The Contribution of DNA Ligase 4 Polymorphisms to Colorectal Cancer

Abstract

Background/Aim

While numerous biomarkers associated with genetic susceptibility to colorectal cancer (CRC) have been identified and validated through epidemiological studies, the specific influence of DNA ligase 4 (Lig4) genotypes remains unexplored. This study aimed to elucidate the hitherto unexamined relationship between Lig4 genotypes and CRC risk.

Materials and Methods

The genotypes of Lig4 rs1805388 were determined applying the polymerase chain reaction-restriction fragment length polymorphism methodology. The potential association between these genotypes and CRC risk was assessed in a Taiwanese population comprising 362 CRC cases and an equal number of age- and sex-matched controls.

Results

In the genotypic analysis, the distribution of CC, CT, and TT genotypes for Lig4 rs1805388 among CRC cases was 54.7%, 38.1%, and 7.2%, respectively. This distribution was not significantly different from the controls, which exhibited genotypic frequencies of 57.2%, 36.7%, and 6.1%, respectively (p for trend=0.7314). Analysis of allelic distribution indicated that individuals carrying the T allele of Lig4 rs1805388 displayed a slightly elevated CRC risk compared to those carrying the C allele (odds ratio=1.10, 95% confidence interval=0.87-1.39, p=0.4685).

Conclusion

The variant genotypes of Lig4 rs1805388 may not serve as predictive markers for CRC risk in the Taiwanese population.

Colorectal cancer (CRC) constitutes approximately 11% of all newly diagnosed cancer cases and stands as the third most prevalent cancer worldwide, leading to the second highest number of cancer-related fatalities (1). The pathogenesis of CRC encompasses a myriad of factors that contribute to intricate genetic and epigenetic mechanisms, culminating in the conversion of normal colonic mucosa into malignant tissues (2). Deficient DNA repair capacity is closely linked with genomic instability and heightened susceptibility to cancer (3-5). Furthermore, emerging evidence underscores the strong correlation between genetic variations in the DNA damage responses, microsatellite instability status and mitochondrial copy numbers and other pathways, serving as crucial determinants for CRC patients (6-11).

The human genome is incessantly subjected to attacks from both endogenous and exogenous DNA damaging agents. Rapid repair of DNA adducts is imperative to avert genomic instability and prevent the emergence of malignant changes. To achieve this, the DNA repair machinery promptly initiates corrective actions upon lesion detection, aiming to rectify altered bases or any other anomalies. In the literature, several well-defined DNA repair pathways exist, encompassing direct reversal, excision repair [including base excision repair (BER) and nucleotide excision repair (NER)], mismatch repair (MMR), and double-strand break (DSB) repair pathways [including homologous recombination repair (HR) and non-homologous end joining (NHEJ)] (12,13). Among these pathways, DSBs represent the most severe form of DNA damage, stemming from both endogenous and exogenous factors, such as replication errors, ionizing radiation exposure, free radicals, and telomere dysfunction (14,15). Timely repair of DSBs is crucial, as failure to do so in cells can lead to severe human disorders, including various types of cancer.

The Lig4 gene, located on chromosome 13q33, encodes a nuclear protein which plays a role in NHEJ (16,17). Clinically, the significance of the Lig4 protein in maintaining genomic stability is underscored by the fact that deficiency in this gene is responsible for Lig4 deficiency syndrome (OMIM 606593 or ORPHA99812), characterized by severe disorders including combined immunodeficiency, bird-head-like facial features, bony deformations, skin conditions, and susceptibility to malignancy (18). Cells from Lig4-deficient patients display increased radiosensitivity and exhibit defective NHEJ repair capacity (19-21). Recently, this syndrome has been effectively corrected through allogeneic hematopoietic stem cell transplantation (22). In a mouse model, Lig4 deficiency leads to late embryonic lethality with numerous neuronal cells undergoing apoptosis, along with developmental arrest of lymphocytes (23,24). Collectively, this evidence underscores the critical role of Lig4 in maintaining the integrity of the human genome. Furthermore, subtle genomic variations in Lig4 may be associated with an increased risk of human disorders.

In comparison to other members of the NHEJ sub-pathway, studies focusing on Lig4 genotyping have been relatively rare. Among the various cancer types studied, breast cancer has received the most frequent attention (25-29). Within the realm of polymorphic sites, Lig4 rs1805388 has emerged as the most investigated variant, involving a C to T shift. This polymorphic site has also undergone scrutiny for its potential associations with glioma (30-33), prostate cancer (34,35), lung cancer (36-39), nasopharyngeal carcinoma (3). However, its potential role in CRC has yet to be explored. Thus, we are motivated to initiate a preliminary hospital-based case-control study aimed at unraveling the potential contribution of Lig4 rs1805388 to CRC.

Materials and Methods

Recruitment of colorectal cancer cases and matched controls. The selection of CRC cases and healthy controls adhered to the methodology delineated in our previous publications (40,41). In brief, CRC cases were sourced from patients seeking care at the Department of General Surgery within China Medical University Hospital (CMUH), with comprehensive pathological data meticulously recorded for each individual. Control subjects were meticulously matched with a 1:1 ratio to the cases, taking age and sex into consideration. Informed consent was obtained from all participants, who contributed blood samples for the study. The research protocols were granted approval and oversight by the Institutional Review Board of CMUH (approval code: DMR99-IRB-108), and the study was carried out in alignment with the principles outlined in the Declaration of Helsinki. Notably, both cases and controls were of Taiwanese origin, and Table I concisely presents the salient characteristics of the study population.

Table I. Selected characteristics of the 362 patients with colorectal cancer and 362 non-cancer controls,

SD: Standard deviation; BMI: body mass index; ^abased on Chi-square test with Yates’ correction.

Genotyping procedure for Lig4 rs1805388. Genomic DNA was isolated from peripheral blood leukocytes of each participant within 24 h of blood collection, utilizing the QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan, ROC), as detailed in standard biomedical practices (40,42,43). The extracted DNA was quantified, preserved at -80˚C for long-term storage, and apportioned as working stock at -20˚C for genotyping purposes. The genotyping protocol for Lig4 rs1805388 was developed by the Terry Fox Cancer Research Lab, Taichung, Taiwan. The primer sequences used were as follows: forward 5’-TCTGTATTCGTTCTAAAGTT-3’ and reverse 5’-TGCTTTACTAGTTAAACGAG-3’. The polymerase chain reaction (PCR) cycling conditions consisted of an initial denaturation step at 94˚C for 5 min, followed by 35 cycles of denaturation at 94˚C for 30 s, annealing at 52˚C for 30 s, extension at 72˚C for 30 s, and a final extension at 72˚C for 10 min. Following PCR amplification, the products were digested with the HpyCH4 III restriction endonuclease and resolved using 3% agarose gel electrophoresis for a duration of 25 min. The Lig4 rs1805388 genotypes were distinguished as follows: wild-type CC genotype with a 121-bp product, heterozygous variant CT genotype with products of 121 bp, 65 bp, and 56 bp, and homozygous variant TT genotype with products of 65 bp and 56 bp. Genotyping was independently repeated by at least two researchers, who are acknowledged contributors, in a double-blind fashion. Notably, all genotyping outcomes achieved a 100% success rate and demonstrated complete concordance among the results.

Statistical analysis. The assessment of Hardy-Weinberg equilibrium among the distinct Lig4 rs1805388 genotypes in 362 non-cancer controls was conducted using the goodness-of-fit chi-square test. The distributions of age, sex, personal habits, BMI, different Lig4 rs1805388 genotypes, and alleles within subgroups were evaluated using Pearson’s chi-square test. Logistic regression analysis was employed to compute odds ratios (ORs) along with their corresponding 95% confidence intervals (CIs) for discerning associations between Lig4 rs1805388 genotypes and CRC risk. A p-value below 0.05 was deemed statistically significant for all comparisons.

Results

The demographic profiles of the 362 CRC patients and their corresponding 362 matched controls are presented in Table I. As the controls were meticulously paired with the cases at a 1:1 ratio, based on age and sex, noteworthy dissimilarities in the distribution of these two variables between the case and control cohorts were absent. Furthermore, notable disparities in the prevalence of smoking (p=0.543), alcohol consumption (p=0.441), and individuals categorized with lower (<24) or higher (≥24) BMI (p=0.181) did not manifest between the case and control groups.

Table II illustrates the distribution of Lig4 rs1805388 genotypes within the cohort of 362 non-cancer controls as well as among an equivalent number of CRC patients. The genotypic frequencies of Lig4 rs1805388 among the control subjects were determined to be in adherence with the Hardy-Weinberg equilibrium (p=0.9175, Table II). Moreover, the genotypic distributions of Lig4 rs1805388 were found to be equivalently distributed between the CRC patients and the non-cancer control group (p for trend=0.7314, Table II). Additionally, neither the heterozygous CT nor the homozygous variant TT genotypes of Lig4 rs1805388 exhibited associations with an altered risk of CRC (OR=1.08 and 1.24, 95%CI=0.80-1.48 and 0.68-2.25, p=0.6601 and 0.5900, respectively; Table II). Similar assessments were carried out in both recessive and dominant models. In the recessive model, a non-significant 1.20-fold increased CRC risk was observed for carriers of the Lig4 rs1805388 TT genotype in comparison to those possessing CC+CT genotypes (95%CI=0.66-2.15, p=0.6541, Table II). In the dominant model, carriers of the CT+TT genotypes of Lig4 rs1805388 exhibited a non-significant 1.11-fold increased CRC risk in comparison to those harboring the CC genotypes (95%CI=0.82-1.48, p=0.5493, Table II).

Table II. Distribution of Lig4 rs1805388 genotypes among the 362 patients with colorectal cancer and 362 non-cancer controls.

N: Νumber; OR: odds ratio; CI: Confidence interval; ^aBased on Chi-square test with Yates’ correction; p_HWE: whether the frequencies in controls are consistent with Hardy-Weinberg Equilibrium.

To corroborate the findings presented in Table II, an analysis of allelic frequency distribution for Lig4 rs1805388 was conducted, with the outcomes presented in Table III. The results of the allelic frequency distribution analysis further reinforce the notion that the Lig4 rs1805388 genotype is not associated with CRC risk. This assertion is substantiated by the observation that the prevalence of the variant T allele was 26.2% among the CRC patients and 24.4% within the cancer-free control group (OR=1.10, 95%CI=0.87-1.39, p=0.4685).

Table III. Distribution of Lig4 rs1805388 allelic frequencies among the 362 colorectal cancer cases and the 362 non-cancer controls.

N: Νumber; OR: odds ratio; CI: Confidence interval; ^aBased on Chi-square with Yates’ correction test.

Discussion

Genotypes of DNA repair genes are frequently regarded as crucial markers, given their potential involvement in genomic stability alterations that can instigate or advance carcinogenesis (44-46). In the human DNA repair system, the principal mechanisms encompass direct reverse, BER, NER, MMR, and DSB repair. Genotypes within these DNA repair genes can potentially dictate the cellular DNA repair capacity and, consequently, exert an influence on an individual’s genetic predisposition to specific types of cancer. Among these various genes, Lig4 potentially holds a pivotal role in mediating the repair of DSBs through the NHEJ pathway. However, to date, the role of Lig4 genotypes in the context of colorectal carcinogenesis remains unexplored (47). In the literature, the Lig4 protein has been shown to interact with another NHEJ protein, XRCC4, culminating in the final ligation of the break through an ATP-dependent step (48,49). In recent years, several studies have reported significant associations between genotypes in DSB repair genes and CRC, such as ataxia telangiectasia mutated (ATM) (50,51), and X-ray repair cross-complementing group 4 (XRCC4) (52-56). Nonetheless, comprehensive investigations into the association between Lig4 genotypes and CRC risk remain rare.

Consequently, we embarked on the pioneering investigation of Lig4 rs1805388 genotypes within the Taiwanese population. Notably, we conducted a comparative analysis between the distribution of Lig4 rs1805388 genotypes among our cancer-free controls and the data documented on the National Center for Biotechnology Information (NCBI) website, encompassing diverse populations worldwide. With a dataset involving 3,818 Asian subjects and 2,450 East Asian subjects, the minor allelic frequencies at Lig4 rs1805388 were calculated to be 0.212 and 0.206, respectively (Table IV, updated as of 2023/08/15). Within our dataset, the variant T allele exhibited a frequency of 0.244, a representation of the Taiwanese population. It is evident that Asians exhibit distinct Lig4 rs1805388 genotypic patterns compared to Caucasians (Table IV), despite the vital role of Lig4 in NHEJ.

Table IV. Variant (T) allele frequencies of Lig4 rs1805388 in different populations.

Data were extracted from https://www.ncbi.nlm.nih.gov/snp/

Subsequently, an exploration of the correlation between Lig4 rs1805388 genotypes and CRC risk transpired in a genetically homogeneous population in Taiwan, encompassing 362 healthy individuals and 362 CRC cases. Our findings unveiled that neither the CT nor the TT genotypes of Lig4 rs1805388 were associated with statistically significant modifications in CRC risk (Table II and Table III). Almost two decades ago, reports indicated that the T allele at Lig4 rs1805388 is linked with diminished adenylation and ligation activities of the Lig4 protein (57). Our current data implies that Lig4 rs1805388 might not be the principal determinant influencing the quantity and/or quality of Lig4 protein, thereby potentially leading to a considerably reduced capability for DSB repair and an increased CRC risk. Consequently, there could exist other polymorphic sites, such as Lig4 rs1805386, that warrant thorough investigation in order to uncover genetic markers pertinent to the prediction of CRC risk.

In conclusion, this study has illuminated that the presence of the variant T allele at Lig4 rs1805388 might not serve as a reliable indicator for predicting susceptibility to CRC. Subsequent investigations should be directed towards comprehending the ramifications of other polymorphic sites within Lig4, while also exploring the interplay between genotypes and phenotypes. Furthermore, a reassessment of the correlation between genotypes and/or the overall capacity of the NHEJ pathway is imperative. Above all, the role of Lig4 in the context of colorectal carcinogenesis warrants thorough and continued exploration.

Conflicts of Interest

The Authors declare no conflicts of interest regarding this study.

Authors’ Contributions

Conceptualization: D.Y., D.T.B., C.W.T. and W.S.C.; Collection: T.W.K. and Y.C.H.; Data curation: M.C.M. and C.W.T.; Genotyping: Y.C.W., Y.T.C. and W.S.C.; Statistics: W.T.W., Y.C.Y. and C.W.T.; Phenotyping: D.T.B. and W.S.C.; Project administration: T.C.Y. and D.T.B.; Supervision: D.T.B., W.S.C. and C.W.T.; Validation: T.W.K. and J.G.; Writing—original draft: D.Y., J.G., and C.W.T.; Writing—review and editing, D.T.B., D,Y. and C.W.T.; All Authors have read and agreed to the published version of the manuscript.

Acknowledgements

The Authors would like to acknowledge the Tissue-Bank of China Medical University Hospital for their invaluable technical support. Furthermore, the authors would like to extend their gratitude to all the study participants, as well as the doctors, nurses, and colleagues who contributed to the study. The technical assistances from Yu-Hsin Lin, Yi-Wen Hung and Hou-Yu Shih are appreciated by all the Authors. This study was supported by the grant from China Medical University and Asia University (CMU111-ASIA-03 and CMU112-ASIA-02).