Revealing the Association of LIG1 Genetic Variants With Pterygium Susceptibility and Demographic Characteristics in a Taiwanese Population

Age and sex distribution between case and control groups . The demographic characteristics of the study population are summarized in Table I. No significant difference was found between the pterygium group and the control group regarding age distribution ( p =0.9047), age stratification ( p =0.3820), or sex distribution ( p =0.7903), indicating that the two groups were well matched to each other (Table I). Association between LIG1 genotypes and pterygium risk. The genotype distributions of LIG1 rs20579 among controls and pterygium patients are presented in Table II . The genotypic frequencies in the control group conformed to HWE ( p =0.1239). Compared with individuals carrying the wild-type GG genotype, those harboring the homozygous variant AA genotype exhibited a significantly increased susceptibility to pterygium (OR=2.74, 95% CI=1.17-6.43, p =0.0305). Moreover, a significant genotype-dose effect was identified by trend analysis ( p trend =0.0474), although there was no significant association with heterozygous AG genotype. In the recessive inheritance model, subjects carrying the AA genotype had a 2.65-fold elevated risk of developing pterygium compared with those carrying the GG+AG genotypes (95% CI=1.14-6.18, p =0.0350). In contrast, no significant association was observed under the dominant model (AG+AA versus GG, p =0.2051) analysis (Table II). N: Number; OR: odds ratio; CI: confidence interval; P trend : p -Value evaluated using Cochran-Armitage trend test; P HWE : p -Value for Hardy-Weinberg equilibrium analysis for the control group; a Based on chi-square test with Yates’ correction; *Statistically significant with a p -Value less than 0.05. Contributions of LIG1 variant alleles to pterygium risk. Allelic frequency analysis further demonstrated that the variant A allele was significantly overrepresented among pterygium patients compared with controls (19.7% versus 14.4%). Individuals carrying the A allele exhibited a significantly elevated risk of pterygium relative to those carrying the G allele (OR=1.46, 95% CI=1.03-2.07, p =0.0413) ( Table III ). N: Number; OR: odds ratio; CI: Confidence interval; a Based on chi-square test with Yates’ correction; *Statistically significant with a p -Value less than 0.05. Joint effects of LIG1 genotypes and age on pterygium susceptibility. To further investigate whether age modified the association between LIG1 rs20579 and pterygium susceptibility, stratified analyses were performed ( Table IV ). Among participants younger than 60 years, neither genotype nor allele frequencies differed significantly between cases and controls. However, among individuals aged 60 years or older, carriers of the AA genotype exhibited a markedly elevated risk of pterygium compared with GG carriers (OR=5.64, 95% CI=1.67-19.04, p =0.0039, Table IV). Consistently, the A allele was associated with a two-fold elevated risk of pterygium in this age group (OR=2.00, 95% CI=1.27-3.14, p =0.0034, Table IV). These findings suggest that the genetic effect of rs20579 may be more obvious among elder citizens in Taiwan. N: Νumber; OR: odds ratio; CI: confidence interval; Ref: reference group; a Based on Chi-square test with Yates’ correction; b Based on Fisher’s exact test; P trend : p -Value for trend analysis; *Statistically significant with a p -Value less than 0.05. Joint effects of LIG1 genotypes and sex on pterygium susceptibility. We next evaluated sex-specific interactions and the results are shown in Table V . Among the males, carriers of the AA genotype on LIG1 rs20579 had a significantly increased susceptibility to pterygium compared with GG carriers (OR=5.23, 95% CI=1.74-15.73, p =0.0034). Similarly, the A allele conferred a significantly elevated risk (OR=2.11, 95% CI=1.34-3.30, p =0.0016). On the contrary, neither significant difference in genotype nor in allele distribution was observed among female participants (Table V). These results indicated that the contribution of the LIG1 rs20579 polymorphism to pterygium susceptibility may be particularly evident in males. N: Νumber; OR: odds ratio; CI: confidence interval; Ref: reference group; a Based on Chi-square test with Yates’ correction; b Based on Fisher’s exact test; *Statistically significant with a p -Value less than 0.05.

Investigated pterygium and control cohort. This hospital-based case-control investigation was performed under the supervision of the Institutional Review Board of China Medical University Hospital (CMUH111-REC1-176). All study procedures complied with the ethical standards outlined in the Declaration of Helsinki, and written informed consent was obtained from every participant before our enrollment. A total of 485 unrelated Taiwanese subjects were consecutively recruited for the present study, comprising 165 patients diagnosed with pterygium and 320 non-pterygium healthy control individuals. All participants were of Taiwanese ethnicity. Compared with previous studies with 160 pterygium cases ( 13 , 14 ), we have recruited five more samples in the current study, so the total case number has been enlarged to 165. Demographic variables were collected using structured questionnaires administered by trained colleagues. Peripheral venous blood samples were collected from each participant after their agreement of joining the study. To reduce potential bias arising from concomitant disorders, extremely rigorous selection criteria were implemented for the control group. For instance, individuals with a previous history of eye diseases (including glaucoma, dry eye disease, cataract), endometriosis, uterine leiomyoma, or any malignant neoplasm were excluded. Most important, all control subjects underwent clinical assessment to confirm the absence of pterygium at enrollment. The demographic characteristics of the study population, including age and sex distributions, are summarized in Table I . N: Number; SD: standard deviation; a based on unpaired Student’s t-test; b based on Chi-square test with Yates’ correction. LIG1 genotyping design and settings. Genomic DNA was isolated from peripheral blood samples using a standardized extraction protocol identical to that employed in our previous studies ( 31 , 32 ). Genotyping of the LIG1 rs20579 polymorphism was conducted following previously validated PCR-restriction fragment length polymorphism (PCR-RFLP) method ( 22 , 29 ). In brief, a genomic fragment containing the LIG1 rs20579 locus was amplified by PCR using the forward primer 5′-AATCTCCAGACGCTGCCAGA-3′ and the reverse primer 5′-CTCTGCACAACCAATCACCT-3′. The resulting amplicons were then digested with BsaH I restriction endonuclease (New England BioLabs, Ipswich, MA, USA) at 37°C for 16 h. Following enzymatic cleavage, the digestion products were resolved on 3% agarose gels and electrophoresed at 100 V for 25 min using a Mupid-2 Plus electrophoresis system. Distinct fragment patterns were used to recognize the different genotypes. Samples harboring the homozygous AA genotype produced an undigested fragment of 402 bp. Individuals carrying the heterozygous AG genotype generated three bands of 402, 303, and 99 bp, whereas homozygous GG carriers yielded two digestion products of 303 and 99 bp. Genotype assignments were performed independently by at least two trained investigators. The call rate and consistent percentages among different investigators were both 100%. Statistical analysis. Continuous variables such as age were analyzed by the unpaired Student’s t -test, whereas categorical variables were assessed using Pearson’s chi-square test with Yates’ correction. Hardy-Weinberg equilibrium (HWE) for the LIG1 rs20579 polymorphism in the control population was evaluated using a goodness-of-fit chi-square test. Differences in genotypic and allelic frequencies between pterygium cases and non-pterygium controls were determined using Pearson’s chi-square test (when all cell count ≥5) or Fisher’s exact test (when any cell count <5). Trend effects across the three genotypes were examined using the Cochran-Armitage trend test. To investigate potential interactions between genetic variants and demographic factors, such as age and sex, stratified analyses were conducted. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to evaluate the associations between LIG1 rs20579 polymorphisms and pterygium susceptibility; the CIs signified the appearance of significance. All analyses were two-tailed, and a p -value less than 0.05 was considered statistically significant.

No artificial intelligence tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

Research design: Hu PS, Mong MC, and Bau DT; patient and questionnaire summaries: Hsia NY and Hu PS; experimental work: Hsia NY, Chang WS, and Tsai CW; statistical analysis: Chen JC, Mong MC, and Hsia TC; Tables and Figures organization: Hu PS and Hsia NY; manuscript writing: Hu PS, Hsia NY, Chang WS, and Bau DT; manuscript checking and discussing: Hu PS, Hsia NY, Mong MC, Hsia TC, Chen JC, Tsai CW, Bau DT, and Chang WS.

In the present study, we investigated the association between LIG1 rs20579 genotypes and pterygium susceptibility in a representative Taiwanese cohort, consistent of 165 pterygium patients and twice as many matched healthy controls (Table I). We found a significant association of homozygous variant AA genotype with elevated risk of pterygium (Table II), which was validated with its allelic frequency analysis results (Table III). In addition, stratified analyses revealed AA genotype at LIG1 rs20579 was associated with a significantly elevated risk of pterygium among individuals aged ³60 years, whereas no protective effect was evident in younger subjects (Table IV). A similar pattern emerged in sex-stratified analysis, where the homozygous variant AA genotype of LIG1 rs20579 had an elevated effect in males but not in females (Table V). These novel findings provide extremely clinically valuable insights for the prediction for pterygium risk from a genomic viewpoint. They also provide further supporting evidence for the hypothesis that DNA repair capacity plays a critical role in the etiology of pterygium. Nevertheless, further studies in the near future are needed to elucidate the underlying mechanisms. Accumulating evidence supports the idea that DNA repair machinery may contribute to the pathogenesis of pterygium, particularly in response to oxidative stress- and/or ultraviolet (UV)-induced DNA damage. Human 8-oxoguanine DNA N-glycosylase 1 (hOGG1), encoded by the hOGG1 gene on chromosome 3, functions as a key DNA glycosylase in the BER repair pathway by removing 8-hydroxy-2′-deoxyguanine lesions, which are most frequently generated during oxidative DNA damage ( 33 ). As early as 2005, Tsai and his colleagues reported an obvious elevation of 8-hydroxydeoxyguanosine (8-OHdG), a well-established marker of oxidative DNA injury, in pterygium tissues and increased hOGG1 expression ( 34 ). Furthermore, the elevated levels of 8-OHdG observed in pterygium specimens provide evidence of UV-mediated DNA damage ( 34 ). Genetic association studies have also indicated hOGG1 polymorphisms in pterygium susceptibility. In a Chinese population, the hOGG1 Ser326Cys variant was found by Kau and his colleagues to be associated with an elevated risk for pterygium ( 35 ). However, subsequent research conducted in another Chinese cohort failed to validate a significant correlation between hOGG1 genetic variants and pterygium risk ( 36 ). Another DNA repair gene, X-ray repair cross-complementing group 1 ( XRCC1 ), participating in the BER pathway, has been linked to pterygium susceptibility ( 36 ). Located on human chromosome 19, XRCC1 and its encoding protein play a pivotal role in coordinating DNA repair processes ( 37 - 40 ). Among a Chinese cohort comprised 83 pterygium patients and 206 controls, the XRCC1 Arg399Gln polymorphism was found to be associated with pterygium and was proposed as a potential genetic marker for disease prediction ( 36 ). Individuals who carried the AG or GG genotype had a 1.71-fold elevated risk of developing pterygium compared to those who carried the AA wild-type genotype ( 36 ). Consistent with these findings, a Taiwanese study demonstrated that individuals carrying the XRCC1 Arg399Gln (rs25487) variant exhibited an elevated risk of pterygium, whereas the Arg194Trp (rs1799782) polymorphism appeared to confer a protective effect against disease development ( 19 ). Beyond BER pathway DNA repair genes, components involved in the repair of DNA double-strand breaks have also been implicated in pterygium pathogenesis ( 20 ). X-ray repair cross-complementing group 6 ( XRCC6 ) encodes Ku70, a DNA end-binding protein that heterodimerizes with XRCC5 to form the Ku complex, a critical initiator of non-homologous end joining-mediated DNA double-strand break repair. Using the same Taiwanese cohort, Tsai and colleagues reported that carriers of the variant C allele at the XRCC6 promoter T-991C (rs5751129) locus had a significantly increased susceptibility to pterygium, whereas no significant association was observed for the promoter G-57C (rs2267437) polymorphism ( 20 ). Overall, these findings support the hypothesis that genetic variations within DNA repair pathways, including hOGG1 , XRCC1 , and XRCC6 , contribute to the molecular etiology of pterygium. Nevertheless, the reported associations have not been entirely consistent across studies and populations. Therefore, further investigations in diverse ethnic groups in addition to larger sample sizes are warranted to clarify the contribution of DNA repair genotypes and phenotypes to pterygium risk prediction and disease progression. In summary, our findings demonstrate that the genotypes of LIG1 rs20579 are significantly associated with pterygium susceptibility in the Taiwanese population. Individuals carrying the homozygous variant AA genotype or the A allele exhibited an elevated risk of developing pterygium, particularly among males and those aged 60 years or older. These results suggest that impaired DNA repair capacity mediated by LIG1 genetic variation may contribute to pterygium pathogenesis. Together with previous evidence implicating DNA repair genes in pterygium development, our study identifies LIG1 rs20579 as a potential novel genetic biomarker for risk assessment and early detection of pterygium.

Pterygium is characterized by aberrant growth of conjunctival epithelial cells and fibrovascular connective tissue extending onto the corneal surface, ultimately compromising ocular integrity and visual function ( 1 , 2 ). Globally, the overall prevalence of pterygium has been estimated at approximately 12 percent ( 3 ). Among the environmental determinants for pterygium, chronic ultraviolet (UV) exposure is recognized as the most effective factor and has consistently been associated with pterygium formation across diverse populations ( 4 - 6 ). In addition, long-term exposure to airborne irritants, including dust and sand particles, has also been proposed to induce persistent ocular surface inflammation and thereby facilitate disease progression ( 7 , 8 ). Furthermore, male individuals exhibit a higher likelihood of developing pterygium than females ( 9 , 10 ), while advancing age has also been consistently recognized as an independent risk factor ( 9 , 11 ). Last, occupations requiring prolonged outdoor activity, particularly in rural environments, are associated with elevated disease incidence ( 11 ). However, in clinical practice, the useful markers for pterygium remained lacking. Beyond environmental and demographic influences collected from epidemiological studies, more and more evidence supports the hypothesis that genetic factors contribute to pterygium pathogenesis. Early observations demonstrating familial aggregation have suggested the existence of inherited susceptibility factors ( 12 ). From a molecular medicine viewpoint, accumulating genomic studies have highlighted the potential role of inherited genetic variants, particularly single-nucleotide polymorphisms (SNPs), as biomarkers for identifying individuals at increased risk of pterygium ( 13 - 18 ). Although translational genomic research has expanded our understanding of the disease, the definitive genetic determinants responsible for pterygium susceptibility have not yet been fully characterized. Therefore, continued investigations of molecular and genomic factors are urgently needed for improving risk stratification, facilitating earlier diagnosis, and identifying individuals who may benefit from personalized interventions. Accumulating evidence suggests that impaired DNA repair capacity plays a critical role in the pathogenesis of pterygium, particularly in response to chronic ultraviolet-induced DNA damage. Consistent with this hypothesis, several DNA repair-related genes have been reported to influence individual susceptibility to pterygium, including XRCC1 , hOGG1 , and XRCC6 ( 19 , 20 ). These findings support the notion that inherited variations in DNA repair pathways may contribute to disease development. Among the genes involved in maintaining genomic integrity, DNA ligase I ( LIG1 ) represents a particularly important candidate, but never been studied regarding pterygium etiology yet. Located on chromosome 19q13.2-q13.3, LIG1 encodes LIG1, which is responsible for sealing Okazaki fragments during lagging-strand DNA synthesis and restoring DNA strand continuity during the base excision repair (BER) process ( 21 ). One of the widely-studied polymorphisms in LIG1 , rs20579 (merged from rs117149496, rs17402855, rs16981751 and rs3730848), has been implicated in susceptibility to several human diseases, including lung cancer ( 22 - 27 ), endometrial cancer ( 28 ), leukemia ( 29 ) and glioma ( 30 ). Given the critical involvement of LIG1 in maintaining genomic integrity through its essential functions in DNA replication and repair, genetic variation at the rs20579 locus may influence DNA repair capacity and thereby contribute to the development of pterygium. Nevertheless, the potential role of LIG1 polymorphisms in pterygium susceptibility has never been investigated before. To address the gap, the present study was conducted to reveal the association between the LIG1 rs20579 polymorphism ( Figure 1 ) and pterygium risk in a Taiwanese population. A total of 485 individuals, including 165 patients with pterygium and 320 age- and sex-matched non-pterygium healthy controls were enrolled for genetic analysis. In addition, stratified analyses were performed to determine whether the relationship between LIG1 rs20579 genotypes and pterygium susceptibility was modified by demographic characteristics, particularly age and sex. Through these research design and approach, we sought to clarify the contribution of LIG1 genetic variation to pterygium pathogenesis and to assess its potential utility as a biomarker for identifying individuals at elevated risk of developing pterygium. The polymorphic sites of LIG1 rs20579 on chromosome 19. The rs20579 located in exon 2 of LIG1, with an alteration from wide-type G to variant A.

All the Authors declare no conflicts of interest regarding this study.

The Authors are grateful to the Tissue-bank of China Medical University Hospital and doctors/nurses for their blood sample and questionnaire collection. The assistance from Yu-Hsin Yen and Ai-Chia Tung are appreciated. This study was supported by research grants from Asia and China Medical University (CMU114-ASIA-14).