The OGG 1 Ser326cys gene Polymorphism and breast tumor risk of Iraqi Women

 

Ghufran Saad Nsaif¹, Amer Hassan Abdallah², Najwa Shehab Ahmed³

^1.2Chemistry Department, College of Science, University of Mustansiriyah, Baghdad-Iraq

³Biotechnology Research Center, University of Al-Nahrian, Baghdad-Iraq

 

ABSTRACT:

 

 

INTRODUCTION:

Breast cancer (BC) counts the most well-known reason for cause of cancer-related death among women. The studies convey about 235303 new cases from BC and about 40430 death records per year in the United States (1). BC is the result of a collaboration of many variables including environmental, reproductive, lifestyle, and genetic related factors (2) and approximately 10% of BC cases are attributed to genetic factors (3). 8-Oxoguanineglycosylase So. called. (OGG1, HMMH, MUTM, OGHI, HOGG1) is DNA glycosylase enzyme that, in humans, is encoded by the OGG1 gene. It is contributed base excision repair. It is. Existing in bacterial, archaeal and eukaryotic species. The (OGG1) gene has an important role in DNA base excision repair and the polymorphism of the gene (4). Studies conducted by Pavel with others showed the association between three genetic polymorphisms in OGG1 (Ser326Cys, 7143A/G, and 11657A/G) and breast cancer risk among 1,058 cases and 1,102 controls. The role from oxidative stress in breast cancer risk is still unclear (5).

 

OGG1 that catalyzes the removal of 8-oxodeoxyguanosine from DNA. 8-oxodeoxyguanosine, the most abundant lesion generated by oxidative stress, is highly mutagenic. Environmental sources of oxidative stress, such alcohol consumption, cigarette smoking, high body mass index (BMI), and low fruits and vegetable intake may adjust the associated from genetic polymorphisms with breast cancer risk (5). The aim of the study to compare the distribution of genotypes of DNA repair gene OGG1 of malignant breast tumor patients with the control group by PCR-RFLP technology.

 

MATERIAL AND METHODS:

Samples collection:

One hundred-three Samples were obtained from patients with breast cancer (28 women malignant breast tumor patients group), (28 control group), (28 benign group) and (2 Radiotherapy group) of (30-59) years old in Al-Eluia hospital for woman care, oncology teaching hospital between September 2016 to January 2017. The complete physical examination was done to every patient. The final diagnosis was established by aspiration of cysts using (FNA) to check cytology, histology (biopsy) and mammography.

 

 

DNA extraction:

Extraction of DNA from 103 samples collected, Genomic DNA was extracted by using DNA mini kit that was supplied by The wizPrep™ gDNA extraction kit, Korea according to manufacturer’s instructions, primer were used in this study were obtained from Alpha DNA company (OGG1 (Ser326Cys)) Forward strand primers 5’- GGA AGG TGC TTG GGG AAT-3’ and Reverse strand primers 5’- ACT GTC ACT AGT CTC ACC AG -3’(6).

 

PCR amplification of OGG1 genes:

Partial OGG1gene sequences were amplified by PCR using primers OGG1 Forward strand primers 5’- GGA AGG TGC TTG GGG AAT -3’ and Reverse strand primers 5’- ACT GTC ACT AGT CTC ACC AG -3’ (6).The PCR reaction mixture included 1.5 μl genomic DNA, 16.5 of ddH2O, 5 μl Master Mix PCR (intron, korea) , and l μl each primer in a fina lreaction volume of 25 μl. Amplifications were performedas follows: initial denaturation at 3 min at 95°C, followed by 35 cycles of denaturation 95 °C for 40 sec, annealing at 61°C for 35sec , extension at 72 °C for 45 sec and a final extension of 72°C for 7 min. The PCR products were separated on 1% agarose gel. The gel is left to run for 90min with a 70volt/65Am current. Following electrophoresis, visualization was conducted with a UV transilluminator after red stain staining.

 

RESULTS AND DISCUSSION:

Polymerase Chain Reaction (PCR) was used to amplify a special region of the genome for a million time in vitro. PCR technique does not need highly purified DNA preparation as it works well with partially purified DNA samples (7). Genomic DNA of white blood cells was used for amplification of the OGG1 gene using PCR specific primers for OGG1gene. The amplified fragment which is yielded of single band of the desired product with a molecular weight of 200 base pair appeared sharp in agarose gel through gel electrophoreses technique and loaded with 100bp DNA ladder, OGG1(rs1052133) polymorphisms were genotyped with a PCR method by confronting primers briefly, rs1052133 genotyping was also performed by PCR-restriction fragment length polymorphism (PCR-RFLP). Genomic DNA was amplified by up strand, 5'-GGA AGG TGC TTG GGG AAT -3' and down the strand, 5'-ACT GTC ACT AGT CTC ACC AG -3', followed by digestion with restriction enzyme (Fnu4HI) . After PCR, amplification product was digested and the reaction was conducted in 10μl final volume; at 37°C for 30 min, for genotyping of studied samples,. The CC and GG genotype (Homozygous) was indicated by a single band of (200bp) and (100bp) respectively, the GC genotype (Heterozygous) was indicated by two band of (200/100bp),as seen in figure (1).

 

Figure(1): Electrophoresis pattern of 200 bp PCR product of healthy digested with Fnu4HI restriction enzyme (2.5% agarose gel). Lane’s 1,3,4 homozygous: GG genotype(100 bp); Lane’s 6,7,8,11,12,13,18,21,28 homozygous: CC genotype (200 bp). Lane’s 2,5,9,10.14,15,16,17,19,20,22,23,24,25,26,27 heterozygous: GC genotype (100/200bp). M: DNA molecular marker 100bp size. By redstain stained bands in the gel.

 

The frequency distribution of the OGG1 gene polymorphism of the malignant group compared with a control group was represented in a table (1) and figure(2).

 

 

Figure (2): Electrophoresis pattern of 200 bp PCR product of malignant group digested with.Fnu 4HI.restriction.enzyme (2.5%.agarose.gel). Lane’s 7 homozygous: GG.genotype (100bp);.Lane’s1,2,3,4,5,6,8,9,10,11,12,13,14,15,16,20,21,22,23,24,27,28homozygous:CC genotype (200bp). Lane’s 17,18,19,25,26 heterozygous: GC genotype (100/200bp). M: DNA molecular marker 100bp size. By red stain stained bands in the gel.

 

Patients with an allele CC (homozygous) genotype with a risk factor of 71% were more likely to have a disease compared to patients who carry allele GC (heterozygous) genotype and allele GG (homozygous) genotype. Total statistical manual allele frequency. The ratio was allele C for the patients 88%, while allele G 12%, whilst control allele C 60%, allele G 40%. The allele C is common in patients compared to control. A non-significant change was observed in the comparison between the malignant group and the control group. The allele CC (P<0.001), while the allele CG (P<0.001) on the other hand, the allele GG was a non- significant (p>0.05). To determine odds ratios and 95% confidence intervals for malignant breast cancer risk, various comparisons of genotypes as well as C vs. G alleles were analyzed as shown in table (1). The odds ratios of specific malignant breast cancer subpopulations for CC vs.CG and GG genotypes. The odds ratio for compared control with malignant breast cancer for CC subjects was 9.33 (95% CI, 2.9–30.0), CG subjects was 0.13 (95% CI, 0.04–0.44) and GG subjects was 0.31 (95% CI, 0.03–0.05).

 

 

 

Table (1): Genotype distribution of OGG1 gene CG & CC &GG polymorphism in healthy control and malignant group patients.

* X2: chi-square.                 * CI 95%: confidence intervals.                                          * PF: Protective factor.

* OR: odds ratios.                                              * RF: Risk factor.

 

 

 

The frequency distribution of OGG1 gene polymorphism of radiotherapy group compared with healthy control was represented in table (2) and figure (3).

 

 

 

Figure(3): Electrophoresis pattern of 200 bp PCR product of radiotherapy grop digested.with.Fnu4HI.restriction.enzymes (2.5%agarosegel):Lane’s1,2,3,4,5,6,7,10,11,12,13,14,15,16,17,18,19,20 homozygous:GG genotype (100bp); Lane’s 8,9 homozygous: CC genotype (200bp). M: DNA molecular marker 100bp size. By redstain stained bands in the gel.

Patients with an allele GG (homozygous) genotype with a risk factor of 89% were more likely to have a disease compared to patients who carry allele GC (heterozygous) genotype and allele CC (homozygous) genotype. Total statistical manual allele frequency. The ratio was allele C for the patients 10%, while allele G 90%, whilst control allele C 60%, allele G 40%. The allele G is common in patients compared to control. A significant change was observed in the comparison between the radiotherapy group and the control group. The allele CG (P<0.001), while the allele GG (P<0.001) on the other hand, the allele CC was a non-significant (p>0.05). To determine odds ratios and 95% confidence intervals for radiotherapy breast cancer risk, various comparisons of genotypes as well as C vs. G alleles were analyzed as shown in table (2).The odds ratios of specific radiotherapy breast cancer subpopulations for CC vs. CG and GG genotypes. The odds ratio for compared control with radiotherapy breast cancer for CC subjects was 0.26(95% CI, 0.05–1.31), CG subjects was 0.02(95% CI, 0.00–0.28) and GG subjects was 81.0 (95% CI, 12.8–512.4).

 

 

 

 

 

Table (2): Genotype distribution of OGG1 gene CG & CC &GG polymorphism in healthy control and radiotherapy patients.

*X2: chi-square.* OR: odds ratios.             *CI 95%: confidence intervals.* RF: Risk factor.          *PF: Protective factor

 

The frequency distribution of OGG1 gene polymorphism of benign group compare with healthy control was represented in table (3) and figure (4).

 

Figure (4): Electrophoresis pattern of 200bp PCR product of benign group digested with.Fnu4 HI.restriction.enzyme (2.5%.agarose.gel). .Lane’s7,8,21,24,27 homozygous: GGgenotype (100bp); Lane’s1,2,5,6, 11,14, 15,16,17,18,20,22,23,25,26,28 homozygous:CC genotype (200bp). Lane’s 3,4,9,10,12,13,19 heterozygous: GC genotype (100/200bp). M: DNA molecular marker 100bp size. By redstain stained bands in the gel.

Patients with an allele CC (homozygous) genotype with a risk factor of 43% were more likely to have a disease compared to patients who carry allele GC (heterozygous) genotype. Total statistical manual Allele frequency. The ratio was allele C for the patients 72%, while allele G 28%, whilst control allele C 60%, allele G 40%. The allele C is common in patients compared to control. A significant change was observed in the comparison between the benign group and the control group. The allele CC shows (P<0.05), while the allele CG shows (P<0.001) on the other hand, the allele GG shows a non- significant (p>0.05). To determine odds ratios and 95% confidence intervals for benign breast cancer risk, various comparisons of genotypes as well as C vs. G alleles were analyzed as shown in table (3). The odds ratios of specific benign breast cancer subpopulations for CC vs. CG and GG genotypes. The odds ratio for compared control with benign breast cancer for CC subjects was 3.5 (95% CI, 1.22–10.01), CG subjects was 0.2(95% CI, 0.07–0.61) and GG subjects was 1.8 (95% CI, 0.4–8.11).

 

Table (3):Genotype distribution of OGG1 gene CG & CC &GG polymorphism in healthy control and benign patients.

The current study investigates the breast cancer and polymorphism of OGG1 genes among group patients from different regions of Baghdad location geography, and the association among the OGG1 gene polymorphisms and various factors was studied.The results showed that there was significant difference when comparing the control with benign and the radiotherapy patients, and there was a non- significant change in the comparison between the control and the malignant patient. The study was conducted to find the association.between.a.polymorphism.OGG1.gene (SNP: .rs.1052133 (Ser326Cys)) on exon 7 with Iraqi women patients of breast cancer, most studies analyzing the association of genetic variability in OGG1 with cancer susceptibility and gene-environment interactions have concentrated on a single nucleotide polymorphism (SNP) located at codon 326 in exon 7 (Ser326Cys)(8). For example, the genetic place of OGG1 (3p26.2) is frequently missing in a range of human tumors including cancers; while OGG1 mutations are rare in most cancer patients (ordinarily<1%), suggesting that this gene is not in charge of for tumor genes in the 3p26 chromosome region may be accountable for the observed disease development (9). Various mutations in gene sequences may result in no effect at all, because either slight variation in the gene product or may result in the inhibition of the gene function. Most of these variations, which affect the gene function, may result in several diseases including cancer. Majority of the disease-causing variants are usually found among the exons of gene as single nucleotide polymorphisms (SNPs). However, DNA repair pathways to maintain the genome integrity repair a large number of these mutations. The cellular DNA repair process stabilizes the genome by reducing the number of mutations caused by the carcinogens (10). A study showed that polymorphism OGG1 gene (rs 1052133) had a dangerous association with breast cancer and it may be a risk factor for breast cancer of Iraqi women patients, recent studies have revealed that variation at OGG1 gene in exon 7 plays a key role in carcinogenesis, although several epidemiological studies have reported the association between OGG1 Ser326Cys polymorphism and breast cancer (11). These results were in agreement with the results reported by Rodrigues et.al, (8). In Spanish population, which showed that, the OGG1 Ser326Cys polymorphism is associated with high risk of breast cancer. Moreover, sangrajrang et.al. (12). Have also reported that Thai women with variant allele of OGG1 Ser326Cys were likely to have an increased risk to breast cancer (10). However,other studies have shown genotype distribution of the OGG1 Ser326Cys polymorphism may not play a substantial role in the risk of breast cancer for women Korean, Japanese, Denmark and Chinese patients (13). The polymorphism allele frequency (rs1052133; CG) was extensively investigated for its association with the risks of several cancers (14). Such as nasopharyngeal carcinoma (15), prostate cancer risk (17), lung can­cer, colorectal cancer hepatitis B related advanced liver disease (14) and type 2 diabetes mellitus (16).

CONCLUSION:

OGG1 gene polymorphisms associated with breast tumor in Iraqi women patients in benign and radiotherapy groups,and there was a non- significant change in the comparison between the control and the malignant patients.

 

ACKNOWLEGDEMENT:

The authors are grateful to the staff of wahj DNA company.

 

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Accepted on 20.12.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):745-749.