Association of Different Variants in ERCC2 Gene with Susceptibility to Lung Cancer

 

Haider Andul-Kareem Abdulla¹, Abeer Hazim Mustafa¹, Qasim Sharhan Al-Mayah²

¹Department of Chemistry, College of Science, Al-Mustansiriyah University

²Medical Research Unit, College of Medicine, Al-Nahrain University

 

ABSTRACT:

Lung cancer (LCa) is a multifactorial disease with exogenous and endogenous causes. Different variants in DNA repair gene, ERCC2, may influence individual’s susceptibility to this disease. This study aims to assess the impact of two single nucleotide polymorphisms (SNPs) in ERCC2 gene (r rs1799787 and s1799788) on susceptibility to LCa in a sample of Iraqi patients. A total of 60 his to logically confirmed patients with LCa and 40 family-unrelated, age- and sex-matched healthy individuals as a control group were recruited for this study. DNA was isolated from blood samples and ERCC2 gene was amplified with specific primers using conventional PCR. Genotyping was performed by direct sequencing. The mutant homozygous genotype (TT) of rs1799787 polymorphism was more frequent in patients than controls (12.67% vs 2.5%) with significant difference (OR=2.45, 95%CI= 1.07-5.92, P=0.046). Model analysis revealed a significant effect of the recessive model where CT+TT genotype was significantly higher in patients than controls (58.33% vs 30%). Furthermore, the frequency of the mutant allele (T allele) was significantly higher in patients than controls (OR=2.775, 95%CI=1.375-5.603, P=0.004). On the other hand, the SNP rs1799788 appeared no to have any association with LCa. These results strongly suggested the role of rs1799787 polymorphism on the susceptibility to LCa among the Iraqi population.

 

 

 

1. INTRODUCTION:

Lung cancer is the leading cause of cancer-related deaths worldwide.  According to recent statistics, there are more than 25.9 million cases of bronchus and LCa causing 1.2 million deaths during the period from 1990-2015; 30-40% of whom were reported in the developing countries [1]. According to the Iraqi Cancer Registry [2], LCa is the second prevalent cancer among Iraqi population and accounted for 8.94% of all cancers, and the first cancer among the males accounting for 16.76% of all male cancers.

 

Like other cancers, LCa is a multifactorial disease with endogenous and exogenous (environmental) causes. The most documented environmental cause of LCa is smoking which is responsible for up to 90% of cases in men and 65% in women [3]. However, not all smokers encounter LCa, neither those who never smoked are safe from the disease. This implies the important role of endogenous factors besides, of course, other environmental factors.

 

Studies have shown a high rate of spontaneous and carcinogen-induced DNA damage in patients with LCa[4]. Thus, DNA repair genes have a significant protecting role. Among the most effective DNA repair genes are excision repair cross complementing-2 (ERCC2) or as formerly known as xeroderma pigmentosum group D (XPD). The product of this gene is involved in nucleotide excision repair (NER) of DNA damage [5]. Genetic variations in this gene are thought to influence the DNA repair efficiency and are suggested to be related to LCa risk. Large numbers of single nucleotide polymorphism (SNPs) were detected in this gene. Some of these SNPs were found to be positively associated with different cancers in different ethnic groups [6,7].

 

In Iraq, there is no previous study addressed the association of ERCC2 gene polymorphism and serum levels of antioxidants with LCa. Therefore. this study was conducted and aimed to investigate the association of two SNPs in ERCC2 gene with LCa.

 

SUBJECTS AND METHODS:

Study Population:

A total of 60 patients with lung cancer (37 male and 23 female, age range 35-79 years) who were attending Oncology Teaching Hospital/Medical City during the period from October 2016 to February 2017 were enrolled in this case/control study. The inclusion criterion was positive for his to pathological results, while the exclusion criterion was the presence of other cancers.

 

Family unrelated, age- matched apparently healthy 40 individuals were selected to represent the control group. Once written informed consent was provided, the subject’s demographic information including age, smoking, dwelling, diabetes mellitus and first relative family history of lung cancer were obtained.

 

Samples, DNA Extraction and Gene Amplification:

From all subjects about 3 ml of venous blood was collected in EDTA tubes. A commercial ready kit (gSYNCTM DNA Mini Kit Whole Blood Protocol/ Geneaid/ Korea) was used for DNA extraction according to the manufacturer’s instructions. Two SNPs in the RCC2 gene were targeted in this study. These are rs1799787 and rs1799788. One primer pair was used to amplify the gene fragment of 450bp which involves these SNPs. The forward primer was 5’-GAGGGAATCGACTTTGGTGA-3’ and the reverse primer was 5’- TGCAGATACGGAGGATGAGA-3’. The reaction tube (Bioneer/Korea) contains 0.4 µmol-l forward primer, 0.4 µmol-l reverse primer, DNA template (2 ng), 4 mmol-l MgCl2, Taq DNA polymerase (0.05 µl), dNTPs (dATP, dCTP, dGTP and dTTP, 0.4 mmol each. A final volume of 50 µl was obtained by adding ddH2O. The PCR conditions were as mentioned before [8].

 

Polymerase chain reaction products of RCC2 were sent to Macrogen company/Korea for DNA sequencing. The obtained sequences were aligned using "Chromas pro" with the normal sequence from GenBank (GenBank accession number NC_00009.10), and examined for the presence of SNPs.

 

Statistical Analysis:

The Statistical Package for the Social sciences (SPSS, version 14) was used for statistical analysis.  The association between different genotypes and susceptibility LCa was estimated by the calculation of adjusted odds ratio (OR) and 95% confidence intervals (CI) using logistic regression. For this analysis, subjects who were homogeneous for the wild type allele were considered as a reference, and polymorphisms as dependent variables. Chi- square test was used for testing the deviation from Hardy- Weinberg equilibrium, the distribution of different alleles between patients and control. A p-value < 0.05was considered statistically significant.

 

RESULTS:

Demographic Data:

Table 1 shows the baseline characteristics of the study population. The age and sex of the control group were intentionally selected to match the control. So there were no significant differences in these two parameters. Smoking is the most prominent factor affecting lung cancer. About three-fourths of patients were either ex-smokers or current smokers compared with only 37.5% of controls with a highly significant difference (OR=4.583, 95%CI=1.942-10.816, P<0.001). Diabetes mellitus is more frequent among patients (15%) than controls (10%) but with no significant difference (1.588(0.454-5.558, P=0.555). Six patients (10%) had a first relative family history of lung cancer compared to none among controls; however, Chi-square test revealed no significant difference (P=0.078).

 

The other factor which has a significant impact on lung cancer susceptibility is the dwelling. Over double percentage of the patients are rural residents compared to controls (OR=2.729, 95%CI=1.035-7.20, P=0.045).

 

 

 

 

 

 

 

 

 

Table 1: demographic data of the study population

Characteristic	Cases (60)	Controls (40)	P-value	OR(95%CI)
Age (years)	55.63±11.85	52.25±9.50	0.802	ـــــــــــــــــــ
Sex Male Female	37(61.67%) 23(38.33%)	28(70%) 12(30%)	0.521	1.0(reference) 1.45(0.618-3.405)
Smoking Never Ex/current	16(26.67%) 44(73.33%)	25(62.5%) 15(37.5%)	<0.001	1.0(reference) 4.583(1.942-10.816)
Diabetes No Yes	51(85%) 9(15%)	36(90%) 4(10%)	0.555	1.0(reference) 1.588(0.454-5.558)
Family History No Yes	54(90%) 6(10%)	40(100%) 0(0%)	0.078	ـــــــــــــــــــــ
Dwelling Rural Urban	22(36.67%) 38(63.33%)	7(17.5%) 33(82.5%)	0.045	1.0 (reference) 2.729(1.035-7.20)

 

The Association of Different SNPs with Lung Cancer:

The SNP rs1799787 appeared in three genotypes: wild type homozygous (CC), heterozygous (CT) and mutant homozygous (TT) in both patients and controls (Figure 1).

 

Figure 1: Different genotypes of the SNP rs1799787. A: wild homozygous genotype (CC), B: mutant homozygous genotype (CT), C: heterozygous genotype (TT).

 

The frequency of different genotypes of this SNP in patients and controls was within HWE. Both heterozygous (CT) and mutant homozygous (TT) were more frequent in patients (46.67% and 12.67% respectively) than controls (27.5% and 2.5% respectively) with a significant difference for the heterozygous genotype (OR=2.451, 95%CI=1.016-5.916, P=0.046) as shown in table 2.

 

Table 2: The frequency of different genotypes and allele of rs1799787polymorphism in lung cancer patients and controls

Analyzing the different models revealed that the influence of this SNP is through a recessive model where the genotypes involving the minor frequency allele (CT +TT) are more frequent among patients (58.33%) than controls (30%) with a highly significant difference (OR=3.267, 95%CI=1.398-7.635, P=0.008). At allelic level, the minor allele (allele T) is more frequent among patients than controls with a highly significant difference (OR=2.775, 95%CI=1.375-5.603, P=0.004).

 

The SNP rs1799788 appeared in only two genotypes (CC and CT) in both patients and controls(Figure 2).

Figure 2: Different genotypes of the SNP rs1799788. A: wild homozygous genotype (CC), B: mutant homozygous genotype (CT).

 

The frequency of genotypes in patients and controls is within HWE. Although the heterozygous genotype (CT) is more frequent in patients (10%) than controls (2.5%), the difference was non-significant (OR=4.333, 95%CI=0.501-37.401, P=0.283). Furthermore, statistical analysis revealed no significant differences in allele frequency between patients and controls although allele T was more frequent in patients (Table 3).

 

Table 3: The frequency of different genotypes and allele of rs1799788polymorphism in lung cancer patients and controls

 

DISCUSSION:

The SNP rs1799787 showed significant association with LCa susceptibility in this study, where heterozygous genotype (CT) associated with a 2.45-fold increase in the risk of LCa, while the minor allele (allele T) was significantly associated with 2.775–fold increase in the risk of the disease. These results are in accordance with that reported by Mei et al. [7] who found that this SNP was significantly associated with the risk of LCa among the Chinese population. However, Sakoda et al. [9] reported null association for the same SNP with LCa among the American population. These disparities in the result may be attributed to ethnic differences which mean that the significance of the polymorphism appears only in certain ethnicities [10].

 

Interestingly, this SNP is located in non–coding sequence (intron 19) of the ERCC2 gene and there will be no change in the amino acid structure of the encoded protein. It is not uncommon to find a significant effect of SNP located in an intron region with certain cancers. For example, Sakoda et al. [9] reported that the SNP rs156641 in the intron of one of the DNA repair gene was significantly associated with a 23% increase in LCa risk in the American population.

 

Several explanations have been proposed for this effect. Firstly, intronic variants might affect the alternative splicing of mRNA, or trigger abnormal splicing spectrum [11]. Secondly, these variants may activate novel promoters (that may direct sense or antisense transcription) causing alterations in mRNA [12]. Finally, some evidence suggested that large numbers of introns encode for micro RNAs which have a crucial role in the regulation of gene expression [13]. Whatever the mechanism employed, there is an eventual disruption in gene expression or in the ERCC2 protein.

 

It is well known that ERCC2 is belongs to the NER pathway which removes the helix distorting lesion and unusual structures in DNA that can occur in the genome. In fact, there are two sub–pathways in NER which are global genomic repair(GGR), and transcriptional coupled repair (TCR). These remove lesions from the genome overall and from actively transcribed DNA strand respectively [14]. Both of these sub– pathways require the transcription Factor IH (TFIH), a protein complex with 10 subunits. Among the most important subunit is ERCC2(XPD) which unwinds the DNA around the lesions and verifies the strand containing the lesion to facilitate the repair. Furthermore, XPD involves in maintaining the integrity of the TFIH complex [15].

 

Several studies have indicated that alteration in DNA sequence (either as mutation or polymorphism) of XPD gene could result in DNA repair– deficient diseases such as xeroderma pigmentosum and Cockayne syndrome [16]. Thus, it is reasonable to assume that the variant rs1799787 can disrupt the efficiency of TFIH and accordingly imperfect NER will result which predispose for LCa in the presence of harmful environmental factors.

 

  On the other hand, the SNP rs1799788 appeared in only two genotypes and had no significant association with LCa. So far, no previous published data suggested the association of this SNP with any malignancy, and it is considered to have no clinical significance according to the National center for Biotechnology information. These data strongly suggest the role of T allele of rs1799787 polymorphism in the susceptibility to LCa cancer among the Iraqi population. However, further studies are required to draw a solid conclusion.

 

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Received on 12.12.2017          Modified on 03.01.2018

Accepted on 25.02.2018         © RJPT All right reserved