Interactive Association of XRCC1, XRCC2, XRCC3, and TP53 Gene Polymorphisms With Gastrointestinal Cancer Risk: Insights From a Hospital-Based Case-Control Study

Background and aim: Gastrointestinal (GI) cancer presents a significant worldwide health burden, influenced by a combination of genetic and environmental factors. This study endeavors to explore the combined effects of the XRCC1, XRCC2, XRCC3, and TP53 genes that contribute to the heightened risk of GI cancer, shedding light on their combined influence on cancer susceptibility. Materials and methods: A total of 200 histologically confirmed cases of GI cancer and an equal number of controls were selected to examine genetic polymorphisms within the XRCC1, XRCC2, XRCC3, and TP53 genes using the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Odds ratios (OR) with 95% confidence intervals (CI) were calculated to assess the association of these polymorphisms with GI cancer susceptibility, with statistical significance (p ≤ 0.05). Results: Logistic regression analysis confirmed strong evidence of synergistic interactions among specific variant genotypes. Notably, combinations such as heterozygous Arg/Ser+Ser/Ser genotype of TP53 Arg249Ser polymorphism with Arg/Trp+Trp/Trp genotype of XRCC1 Arg194Trp polymorphism (OR=2.64; 95% CI: 1.35-5.18; p=0.004), Arg/Gln+Gln/Gln genotype of XRCC1 at codon 399 (OR=5.04; 95% CI: 2.81-9.05; p=0.0001), Arg/His and His/His genotypes of XRCC2 Arg188His (OR=2.16; 95% CI: 1.06-4.39; p<0.032), and Thr/Met+Met/Met genotype of XRCC3 Thr242Met (OR=3.48; 95% CI: 1.79-6.77; p=0.0002) showed significant associations with GI cancer risk in the study population. Conclusions: The findings indicate a notable association between the combined effect of heterozygous variant genotypes of TP53 and variant genotypes of XRCC1, XRCC2, and XRCC3 on GI cancer risk. However, further research with a larger sample size and broad single nucleotide polymorphism (SNP) spectra is necessary to understand the interaction between genetic variations and environmental factors influencing GI cancer susceptibility.


Introduction
Gastrointestinal (GI) cancer is one of the most pressing global health concerns and the fourth leading cause of cancer-related deaths worldwide.In 2022, there were 5.1 million new cases and 3.4 million deaths from GI cancer, with projections to increase by 60% to 75% in the next two decades [1].Understanding the presentation and progression of GI cancer requires a comprehensive grasp of the complex interplay between genetic and environmental factors, which leads to the accumulation of genetic alterations disrupting normal cellular functions in individuals.The X-ray cross-complementing (XRCC) genes form vital components of the DNA repair mechanism, addressing both single-strand breaks (SSBs) and double-strand breaks (DSBs), thereby contributing to maintaining genomic stability.XRCC1, XRCC2, and XRCC3 are important regulators of the base excision repair (BER) pathway, where XRCC1 is an integral part of SSB repair, XRCC2 contributes to DSB repair, and XRCC3 is connected to double-strand breaks via the homologous recombination repair (HRR) pathway.Their roles are deemed essential for cancer prevention.The XRCC genes are highly polymorphic, where three polymorphisms, including Arg>Trp at codon-194 in exon-6 (rs1799782), Arg>His at codon-280 in exon-9 (rs25489), and Arg>Gln at codon-399 in exon-10 (rs25487), are most commonly studied polymorphisms of the XRCC1 gene [2].
Additionally, the commonly studied polymorphisms associated with cancer susceptibility include the substitution of histidine for arginine at codon 188 of exon 3 of the XRCC2 gene (rs3218536) and the transition of C to T at codon 241 of exon 7 of the XRCC3 gene (rs5861539), resulting in the substitution of threonine with methionine [3].The tumor suppressor (TP53) genes are recognized for their involvement in DNA repair mechanisms, cell cycle regulation, maintenance of genomic stability, and other essential cell signaling functions, including the induction of apoptosis.Similarly, TP53 exhibits high polymorphism, with 13 identified single nucleotide polymorphisms (SNPs) to date, the most commonly studied being Arg72Pro at codon 72 in exon-4 (rs1042522), characterized by a G to C transversion, and Arg249Ser at codon 249 in exon-7 (rs28934571), where a base substitution of G to T at the third position results in the replacement of arginine with serine amino acid [4].An individual's susceptibility to cancer development is significantly influenced by their genetic background.
The role of genetic polymorphisms in key DNA repair genes such as XRCC1, XRCC2, and XRCC3, along with their respective SNPs, has been extensively studied for their association with cancer risk across different types of cancers, such as lung [5], breast [6], and cervical cancer [7].Similarly, the tumor suppressor TP53 gene with polymorphisms at codon 72 and codon 249 is recognized for its association with lung [8], ovarian [9], and cervical cancer risk [10].However, the combined effects of polymorphic variants of various SNPs may also be significant in influencing their impact on cancer development across different populations [11].The combined interactions between SNPs in the XRCC1, XRCC2, and XRCC3 genes, along with TP53 gene polymorphisms, have also been reported and are associated with gastric [12], cervical [13], and breast cancer susceptibility [14,15].Despite studying multiple polymorphisms in various genes for their association with cancer risk, the understanding of the combined effects of SNP-SNP interactions between these genes, which contribute to an increased risk of GI cancer, remains insufficient.We found limited data on the genetic polymorphisms and interactions between the XRCC1, XRCC2, XRCC3, and TP53 genes involved in cancer development, as well as their association with the risk of GI cancer or other carcinomas documented in India.
There is a need to further explore this area to comprehend their cumulative effects on GI carcinogenesis.Therefore, in this hospital-based case-control study, we aimed to assess the impact of SNPs of DNA repair genes including Arg194Trp, C26304T, at exon-5, (rs1799782); Arg399Gln, G28152A at exon-10, (rs25487) of XRCC1; Arg188His, G31479A at exon-3, (rs3218536) of XRCC2, and Thr241Met, C18067, at exon-7, (rs861539) of XRCC3 and Arg72Pro at exon 4, (rs1042522) and Arg249Ser at exon-7, (rs28934571) SNPs of tumor suppressor TP53 gene and their collective association with risk of GI cancer among the population residing in rural Maharashtra, India.

Study population
This

Blood sample collection and genomic DNA extraction and purification
After obtaining informed consent, sterile EDTA-containing vacutainers were used to collect 5 milliliters (mL) of whole blood from 200 patients and healthy controls.Genomic DNA extraction from peripheral blood samples was performed using the HipurA® Blood genomic DNA miniprep purification kit (Cat no.MB504-250PR, HiMedia Laboratories, Thane, India), following the manufacturer's instructions.Subsequently, the purified genomic DNA was utilized for genotyping assays employing polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP).
Additionally, DNA samples were genotyped for SNPs at TP53 codon 72 and TP53 codon 249 loci using PCR-RFLP based on previous research [16].The PCR cycling conditions for the amplification of 309 bp of cd-72 of TP53 involved an initial denaturation at 95 °C for 5 min followed by 30 cycles of 95 °C -30 sec, 52 °C -30 sec, 72 °C -30 sec, and final extension at 72 °C -10 min.Similarly, for amplifying 286 bp of cd-249 of TP53, the PCR cycling conditions involved an initial denaturation at 95 °C for 5 min followed by 30 cycles of 95 °C -20 sec, 60 °C -30 sec, 72 °C -20 sec, and final extension at 72 °C -10 min.The PCR products were digested with 1 U of BstuI and HaeIII for cd72 and cd249 of TP53, respectively.The digestion products were then separated on 2%-3% low electroendosmosis (EEO) agarose gel stained with ethidium bromide at 100 V for 30 min and photographed in a gel documentation system.The validation of PCR-RFLP results was performed by DNA sequencing of randomly selected samples.The PCR products were purified using a PCR purification kit (cat.no.#K0701; Thermo Scientific, Waltham, Massachusetts).The DNA sequencing was carried out using the ABI PRISM 310 Analyzer (PathCare Laboratories Private Limited, Hyderabad, India) by a dideoxy nucleotide chain termination method.

Statistical analysis
The study investigated the association between XRCC1, XRCC2, XRCC3, and TP53 genotypes and the risk of developing GI cancer by calculating the odds ratio (OR) with a 95% confidence interval (CI) through Chisquare analysis.Confirmation of the SNP-SNP interactions was conducted using logistic regression analysis with IBM SPSS Statistics for Windows, Version 20 (Released 2011; IBM Corp., Armonk, New York).A significance level of p ≤ 0.05 was considered indicative of association.

Investigating the combined effects of XRCC1, XRCC2, XRCC3, and TP53 gene polymorphisms with GI cancer risk
The combined effects of polymorphism of XRCC1, XRCC2, and XRCC3, along with TP53 gene polymorphism, were investigated for their association with GI cancer risk in the rural population of Maharashtra.Specifically, this analysis includes the evaluation of Arginine72Proline and Arginine249Serine polymorphism of TP53 and their association with BER gene polymorphisms.The findings of the analysis revealed that the combination of variant Gln/Gln genotype of XRCC1 Arg399Gln and variant TP53 Arg72Pro genotype increased the risk of GI cancer with 2.80-fold with OR=2.80; 95% CI:

Discussion
Recent advancements in cancer genetics have deepened our understanding of the significance of SNPs, which serve as decisive signatures for assessing an individual's susceptibility to cancer.Numerous studies have emphasized the significance of SNPs in various pathway genes, elucidating their involvement in the carcinogenesis process.Among these pathways, BER stands out as a crucial mechanism in DNA repair, with the XRCC1, XRCC2, and XRCC3 genes being extensively studied for their SNPs.Particularly, the Arg194Trp and Arg399Gln variants of XRCC1, the Arg188His variant of XRCC2, and the Thr241Met variant of XRCC3 have been studied for their associations with various types of cancer [5][6][7].Similarly, the SNPs Arg72Pro and Arg249Ser in TP53 have been frequently investigated for their correlation with cancer risk [8][9][10].However, there is a dearth of literature exploring the combined effects of genetic polymorphisms in the XRCC1, XRCC2, XRCC3, and TP53 genes for their involvement in cancer risk.
In this regard, the retrospective analysis of the current study revealed a significant association between the XRCC1 399Gln variant genotype and GI cancer risk, aligning with previous research in Chinese [17] and Turkish populations [12].Several epidemiological studies have shown a positive correlation between the XRCC1 399Gln polymorphism and colorectal [18] and hepatocellular carcinoma [19].Conversely, some studies have reported no association between the 399Gln variant genotype and GI cancer risk [20,21].In our investigation of the XRCC2 Arg188His and XRCC3 Thr241Met polymorphisms and their role in cancer development, we noted no association with the XRCC2 polymorphism, while the XRCC3 polymorphism exhibited a negative association, indicating a protective effect against GI cancer risk in the studied population.These findings were in accordance with previous Indian studies that reported no association of XRCC2 Arg188His polymorphism with cervical [22], nasopharyngeal [23], or breast cancers [24].Similarly, the rs1042522 and rs28934571 SNPs of TP53 were comprehensively studied in relation to GI cancer risk [25].However, others have reported conflicting results or found no association [26].
Based on the genotype frequency of TP53 (rs1042522), our study found no obvious association between the Arg72Pro polymorphism and GI cancer risk, contrary to the findings in other Indian studies [27,28].The major finding of this study indicates that the polymorphism in the Ser/Ser allele of codon 249 of exon-7 of TP53 (rs28934571) contributes to a 3.54-fold increased risk for GI cancer in the study population, which is supported by similar findings with strong association between the 249(Ser) TP53 polymorphism in colorectal [29], hepatocellular [30], and cervical cancer risk [31].There is limited literature available regarding the combined effects of the SNP-SNP interaction between genetic polymorphisms of the XRCC1, XRCC2, XRCC3, and TP53 genes in breast [14,15,32] and cervical cancer susceptibility [13].
In our study, XRCC1 Arg399Gln significantly correlated with GI cancer risk, while XRCC3 Thr241Met showed a negative association.However, the interaction of XRCC1 Arg399Gln and TP53 Arg72Pro polymorphisms did not show an association with gastric cancer [12].The XRCC1 Arg194Trp and XRCC2 Arg188His showed no significant association with GI cancer.Similarly, TP53 Arg72Pro did not increase GI cancer risk, but both homozygous and heterozygous Arg249Ser variants were associated with GI cancer risk.Additionally, our study found significant combined effects of TP53 Arg249Ser variants with XRCC1 Arg194Trp and Arg399Gln, XRCC2 Arg188His, and XRCC3 Thr241Met, indicating increased GI cancer risk, underscoring the importance of the SNP-SNP interactions in determining cancer susceptibility within the studied population.
In this study, we found that the combination of heterozygote Arg72Pro mutant genotype of TP53 with variant genotypes of XRCC1 (Arg194Trp, Arg399Gln), XRCC2 (Arg188His), and XRCC3 (Thr241Met) did not correlate with GI cancer risk.However, it is important to note that our study had certain limitations, including a small sample size, a hospital-based case-control study design, and potential selection bias due to the limited number of SNPs considered.Further research with a larger sample size is necessary to elucidate interactions between genetic and environmental factors and the risk of GI cancer in the studied population.

Conclusions
In summary, our study concludes that the combination of the heterozygous variant genotypes of TP53 with Arg249Ser polymorphism with variant genotypes of XRCC1, XRCC2, and XRCC3 was significantly associated with GI cancer risk in the Maharashtrian population.The current findings lack a clear mechanistic explanation, prompting the need for further confirmation in larger populations to elucidate this aspect.
Validating our findings in larger cohorts will help to elucidate the collective impacts of SNPs in the XRCC1, XRCC2, XRCC3, and TP53 genes on GI cancer risk.
have an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

TABLE 2 : Association between GI cancer risk and genetic variants of XRCC1, XRCC2, XRCC3, and TP53 genes in a recessive model.
*Significant odds ratio (p<0.05);p-value determined based on χ2 Significance: p<0.05 OR: odds ratio; CI: confidence interval; GI: gastrointestinal

TABLE 3 : Association between GI cancer risk and genetic variants of XRCC1, XRCC2, XRCC3, and TP53 genes in a dominant model.
*Significant odds ratio (p<0.05);p-value determined based on χ2 Significance: p<0.05 OR: odds ratio; CI: confidence interval; GI: gastrointestinal Arg188His of XRCC2, and Thr241Met of XRCC3 in combination with Arg72Pro polymorphism of TP53 did not show an increased risk of GI cancer (p>0.05),according to Table4.

TABLE 4 : Distribution and combined effects of XRCC1, XRCC2, and XRCC3 along with codon 72 of TP53 genotype frequencies and their association with GI cancer risk.
In individuals with a combination of Arg/Trp+Trp/Trp genotype of XRCC1 Arg194Trp polymorphism and Arg/Ser+Ser/Ser genotype of TP53 Arg249Ser polymorphism, the risk of GI cancer was found to increase significantly by 2.64 times (OR=2.64;95% CI: 1.35-5.18;p=0.004).Notably, a statistically significant correlation was observed between the presence of polymorphic Arg/Gln+Gln/Gln genotype of XRCC1 at codon 399 and Arg/Ser+Ser/Ser genotype of TP53 at codon 249, showing a substantial increase in GI cancer risk with an OR of 5.04 (95% CI: 2.81-9.05;p=0.0001).Similarly, the combined effects of Arg/His and His/His genotypes of XRCC2 Arg188His with Arg/Ser and Ser/Ser genotypes of TP53 Arg249Ser were associated with an increase in the risk of GI cancer (OR=2.16;95% CI: 1.06-4.39;p<0.032) within the studied population.