1. Introduction
Colorectal cancer (CRC) is one of the most common cancers worldwide, being the third most diagnosed cancer in men (10.6%), after lung cancer (14.3%) and prostate cancer (14.1%), and the second most diagnosed cancer in women (9.4%), after breast cancer (24.5%) [Reference Xi and Xu1]. In 2020, there were 1,930,000 CRC cases and 940,000 CRC-associated deaths, representing 10% and 9.4% of all cancers, respectively [Reference Saraiva, Rosa and Claro2, Reference Sung, Ferlay and Siegel3]. CRC is classified according to its etiology as sporadic, caused by somatic mutations, which represents nearly 70% of the cases, familial (predisposition to CRC), accounting for 10–30%, and hereditary (with Mendelian inheritance), explaining around 5–7% of the cases. Its etiopathogenesis is complex and influenced by the genetic background, mainly by chromosomal and microsatellite instability, abnormal DNA methylation, and DNA repair defects [Reference De Rosa, Pace and Rega4].
High penetrance mutations, such as those in mismatch repair genes and APC gene, comprise about 5% of CRC cases, and their role in CRC pathogenesis is well established. In contrast, low-penetrance variants represent the remaining genetic factors and are poorly understood [Reference Whiffin, Hosking and Farrington5]. In this line, genetic polymorphisms in DNA repair genes, such as XRCC1, may contribute to differences in DNA repair capacity and thus increase susceptibility to CRC [Reference Naccarati, Pardini, Hemminki and Vodicka6]. The XRCC1 gene encodes a scaffold protein that interacts with several enzymes, such as polyadenosine diphosphate (ADP)-ribose polymerase (PARP), DNA ligase III, and DNA polymerase β (polyβ) to facilitate DNA single-strand breaks repair and base excision repair (BER), and thus contribute to DNA maintenance [Reference Caldecott7]. The most studied XRCC1 polymorphisms are R194W (rs1799782, C26304T, and C194T) and R399Q (rs25487, G2815A, and G399A); the former is located between the polyβ and PARP-binding domains, while the latter is in the carboxyl-terminal side of the PARP-interacting domain [Reference Dai, Luo, Li, Huang, Zhou and Yang8]. These variants have been associated with CRC, but the results are inconsistent [Reference Dai, Luo, Li, Huang, Zhou and Yang8–Reference Hashemi, Baghbani-arani and Larijani35].
This study aimed to investigate whether the XRCC1 R194W and R399Q polymorphisms are associated with CRC in a population from northeastern México.
2. Materials and Methods
2.1. Subjects
The study included 155 male patients aged 47–79 years (mean 59.7 years) with histopathologically confirmed CRC who were enrolled at the Oncology Service of the Dr. José María Cantú Hospital in Reynosa Tamaulipas, México. Some women diagnosed with CRC were excluded from the analysis due to the small sample size (less than 20). In addition, 155 cancer-free men over the age of 50 with no history of CRC or other cancers who were seen at the same hospital for other reasons were consecutively recruited as a control group. The mean age of this group was 58.4 years (50–73 years). This research was conducted according to the guidelines of the Helsinki Declaration and approved by the Ethics Committee of the Faculty of Medicine of the Universidad Autónoma de Tamaulipas, Campus Matamoros. In addition, written informed consent was obtained from the patients prior to enrollment.
2.2. Genotyping of the XRCC1 C194T and G399A Polymorphisms
Genotyping was performed using the RFLP method from the DNA extracted from peripheral blood, as previously reported by Meza-Espinoza et al. The description of the methodology partially reproduces their wording [Reference Meza-Espinoza, Peralta-Leal and Gutierrez-Angulo36]. Briefly, the primers used were 5′-GCCCCGTCCCAGGTA-3′ and 5′-AGCCCCAAGACCCTTTCACT-3′ for the C194T polymorphism and 5′-TTGTGCTTTCTCTGTGTCCA-3′ and 5′-TCCTCCAGCCTTTTCTGATA-3′ for the G399A variant. The PCR amplification conditions for both polymorphisms consisted of an initial denaturation at 94°C for 4 min and 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by a final elongation at 72°C for 4 min. The PCR products were 491 base pairs (bp) for C194T and 615 bp for G399A. Digestion was performed with the Hpa II enzyme for both polymorphisms. For C194T polymorphism, cleavage yielded fragments of 292 bp, 178 bp, and 21 bp for the C allele and 313 bp and 178 bp for the T allele (Supplementary Figure 1), whereas for G399A polymorphism, cleavage rendered fragments of 377 bp and 238 bp for the G allele and 615 bp (uncut) for the A allele (Supplementary Figure 2).
2.3. Statistical Analysis
Allele and genotype frequencies were recorded, and Hardy–Weinberg equilibrium was assessed by the chi-squared test using the control group. Pearson’s chi-squared test was used to compare allele and genotype frequencies between both groups, and the association with CRC was estimated by odds ratio and 95% confidence interval (SPSS 25.0). A P = 0.05 was considered significant.
3. Results
The genotype and allele frequencies of XRCC1 C194T and G399A polymorphisms in CRC patients and controls are shown in Table 1. The C194T genotype frequencies were 70.5%, 27.4%, and 2.1% in controls and 73.3%, 26.7%, and 0% in patients for CC, CT, and TT, respectively; the 194T allele frequency was 15.8% in controls and 13.4% in patients. For G399A, the genotypes were 59.4%, 37.4%, and 3.2% in controls and 49.7%, 41.9%, and 8.4% in patients for GG, GA, and AA, respectively, and the 399A allele frequency was 21.9% in controls and 29.4% in patients. Both polymorphisms were consistent with Hardy–Weinberg equilibrium in the controls (C194T: χ 2 = 0.15, P = 0.70; G399A: χ 2 = 1.33, P = 0.25). As shown in Table 1, an association with CRC was found for the 399A allele (OR = 1.48 (1.03–2.13), P = 0.034) and the AA genotype in a codominant model (OR = 3.11 (1.06–9.10), P = 0.031). In contrast, there were no significant differences in the distribution of C194T polymorphism between CRC patients and controls (OR = 0.82 (0.52–1.31), P = 0.41).
Table 1 Analysis of the XRCC1 C194T and G399A polymorphisms in CRC patients and controls from northeastern México.
n: sample size; OR: odds ratio; CI: confidence interval; P: P value. aPearson’s chi-square test. bCodominant model. cDominant model. Bold indicates that the A allele and the AA genotype were associated with colorectal cancer.
4. Discussion
Our results show that subjects carrying the 399A allele have a significantly increased risk of developing CRC. This finding is consistent with observations from studies conducted in other populations, namely, Korean [Reference Hong, Lee and Kim9], Polish [Reference Jelonek, Gdowicz-Klosok and Pietrowska10, Reference Kabziński and Majsterek11], Han Chinese [Reference Zhao, Deng, Wang, Wang and Liu12, Reference Huang, Li and He13], Japanese [Reference Yin, Morita and Ohnaka14], Romanian [Reference Procopciuc and Osian15], Thai [Reference Poomphakwaen, Promthet, Suwanrungruang, Chopjitt, Songserm and Wiangnon16], and Iranian [Reference Hosseini, Mohammadiasl, Talaiezadeh, Alidadi and Bijanzadeh17, Reference Mehrzad, Dayyani and Erfanian-Khorasani18] (Table 2). Similarly, the AA genotype was associated with CRC under a codominant model in these races: Han Chinese (OR = 2.28 (1.52–3.44), P = 0.0001) [Reference Zhao, Deng, Wang, Wang and Liu12] and (OR = 1.93 (1.05–3.54), P = 0.03) [Reference Huang, Li and He13], Japanese (OR = 1.61 (1.05–2.48), P = 0.028) [Reference Yin, Morita and Ohnaka14], Romanian (OR = 3.49 (1.55–8.02), P = 0.001) [Reference Procopciuc and Osian15], Thai (OR = 4.95 (1.99–12.30), P = 0.0005) [Reference Poomphakwaen, Promthet, Suwanrungruang, Chopjitt, Songserm and Wiangnon16], and Iranian (OR = 5.30 (1.90–14.20), P = 0.001) [Reference Hosseini, Mohammadiasl, Talaiezadeh, Alidadi and Bijanzadeh17]. The A allele also showed an increased CRC risk under a dominant model in Korean (OR = 1.61 (1.09–2.39), P = 0.017) [Reference Hong, Lee and Kim9] and Iranian (OR = 1.78 (1.16–2.74), P = 0.009) [Reference Mehrzad, Dayyani and Erfanian-Khorasani18]. Even the GA genotype was associated with CRC under a codominant model in Polish (OR = 2.73 (1.31–5.68), P = 0.006) [Reference Jelonek, Gdowicz-Klosok and Pietrowska10] and (OR = 2.48 (1.75–3.53), P = 0.0001) [Reference Kabziński and Majsterek11], Han Chinese (OR = 1.46 (1.06–2.01), P = 0.02) [Reference Huang, Li and He13], and Romanian (OR = 1.75 (1.09–2.82), P = 0.017) [Reference Procopciuc and Osian15]. However, similar findings have not been replicated in many other studies, especially in Taiwanese [Reference Yeh, Sung, Tang, Chang-Chieh and Hsieh19], Norwegian [Reference Skjelbred, Saebø and Wallin20], Spanish [Reference Moreno, Gemignani and Landi21], Polish [Reference Sliwinski, Krupa and Wisniewska-Jarosinska22, Reference Gil, Ramsey and Stembalska23], Singaporean [Reference Stern, Conti and Siegmund24], Italian [Reference Improta, Sgambato and Bianchino25], Czech [Reference Pardini, Naccarati and Novotny26], American [Reference Curtin, Samowitz and Wolff27, Reference Brevik, Joshi and Corral28], Mexican [Reference Muñiz-Mendoza, Ayala-Madrigal and Partida-Pérez29], Indian [Reference Khan, Pandith and Yousuf30, Reference Nissar, Lone and Banday31], Northeast Chinese [Reference Li, Li and Wu32], Swedish [Reference Dimberg, Skarstedt, Slind Olsen, Andersson and Matussek33], and Malaysian [Reference Lau, Lian and Cheah34] (Table 2).
Table 2 Analysis of the XRCC1 G399A polymorphism in populations worldwide.
aOnly case-control studies with at least 100 patients and 100 controls reporting genotype frequencies were included. Meta-analyses were excluded. bComparisons were made using Pearson’s chi-square test with the G allele as the reference. Bold indicates a significance for the A allele with colorectal cancer.
Regarding the R194W polymorphism, like us, most studies showed no association with CRC, namely, Han Chinese [Reference Huang, Li and He13], Japanese [Reference Yin, Morita and Ohnaka14], Norwegian [Reference Skjelbred, Saebø and Wallin20], Polish [Reference Sliwinski, Krupa and Wisniewska-Jarosinska22], Italian [Reference Improta, Sgambato and Bianchino25], Mexican [Reference Muñiz-Mendoza, Ayala-Madrigal and Partida-Pérez29], and Malaysian [Reference Lau, Lian and Cheah34]. However, some Asian studies reported a risk of CRC in carriers of the 194T allele, specifically in Han Chinese (OR = 1.30 (1.04–1.64), P = 0.023) [Reference Dai, Luo, Li, Huang, Zhou and Yang8], Northeast Chinese (OR = 1.29 (1.07–1.55), P = 0.007) [Reference Li, Li and Wu32], Korean (OR = 2.87 (2.01–4.11), P = 0.0001) [Reference Hong, Lee and Kim9], and Iranian (OR = 4.95, (2.11–11.6), P = 0.001) [Reference Hashemi, Baghbani-arani and Larijani35] (Table 3).
Table 3 Analysis of the XRCC1 C194T polymorphism in several countries.
aOnly case-control studies with at least 100 patients and 100 controls reporting genotype frequencies were included. Meta-analyses were excluded. bComparisons were made using Pearson’s chi-square test with the C allele as the reference. Bold values indicate a significance for the T allele with colorectal cancer.
The discordance observed between studies for both polymorphisms is likely due to multiple factors, but perhaps racial and genetic differences, cancer histology, and inclusion criteria are the most relevant. Even this research differs from another study we have previously reported, in which no association of the R194W and R399Q polymorphisms with CRC was found in a group of patients from western México [Reference Muñiz-Mendoza, Ayala-Madrigal and Partida-Pérez29]. Since both studies analyzed Mexican patients, these contradictory results may be explained by the genetic background between both regions, as genetic differences between Mexican geographic areas have been demonstrated [Reference Castro-Martínez, Leal-Cortés and Flores-Martínez37]. Different dietary and lifestyle habits could also be involved.
Although CRC is complex and multifactorial, oxidative stress influenced by oxidizing agents plays a role in its etiopathogenesis [Reference Kabziński and Majsterek11]. One of the DNA damage principal agents is known to be reactive oxygen species [Reference Cooke, Evans, Dizdaroglu and Lunec38]. This damage is mainly caused by the formation of 8-oxoguanine (8-oxoG), which can cause mispairing with adenine, resulting in guanine-to-thymine and cytosine-to-adenine changes [Reference Cheng, Cahill, Kasai, Nishimura and Loeb39]. Accumulation of DNA damage due to misrepair or incomplete repair can lead to mutagenesis and subsequent transformation [Reference Obtulowicz, Swoboda and Speina40]. In this regard, an increase in oxidatively damaged DNA by 8-oxoG has been reported in leukocytes from CRC patients [Reference Kryston, Georgiev, Pissis and Georgakilas41]. The removal of 8-oxoG from DNA is accomplished by BER, mainly through 8-oxo-guanine glycosylase (OGG1) activity [Reference Colussi, Parlanti and Degan42], which interacts with other proteins, such as XRCC1, to maintain genomic stability. The interaction of XRCC1 with OGG1 leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of this enzyme, which accelerates the overall repair process of oxidized purines and single-strand breaks [Reference Marsin, Vidal and Sossou43]. The R399Q polymorphism has been shown to increase cancer risk in association with lower 8-oxoG cleavage activity and, consequently, increased levels of 8-oxoG [Reference Kabziński and Majsterek11, Reference Janik, Swoboda and Janowska44].
The functional significance of these polymorphisms was evaluated in individuals exposed to mutagenic agents. Regarding R399Q, the A allele (399Q) was found to contribute to ionizing radiation hypersensitivity in subjects exposed to γ-rays [Reference Hu, Smith, Miller, Mohrenweiser, Golden and Case45]; this was also associated with an increase in chromosomal deletions in individuals exposed to X-rays [Reference Au, Salama and Sierra-Torres46]; in addition, among subjects exposed to bleomycin and benzo[a]pyrene diol epoxide, those with the AA genotype had higher levels of chromosomal breaks than those with other genotypes [Reference Wang, Spitz, Zhu, Dong, Shete and Wu47]. In contrast, wild-type CC homozygotes for the R194W polymorphism had increased levels of chromosomal breaks [Reference Wang, Spitz, Zhu, Dong, Shete and Wu47], and subjects carrying the T allele had a reduced risk of chronic benzene poisoning [Reference Zhang, Wan and Jin48]. It is known that benzene, through its metabolites, can induce genotoxicity and, consequently, malignancy through oxidative stress [Reference Mathialagan, Abd Hamid, Ng, Rajab, Shuib and Binti Abdul Razak49]. These studies demonstrate the importance of these genetic variants in the ability of XRCC1 in DNA repair.
5. Conclusion
This study found that the XRCC1 R399Q polymorphism, but not the R194W, is associated with CRC susceptibility in a population from northeastern México. However, further validation of our findings in larger samples is needed.
Data Availability
The datasets generated and analyzed in this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
JPME and ELU conceptualized and designed the study and drafted the manuscript. ABA and JDG collected the samples and data. JPME, ELU, and VPL performed the genetic and statistical analyses and data interpretation. ABA, JDG, VPL, and NMG revised the manuscript and provided critical intellectual input. All authors read and approved the final version of the manuscript.
Acknowledgments
The authors thank all patients who participated in the study and the collaborating clinicians. This study was supported in part by an institutional grant Universidad Autónoma de Tamaulipas (UAT-CA-69 P/PFCE-2018-28MSU0010B-13).
Supplementary Materials
Supplementary Figure 1: photograph showing results of the XRCC1 C194T polymorphism. Lanes 1 and 4–8: wild homozygotes (CC). Lanes 2 and 3: heterozygotes (CT). Lane 10: polymorphic homozygote (TT). Lane 9: 100 bp marker. Supplementary Figure 2: photograph showing results of the XRCC1 G399A polymorphism. Lanes 1, 3, 4, and 7–11: heterozygotes (GA). Lanes 2 and 12: wild homozygotes (GG). Lane 6: polymorphic homozygote (AA). Lane 5: 100 bp marker.
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