of EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with risk of lung cancer as a potentially diagnostic biomarker in Iranian population.

Methods

In this case–control study, A total of 100 patients with lung cancer and 100 age and gender-matched healthy controls were recruited into this study and the association between EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with the risk of lung cancer was investigated by using PCR–RFLP technique and bioinformatics approach.

Results

The rs712829 of EGFR gene show that a significant statistical difference between G allele and risk of lung cancer (P = 0.001, OR = 2.976, CI = 95%, 1.557–5.691), in contrast, the T allele and TT genotype show a protective role against the risk of lung cancer. Result of in silico analysis indicated that the rs712829 alter splicing and promoter regulation of EGFR gene and associated with the risk of lung cancer. PCR–RFLP result for rs1047840 of Exo1 gene showed that the AA genotype and A allele of this polymorphism associated with risk of lung cancer, whereas the GG genotype show a protective effect against the risk of lung cancer (P = 0.004, OR = 5.391, CI = 95%, 1.690–17.200). On the other hand, in silico analysis showed that the existence of rs1047840 in Exo1 gene influence lung cancer susceptibility. For rs7799039 of LEP gene, PCR–RFLP analysis showed that, there is no significant association between this polymorphism and the risk of lung cancer.

Conclusion

The rs712829 of EGFR gene and rs1047840 of Exo1 are associated with risk of lung cancer among Iranian population and can be used as a potentially candidate biomarker for early detection and primary prevention.

Background

Lung cancer (LC) is the cancer with the highest incidence and mortality rate in the over world [1, 2]. LC is the most common cancer that usually diagnosed in an unresectable and advanced stage [3]. Most patients diagnosed with LC presented with distant or metastatic disease; less than 20% were diagnosed with localized [4].

In 2018, 2.09 million people were diagnosed with LC and there were 1.76 million deaths from LC [5]. LC is the third most common type of cancer in Iran and its prevalence is increasing rapidly [6]. LC has been classified into two major categories: first group is non-small cell lung cancer (NSCLC), which collectively comprises adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Second group is small cell lung cancer (SCLC), which is more aggressive and has worse survival rates. Approximately 85–87% of lung cancers are NSCLC [3,4,5, 7]. The etiology of LC was unclear, but the development of LC is known to be multifactorial. Genetic and environmental factors such as addiction to tobacco use, radiation therapy, other lung diseases, race and family history of LC play an important role in disease susceptibility [4, 8, 9]. Rahal et al. study indicated that genetics factor such as single nucleotide polymorphisms (SNPs) may play a role in risk of LC [10].

The finding of genetic markers associated with increase susceptibility to LC is an active area of research [11]. Several reports in different populations have been published suggesting that SNPs of the EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) genes are associated with LC risk [12,13,14]. The fact that numerous histological types of lung cancer responded to medications that inhibit EGFR and/or its downstream effectors demonstrates the function of EGFR in mediating lung cancer etiology. These observations imply the possibility of lung cancer-causing mutations and/or polymorphisms in the EGFR gene. Epidermal growth factor receptor (EGFR), a trans-membrane glycoprotein with tyrosine kinase activity that encoded by a gene located on 7p12 and is a main regulator of different signaling pathways and it belongs to the ErbB family and plays a significant role in regulating many different signaling pathways that include cell proliferation [15, 16]. EGFR is frequently overexpressed in many cancers including LC [12]. Among variants of EGFR gene, − 216G/T (rs712829), 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 [15, 17]. In addition, Bashir et al. has reported the association of − 216G/T polymorphism of EGFR gene with the risk of LC in different populations [12]. Another gene polymorphism that involved in the development of LC is the Exo1 gene. Exonuclease 1 (Exo1) located on human chromosome 1q42-q43 and encoding an 846 amino acid protein that play important role in DNA repair by MMR mismatch repair pathway [18, 19].

Many researches show that there is an association between Exo1 inactivation and increased likelihood of tumorigenesis in lung tissue [19, 20]. Particularly in patients with a history of smoking, a number of EXO1 SNPs have been indicated to be associated with an increased susceptibility to lung cancer and suggest a potential oncogenic role for EXO1. Among SNPs of Exo1 gene, the rs1047840 (K589E) is most important variant associated with the risk of various cancers, including breast cancer, gastric cancer, oral cancer and LC [13, 19, 21,22,23]. The rs1047840 affects Exo1 mRNA expression and cause inefficiency in the DNA repair process as a result can led to LC [13, 24, 25].

Recent studies have showed that leptin is crucial to the development of tumors [26,27,28]. Leptin (LEP) is necessary for adipocyte homeostasis, and leptin resistance causes adipotoxicity and cellular fat death, which raise the risk of neoplasia [29]. LEP located at chromosome 7q31.3, encodes a 16 kDa protein that to be associated with endocrinologic metabolism [30]. LEP participates in the regulations of energy balance, adiposity, endocrine systems, immunity, angiogenesis and oncogenesis [31, 32]. Several studies indicated that LEP − 2548 G/A (rs7799039) are correlated with the development of many cancers such as oral cancer [33], prostate cancer [34], oropharyngeal cancer [35], breast cancer [36], hepatocellular carcinoma [37] and LC [14, 38].

The existence of − 2548 G/A polymorphism in LEP gene associated with high gene expression and twofold LEP secretion. Higher mRNA expression of Leptin may increase risk of LC [14]. The prior reports representing different ethnic population have presented controversial results about studied SNPs. Identification of genetic variants involved in LC can be used as clinical biomarkers for early genetically detection [39]. The use of these clinical biomarkers can play an important role in reducing mortality and also reducing material and spiritual damages caused by this disease. The aim of this study was investigating the association of EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with risk of lung cancer as a diagnostic biomarker in Iranian population. The use of these clinical biomarkers can play an important role in reducing mortality and also reducing material and spiritual damages caused by lung cancer.

Method and materials

In current case–control study, the association between EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms and lung cancer risk were evaluated by using PCR–RFLP technique and bioinformatics approach analysis.

Study subjects

This study designed to assess the association between the EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with 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 from the Ayatollah Khansari Hospital, Arak City, Iran, from January 2019 until April 2020. Lung cancer of patients was diagnosed by an oncologist and was confirmed by histopathology examination. The exclusion criteria of the control group were previous malignancy or genetic diseases. Clinical data were collected from the patient's medical records and demographics information were collected through a questionnaire. The study was approved by the Research Ethics Committees of Baqiyatallah University of Medical Sciences.

PCR–RFLP analysis

DNA extraction of blood samples done with DNG-plus kit (Cinna Gen, Iran) according to the manufacturer’s instructions and stored at – 20 °C until molecular analysis. The SNPs of the EGFR (rs712829), Exo1 (rs1047840) and LEP (rs7799039) genes were analyzed with PCR–RFLP method. The sequences of the primers used for PCR amplification and PCR conditions for each SNP are shown in Table 1. The PCR products were studied after digestion with BseRI (Fermentaz, USA), Mse I (Thermo Fisher Scientific, UK) and HhaI (Fermentaz, USA) restriction enzymes for − 216G/T of EGFR gene (cut from 240 bp G type into 180 + 60 bp T type), K589E of Exo1 gene (cut from 306 bp G type into 196 + 110 bp A type) and − 2548G/A of LEP gene (cut from 281 bp A type into 172 + 109 bp G type), respectively. Digestion reaction conditions were performed per manufacturer recommendations and the fragments obtained from enzymatic digestion were recorded with gel documentation system. Statistical analysis was performed by SPSS version 16. The P < 0.05 was considered as the level of significance. The odd ratios (OR) with their corresponding 95% confidence intervals (CI) were calculated by binary logistic regression to study of the associations of SNPs studied with the risk of lung cancer.

Table 1 The primer pairs for each SNP and their characteristics
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In silico analysis

The NCBI SNP database (https://www.ncbi.nlm.nih.gov/ SNP/) was used to access studied SNPs information including the FASTA format of the DNA and protein sequences of the EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms. The non-synonymous SNP (nsSNP) were analyzed using various bioinformatics tools (SIFT, PolyPhen-2, PhD-SNP, SNPs&GO, I-Mutant, MUpro and Project HOPE). The SNPs at the UTR regions were analyzed using FastSNP and SNP Function Prediction tools. The flowchart of the bioinformatics software's used in this study were shown in Fig. 1.

Fig. 1
figure 1

Diagram of bioinformatics software's used to In Silico analysis process

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GeneMANIA (http://www.genemania.org/) is online tool software which predicts gene function, physical interaction, genetic interactions, gene co-expression, co-localization, shared protein domains, and pathway involved [40]. Several online-based tools were employed to determine prediction of nsSNPs studied including Sorting Intolerant from Tolerant (SIFT) (http://sift.jcvi.org/), Polymorphism Phenotyping-v2 (PolyPhen-2) (http://genetics.bwh.harvard.edu/pph 2/), Predictor of human deleterious single nucleotide polymorphism (PhD-SNP) (http://snps.biofold.org/phd-snp/phd-snp.html), and SNPs&GO (GO-Gene Ontology) (http://snps-and-go.biocomp.unibo.it/snps-andgo/) web-based tools. SIFT gives a probability score. The score less than or equal to the threshold of 0.05 is deleterious, and a prediction greater than the threshold is tolerant [41]. PolyPhen v2 predicts the possible effect of nsSNPs based on sequence, structural conformation. The output of the PolyPhen v2 is a numerical score ranging from 0.0 (benign) to 1.0 (damaging) [42]. PhD-SNP is based on support vector machines (SVMs) which predicts whether a point mutation is a neutral polymorphism or is associated to genetic disorders in humans [43]. SNPs & GO is a method applied to predict whether an amino acid substitution may be related to diseases or not. The prospect score higher than 0.5 detects that the SNP is disease related [44]. For analysis the stability of protein due to the nsSNPs, two online servers were used: I-Mutant 3.0 (http://gpcr.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi) and MUpro (http://mupro.proteomics.ics.uci.edu/). I-Mutant 3.0 gives the output as a DDG value which is categorized into one of the three predictions: largely unstable (DDG <  − 0.5 kcal/mol), largely stable (DDG > 0.5 kcal/mol), or neutral (− 0.5 ≤ DDG ≤ 0.5 kcal/mol). MUpro tool calculates a score between − 1 and 1 as the confidence of prediction. The confidence score < 0 indicates that the mutation decreases the protein stability, while a confidence score > 0 means that the mutation increases the protein stability [45, 46]. In order to analysis the biophysical and structural of protein due to the nsSNPs, Project HOPE (https://www3.cmbi.umcn.nl/hope/) were used [47]. For analyze the SNPs located within the UTR region, SNP function prediction SNPinfo (FuncPred) (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm) and FASTSNP (http://FASTSNP.ibms.sinica.edu.tw) were used [48]. The score is given on the basis of levels of risk with a ranking of 0, 1, 2, 3, 4, or 5. This signifies the levels of no, very low, low, medium, high, and very high effect, respectively [49].

Results

PCR–RFLP results

The frequency distributions of selected characteristics of control and lung cancer group with TNM information are shown in Table 2. The age and sex of patients and controls were matched. The mean age of the lung cancer patients and the controls were 62.45 (standard deviation, SD = 11.47) and 61.5 (SD = 10.70) years, respectively. The results of comparing the age range between the two groups shows that, there was no statistical significance difference in the case and control groups (P = 0.429). Also, there was no significant statistical difference between male or female among two groups (P = 0.753). There was significant statistical difference between the smoking status and the risk of lung cancer among two case and control groups (P = 0.004). According to Table 2, the tumor grade was assessed and the majority of the patients were in grade IV. Sixty percent of the patients were positive for lymph node involvement and 70% of patients were metastatic positive.

Table 2 Characteristics of control and lung cancer groups with TNM information
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The PCR–RFLP results of studied SNPs are shown in Fig. 2. The frequency of the alleles and genotypes for the rs712829 (EGFR), rs1047840 (Exo1) and rs7799039 (LEP) between the lung cancer and controls patients is shown in Table 3. For rs712829 of EGFR gene, there was a significant statistical difference in TT frequency among lung cancer patients compared to controls (P = 0.000, OR = 5.500, CI = 95%, 2.239–13.510). Results of the allele analysis for rs712829 show that there was a significant statistical difference between G allele and risk of lung cancer, in contrast the T allele show a protective role against the risk of lung cancer (P = 0.001, OR = 2.976, CI = 95%, 1.557–5.691).

Fig. 2
figure 2

The PCR–RFLP results of studied SNPs that shown by 3% agarose electrophoresis. A Results PCR–RFLP analysis of the EGFR rs712829 polymorphism with BseRI restriction enzyme at 37 °C/3 h. B Results PCR–RFLP analysis of the Exo1 rs1047840 polymorphism with MseI restriction enzyme at 65 °C/3 h. C Results PCR–RFLP analysis of the LEP rs7799039 polymorphism with HhaI restriction enzyme at 37 °C/2 h

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Table 3 The frequency of the alleles and genotypes for SNPs studied in cases and the controls
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According to Table 3, the frequencies genotypes and allele's analysis for rs1047840 of Exo1 gene show that the frequency of GG genotype in the case and control groups, was 58% and 78%, respectively, versus the frequency of AA genotype in the cases is 4 times more than controls group. The frequency of A allele in the case and control groups, was 31% and 13/5%, respectively. The frequencies genotypes and allele's analysis for rs1047840 of Exo1 gene in cases and the controls show that A allele of this polymorphism associated with risk of lung cancer (P = 0.010, OR = 2.851, CI = 95%, 1.291–6.300), in contrast the GG genotype and G allele show a protective effect against the risk of lung cancer (P = 0.004, OR = 5.391, CI = 95%, 1.690–17.200). For rs7799039 of LEP gene, no statistically significant difference was observed between the genotypes of this polymorphism in the case and control groups. Also, no statistically significant difference was observed between allele frequencies of the rs7799039 polymorphism in the case and control groups (P = 0.519, OR = 0.812, CI = 95%; 0.431–1.530).

The result of association between the genotypes of the SNPs studied and TNM staging system of patients are shown in Table 4. There was no significant statistical difference between the genotypes of rs712829 and rs1047840 polymorphism with tumor grade, in contrast the GA genotype of rs7799039 polymorphism was significantly associated with increase tumor grade in the patients (P = 0.024, OR = 0.222, CI = 95%, 0.060–0.824). According Table 4, the results showed that there was no significant difference between the genotypes of the studied SNPs with lymph node involvement and metastasis in the patients.

Table 4 The analysis association between genotypes of SNPs studied with TNM staging system
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In silico analysis results

We investigated rs712829 (EGFR), rs1047840 (EXO1) and rs7799039 (LEP) gene polymorphisms using NCBI dbSNP database. The rs1047840 (K589E) were a coding and non-synonymous SNP that located on exon12 of the Exo1 gene. The rs712829 (− 216G/T) of EGFR gene and rs7799039 (− 2548G/A) of LEP gene as non-coding SNPs were in the 5′-UTR and 2KB Upstream, respectively.

GeneMANIA builds a multiplex gene–gene functional interaction network. The interaction network of the Exo1, EGFR and LEP genes predicted by GeneMANIA is shown in Figs. 3, 4 and 5, respectively.

Fig. 3
figure 3

Gene–gene interaction network of the Exo1 gene predicted by GeneMANIA

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Fig. 4
figure 4

Gene–gene interaction network of the EGFR gene predicted by GeneMANIA

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Fig. 5
figure 5

Gene–gene interaction network of the LEP gene predicted by GeneMANIA

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In order prediction of SNPs pathogenicity, the rs1047840 of Exo1 gene as nsSNP were predicted by various bioinformatics tools. The result prediction was shown in Table 5.

Table 5 Prediction of rs1047840 of Exo1 by various bioinformatics tools
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The result protein stability analysis of the rs1047840 of Exo1 gene, by I-Mutant 3.0 and MUpro servers were shown in Table 6.

Table 6 Analysis of protein stability the rs1047840 of Exo1 gene by MUpro
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The existence of K589E mutant (rs1047840 of Exo1 gene) and substitution of glutamic acid might harshly disrupt a special conformation of EXO1 protein (Fig. 6).

Fig. 6
figure 6

Schematic structures of the original (left) and the mutant amino acid of K589E by HOPE software

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The results obtained by Project HOPE software for rs1047840 of Exo1 gene were shown in Table 7.

Table 7 Interpretation of the impact of rs1047840 on Exo1 protein structure and stability by HOPE
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Table 8 Analysis of rs712829 and rs7799039 by SNP Function Prediction
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Table 9 Analysis prediction functionally significant rs712829 and rs7799039 by FastSNP
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Discussion

Among all the cancers, lung cancer with over 1.3 million deaths per year. In Iran, lung cancer is the third most common type of cancer and its prevalence is increasing rapidly [7, 16]. SNPs that lead to protein insufficiency can be associated with an increased risk of cancer development [50]. A large number of genes associated with lung cancer contain SNPs. SNPs are located in gene promoters, exons, introns as well as 5' and 3'-UTRs and affect gene expression, structure and function of protein by various mechanisms. Several studies in different populations have been published suggesting that SNPs of the EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) genes are associated with lung cancer risk [12,13,14]. Among the polymorphisms investigated as In vitro analysis, we found that variant genotypes of EGFR − 216G/T and Exo1 K589E were significantly associated with a higher susceptibility of lung cancer, in contrast no significant association was observed between LEP (− 2548G/A) polymorphism and the risk of lung cancer. The result of analysis association between genotypes of SNPs studied with TNM staging system were showed that there is a significant association between the GA genotype of rs7799039 polymorphism with increase tumor grade. Results of in silico analysis showed that the rs1047840 of Exo1 gene as nsSNP associated with risk of lung cancer. Also, in silico analysis showed that the rs712829 and rs7799039 may be associated with risk of lung cancer. For the understanding of cellular processes gene–gene functional interaction network is a fundamental factor. GeneMANIA plays an importance role to description of genes co-expression, co-localization, shared protein domains, physical and genetic interactions. In the present study, this database revealed that the existence of rs712829 on EGFR, rs1047840 on Exo1 and rs7799039 on LEP gene, can alters the biological processes of these genes and disruption of these pathways, so that may be associated with risk of lung cancer. Among variants of EGFR gene rs712829 is a functional polymorphism in the EGFR promoter [16, 17]. The results in our study showed a significant association between rs712829 of EGFR gene and lung cancer risk. Several studies have demonstrated that the expression and activity of the EGFR is highly modified by several polymorphic regions within the EGFR gene [12, 16, 49, 51]. The rs712829 polymorphism is located in the promoter region of the EGFR gene and was shown in previous studies to modify the expression of the EGFR gene and may be associated with risk of lung cancer [12, 51]. According to the results in this study, the frequency of the TT genotype of rs712829 was higher in the control group compared to patients with lung cancer. The presence of the T allele can be protective against lung cancer, so that people who carry two copies of T allele are at a lower risk of susceptibility to lung cancer. In contrast, the presence of the G allele of rs712829 is associated with an increased risk of lung cancer, so that people who carry GG genotype of rs712829 are at a higher risk of susceptibility to lung cancer. This observation is consistent with the results of Nabil et al. [12], who found that the − 216G allele of rs712829 increased lung cancer risk in the Jordanian population. In contrast, a lack of association between the rs712829 of EGFR and lung cancer was reported in a United States [49] and Japanese population [51]. Also, in the study conducted by Torres-Jasso et al. [51] it was found that the TT genotype of rs712829 associated with an increased risk of gastric cancer in a Mexican population. The rs712829 of EGFR gene, a functional variant in the EGFR promoter, is located in the Sp1 recognition site where several protein factors and transcriptional start sites have been identified. Since the Sp1 binding site is a region that is important for the regulation of EGFR transcription, the replacement of G by T at position − 216 increases promoter activity by 30%, thereby resulting in a higher EGFR expression level [15]. The rs712829 polymorphism has been shown to influence the response to EGFR-TKIs therapy in Chinese [52] and Korean lung cancer patients [53]. Result of in silico analysis in this study indicated that the rs712829 is a non-coding SNP which were in the 5′-UTR of EGFR gene. In silico analysis performed by FastSNP and SNP Function Prediction showed that the rs712829 can alter promoter regulation by effect on transcription factor-binding site and alter splicing by effect on exonic splicing enhancer or exonic splicing silencer of EGFR gene and may be associated with the risk of lung cancer [48]. The rs712829 can have important effect on mRNA processing, transcription activity, stability, and protein translation, therefore, it can play a fundamental role in the development of lung cancer [54]. Among SNPs of Exo1 gene, the rs1047840 is most important variant associated with the risk of various cancers [16, 19, 21,22,23]. The rs1047840 of Exo1 is located on exon 12, and its variation leads to a change in the 589th amino acid of the Exo1 protein from lysine to glutamic acid, which might affect Exo1 expression (52). Some studies show that the A allele of the rs1047840 polymorphism may influence Exo1 activity and may be associated with risk of cancer. For a person who has a risk-imparting genetic variant, such as the A allele of the rs1047840 polymorphism, that variant will likely synergistically increase their cancer risk [24, 55]. For the rs1047840 of Exo1 gene, we found that A allele were significantly associated with increased risk of lung cancer, versus individuals who carry GG genotype are at a lower risk of susceptibility to lung cancer, so that G allele of this polymorphism has a protective role against lung cancer. The finding in our study about rs1047840 of Exo1 gene is consistent with the results of Jin et al. [13] in the Chinese population. Also, in the meta-analysis study conducted by Tang et al. [20] was found that the A allele compared to G allele, was associated with 1.18 times increased risk of lung cancer, so that the A allele of Exo1 rs1047840 may confer modulating effects on the risk of lung cancer and can be used as a marker for early detection. In the study conducted in Iranian population by Nasserinejad et al. [56] was found that the A allele associated with increased risk of colorectal cancer that is consistent with result in our study. Luo et al. [52] identified that the A allele of the Exo1 rs1047840 polymorphism was significantly related to an increased cervical cancer risk compared with the G allele. Duan et al. [57] show that the A allele of rs1047840 polymorphism may be applied as a novel biomarker for tumor susceptibility. Similarly, many other studies are consistent with this association of the Exo1 rs1047840 with breast cancer, oral cancer and gastric cancer [21,22,23]. However, Zienolddiny et al. [25] have shown no significant association of the rs1047840 polymorphism and non-small cell lung cancer risk. The inconsistent results related to the association between rs1047840 and lung cancer may be due to different ethnics and explained by the differences in the studied populations. Also, for accurate analysis association of this polymorphism with lung cancer in Iranian population, other information associated with lung carcinogenesis like smoking exposures, family history of cancer in first-degree relatives, occupational exposures and certain dietary factors should be considered. Result of in silico analysis about rs1047840 showed that this SNP is a coding and non-synonymous SNP that located on exon12 of the Exo1 gene. A non-synonymous SNP that changes amino acid sequence of the protein–protein interface can alter protein interactions, affect stability and alter post-translational modifications [58]. The rs1047840 as a non-synonymous SNP result in amino acid substitution (K589E) that can affect Exo1 protein structure and function. The prediction of rs1047840 pathogenicity analysis by SNPs & GO prediction indicated that this SNP associated with the incidence of lung cancer. Protein stability is necessary for the structural and functional activity of a protein [59]. According result of I-Mutant 3.0 server, nsSNP rs1047840 of Exo1 gene showed a DDG value of less than − 0.5, which indicated that they were largely unstable and decreased protein stability. Also, results obtained with MUpro software showed that the existence of rs1047840 in Exo1 gene causes decrease protein stability of the Exo1 protein structure. Protein stability governs the conformational structure of the protein and thus determines the function. Any change in protein stability may cause degradation or aberrant conglomeration of proteins. In other words, the function, activity and regulation of a protein significantly depend on the structural stability of the molecule, therefore decrease in protein stability causes misfolding and aggregation of proteins leading to dysfunction [60]. Result of protein stability about rs1047840 indicated that the existence of rs1047840 in Exo1 gene causes decrease protein stability of the Exo1 protein structure therefore, it can increase the risk of tumorgenesis. Biophysical and structural analysis of nsSNP rs1047840 by Project HOPE software indicated the mutated residue is located in a domain that is important for the main activity of the protein. Mutation of the residue might disturb this function. The mutation introduces a residue with a charge opposite to the wild-type. This can cause repulsion with other residues in the protein or ligands. Also, this mutation might lead to loss of interactions. In other words, the existence of rs1047840 (K589E) in Exo1 gene influence lung cancer susceptibility due to changes in the structure, function and interactions of the Exo1 proteins. A role for leptin in lung cancer etiology is proposed by observations from the different studies, in which an increased risk of lung cancer was associated with the overexpressing genetic variant in LEP gene [14]. Several studies indicated that LEP rs7799039 are correlated with the development of cancers. The existence of rs7799039 polymorphism in LEP gene associated with high gene expression and twofold LEP secretion. Higher mRNA expression of LEP gene may increase risk of lung cancer [14, 29,30,31,32,33]. In our study there was no significant association between rs7799039 polymorphism of LEP gene and the risk of lung cancer that is consistent with the results of meta-analysis by Juan et al. [16] about colorectal cancer [16]. Also, in the study conducted by Hung et al. [33] it was found that there is no association between rs7799039 polymorphism of LEP gene and the risk of oral cancer in Taiwan population. However, this observation in our study is inconsistent with the results of Ribeiro et al. [14], who found that the rs7799039 polymorphism increased lung cancer risk in the Portugal population. Marcello et al. [60] identified that the A allele of the rs7799039 polymorphism was significantly related to an increased thyroid cancer risk in Brazilian population. Moreover, Ribeiro et al. [14] identified that the A allele of the rs7799039 polymorphism was significantly associated to an increase prostate cancer risk. The significant association of rs7799039 polymorphism with the risk of breast cancer has been reported by Mohammadzadeh et al. [50] in the Iranian female population. Our results suggest that individuals with lung cancer that carry GA genotype of rs7799039 are associated with increase grade tumor. This SNP is located in the promoter region, this polymorphism may affect gene expression at a transcriptional level, leading to more or less leptin production [60]. Probably the high expression of leptin gene in patients with GA genotype is associated with an increase in tumor grade. These findings suggest that leptin may have an important role in tumor cells growth and inducing a carcinogenic environment. In Silico analysis indicated that the rs7799039 is a non-coding SNP which were in the 2KB Upstream promoter of LEP gene. Promoter SNPs located in transcription factor binding sites are associated with gene expression changes and regional histone modifications [61]. FastSNP and SNP Function Prediction analysis showed that the rs7799039 can alter promoter regulation by effect on transcription factor-binding site of LEP gene and may be associated with the risk of lung cancer. The rs7799039 in promoter of LEP gene can increase transcription activity (high expression mRNA), therefore, it may be associated with the development of lung cancer. Further studies on this topic are required in order to clarify rs7799039 of LEP gene and lung cancer etiopathogenesis. The inconsistent finding about SNPs investigated in our study in the Iranian population compared to various studies in other populations can be explained by the differences in the genetic background of the studied populations. Other polymorphisms in the region of the EGFR, ExoI and LEP genes in the studied populations that regulate the effect of the SNPs studied. Analysis of the expression of the EGFR, ExoI and LEP genes in the presence of rs712829, rs1047840 and rs7799039 polymorphisms respectively can give more complete results about association of these SNPs with risk of lung cancer.

Conclusion

In conclusion, translational research that discovered polymorphisms with risk of lung cancer previously established in clinical practice. The rs712829 of EGFR and rs1047840 of Exo1 genes associated with lung cancer among Iranian population can be considered as a risk factor of non-small cell lung cancer for early detection and primary prevention.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

LC:

Lung cancer

NSCLC:

Non-small cell lung cancer

SCLC:

Small cell lung cancer

SNPs:

Single nucleotide polymorphisms

EGFR:

Epidermal growth factor receptor

Exo1:

Exonuclease 1

LEP:

Leptin

PCR–RFLP:

Polymerase chain reaction fragment length polymorphism

MMR:

DNA mismatch repair

TKIs:

Tyrosine kinase inhibitors

CI:

Confidence interval

OR:

Odds ratio

SIFT:

Sorting intolerant from tolerant

PHD-SNP:

Predictor of human deleterious single nucleotide polymorphism

SNPs&GO:

GO-gene ontology

SVMs:

Support vector machines

DDG:

Delta Delta G

HOPE:

Have (y) our protein explained

TFBS:

Transcription factor binding sites

ESE:

Exonic splicing enhancers

ESS:

Exonic splicing silencers

nsSNP:

Non-synonymous single nucleotide polymorphism

RI:

Reliability Index

CS:

Confidence Score

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Acknowledgements

The authors would like to thank the Arak University of Medical Sciences, Ayatollah Khansari Hospital, Arak city and Human Genetics Research Center of Baqiyatallah University of Medical Sciences for their support.

Funding

Not applicable.

Author information

Authors and Affiliations

  1. Department of Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, 1435916471, Iran

    Milad Pezeshki & Sayed Mostafa Hosseini

  2. Chemical Injuries Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

    Sayed Mostafa Hosseini

  3. Department of Oncology, Ayatollah Khansari Hospital, Arak University of Medical Sciences, Arāk, Iran

    Jamshid Ansari

  4. Department of Genetics, Infectious Diseases Research Center, Arak University of Medical Sciences, Arāk, Iran

    Azam Ahmadi

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  1. Milad Pezeshki
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  2. Sayed Mostafa Hosseini
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  3. Jamshid Ansari
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  4. Azam Ahmadi
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Contributions

MP and SMH designed the experiments; MP, AA and JA performed experiments and collected data; MP and SMH discussed the results and strategy; SHM Supervised, directed and managed the study; MP, SMH, AA, and JA Final approved of the version to be published.

Corresponding author

Correspondence to Sayed Mostafa Hosseini.

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The study was approved by the Research Ethics Committees of Baqiyatallah University of Medical Sciences, Tehran, Iran (Approval ID: IR.BMSU.REC.1399.312). Informed consent was obtained from all individual participants included in the study.

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Pezeshki, M., Hosseini, S.M., Ansari, J. et al. Investigating the importance of EGFR (− 216G/T), Exo1 (K589E) and LEP (− 2548G/A) gene polymorphisms with risk of lung cancer as potential diagnostic biomarker in Iranian population. Egypt J Med Hum Genet 24, 55 (2023). https://doi.org/10.1186/s43042-023-00436-8

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