Abstract
Background/Aim: The dysregulation of matrix metalloproteinase (MMP) proteins has been reported to be involved in the etiology of pterygium. However, studies about the role of matrix metalloproteinase-11 (MMP-11) are lacking. This study is the first to examine the genomic role of MMP-11 in pterygium. Materials and Methods: The genotypes of MMP-11 rs738791, rs2267029, rs738792, and rs28382575 were determined in 140 pterygium cases and 280 non-pterygium controls by utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing. Results: The genotypic frequencies of MMP-11 rs738792 TT, CT and CC were 40.0%, 50.7%, and 9.3% in the pterygium group, significantly different from those in the non-pterygium group (55.0%, 37.1%, and 7.9%, respectively; p for trend=0.0139). Specifically, individuals carrying the variants CT and CC had a 1.88- and 1.63-fold odds ratio of pterygium risk [95% confidence interval (CI)=1.22-2.89 and 0.77-3.44, p=0.0054 and 0.2834, respectively]. In the dominant model, individuals carrying CT+CC had significantly higher pterygium risk (odds ratio=1.83, 95%CI=1.21-2.77, p=0.0052). No association was found for other MMP-11 polymorphisms. Allelic analysis showed that MMP-11 rs738792 C allele was significantly associated with pterygium risk (odds ratio=1.48, 95%CI=1.08-2.01, p=0.0169). for the variant alleles of other MMP-11 polymorphisms were not associated with pterygium risk. Conclusion: The MMP-11 rs738792 genotypes can serve as a predictive marker for pterygium risk in Taiwanese. Additionally, elucidating the role of MMP-11 in the pathogenesis of pterygium could inform targeted therapies based on MMP-11 modulation.
In 2018, a multinational study revealed that the overall prevalence of pterygium in a cohort of 415,911 participants from 24 countries was approximately 12%. The prevalence was lowest at 3% in the 10- to 20-year-old age group and highest at 19.5% in individuals over 80 years old. Similar prevalence rates were observed in both male and female (1). Pterygium development shares several characteristics with tumorigenesis, including fibrovascular growth, corneal invasion, remodeling of the extracellular matrix (ECM), and a high likelihood of recurrence post-surgery (2, 3). The progression of these wedge-shaped tissues from the bulbar conjunctiva onto the cornea mirrors certain tumorigenic processes observed in solid malignancies (4). In the subepithelial regions of pterygium, extensive ECM deposits with fibrillar and amorphous components are found, contrasting sharply with the normal conjunctiva, where these features are absent. Despite rapid advancements in the integrative analysis of high-throughput transcriptomic and proteomic datasets, pterygium studies remain constrained by consistently inadequate sample sizes (5, 6). Although research into genetic biomarkers shows promise for improving diagnostic and therapeutic options, there remains a significant gap in identifying practical, widely applicable markers for pterygium.
The dysregulation of ECM remodeling is a key aspect of pterygium pathogenesis, yet it remains inadequately understood. Numerous studies have highlighted the involvement of altered matrix metalloproteinase (MMP) activity in the progression of pterygium. Early research identified increased transcriptional and translational expression of MMP-1 and MMP-3 in cultured human pterygium fibroblasts (7). Similarly, MMP-7 over-expression was detected in cultured pterygium tissues using monoclonal antibodies (8). Moreover, the presence of MMP-2 and MMP-9 mRNA and their enzymatic activity, assessed via gelatin zymography, was observed in tissue samples from 15 pterygium patients (9). With a collection of 82 pterygium specimens and 30 normal conjunctivas, Tsai et al. have found that MMP-10, in addition to MMP-9, was highly expressed in pterygium tissues (10). More recent studies have reported elevated levels of MMP-1 and MMP-9 in recurrent pterygium samples (11), as well as up-regulation and over-activation of MMP-14 in 28 pterygium samples (12). The understanding of MMPs’ role in pterygium etiology appears to have been significantly advanced by developments in antibody technology, alongside growing interest from ophthalmologists and translational medical researchers.
Matrix metalloproteinase-11 (MMP-11), also known as matrix decomposin-3 (ST-3) or stromelysin-3, is encoded by a gene located on chromosome 22q11.23, comprising eight exons and seven introns (13). Initially identified in invasive breast tumors, MMP-11 is significantly up-regulated in the blood serum and solid tumor tissues of cancer patients, with minimal to no expression in normal tissues (14). Over-expression of MMP-11 has been observed in various cancers, including those of the lung (15), oral (16, 17), esophageal (18), pancreatic (19), colorectal (20), and ovarian cancer (21). Despite its established role in various malignancies, MMP-11 expression has not been documented in pterygium tissues. Furthermore, no studies have yet investigated the relationship between MMP-11 genotypes and the risk of developing pterygium. To address this gap, we conducted the first hospital-based case-control study to examine the association between MMP-11 genotypes (rs738791, rs2267029, rs738792, and rs28382575) and pterygium risk in a Taiwanese population.
Materials and Methods
Requirement of pterygium and non-pterygium populations. The research framework, hypotheses, and experimental methodologies of this study have been reviewed and approved by the Institutional Review Board of Changhua Christian Hospital (IRB number: 151225). Written informed consent was obtained from one or both parents of all participants. The study recruited 140 individuals diagnosed with pterygium, along with a control group consisting of twice that number of non-pterygium subjects. All participants were Taiwanese citizens who voluntarily completed a questionnaire and provided peripheral blood samples for genetic analysis. The control group was carefully selected to exclude individuals with pterygium, endometriosis, myoma, or any type of cancer. A summary of the demographic characteristics of the participants is provided in Table I.
Demographics of the pterygium cases and the non-pterygium controls.
MMP-11 genotyping processes. Upon obtaining informed consent, all participants provided 3-5 ml of blood, from which genomic DNA was extracted from peripheral blood leukocytes on the same day. The DNA was then diluted and aliquoted to create temporary working stocks stored at −20°C, following our standard procedures (22, 23). For long-term preservation, samples were stored at −80°C or in liquid nitrogen. The method for determining MMP-11 genotypes was developed in Terry Fox Cancer Research Lab, including the design of specific primers and the selection of appropriate restriction enzymes. Details of the primer sequences, restriction enzymes used, and the DNA fragment sizes before and after enzymatic digestion are provided in Table II.
The primer sequences, methodologies for identifying MMP-11 rs738791, rs2267029, rs738792, and rs28382575 genotypes.
The polymerase chain reaction (PCR) was conducted under the following conditions: an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 30 seconds. This was followed by a final extension at 72°C for 10 min. After amplification, the PCR products for MMP-11 rs2267029 and rs738792 were digested and then separated via 3% agarose gel electrophoresis. For MMP-11 rs738791 and rs28382575, direct sequencing PCR was performed. Genotyping was carried out independently by two experienced researchers, with all results showing 100% concordance.
Statistical analyzing methodologies. The ages of the pterygium patients and non-pterygium control subjects were compared using the mean±standard deviation (SD), with statistical significance assessed by an unpaired Student’s t-test. To evaluate the potential contributions of MMP-11 polymorphisms to pterygium risk, Pearson’s chi-square (each number analyzed ≥5) or Fisher’s exact test (any number <5) was employed. Associations were further examined using odds ratios (ORs) along with their corresponding 95% confidence intervals (CIs). A p-Value of less than 0.05 was considered indicative of statistical significance.
Results
Distributions of age and sex between the pterygium and non-pterygium control groups. Initially, we analyzed the age distribution between the pterygium and non-pterygium groups and found no significant difference (p=0.2980). This finding held true even when the data were stratified using a cutoff age of 60 years (p=0.4109). Additionally, as part of our recruitment protocol, we ensured that the pterygium and non-pterygium groups were perfectly matched according to sex, resulting in no disparity (p=1.0000).
Association of MMP-11 genotypes with the risk of pterygium. Figure 1 depicts the physical map showing the positions of the MMP-11 polymorphisms rs738791, rs2267029, rs738792, and rs28382575. Analysis of genotype frequencies for these polymorphisms in the control group adhered to the Hardy-Weinberg equilibrium (p-values=0.0733, 0.5754, 0.4528, and 0.1722, respectively) (Table III). Specifically, in the pterygium group, the frequencies of the MMP-11 rs738792 genotypes (TT, CT, and CC) were 40.0%, 50.7%, and 9.3%, respectively, which differed from the non-pterygium group where the frequencies were 55.0%, 37.1%, and 7.9% (p for trend=0.0139). Individuals with the CT and CC genotypes had a 1.88-fold and 1.63-fold increased risk of developing pterygium, respectively (95%CI=1.22-2.89 and 0.77-3.44, p=0.0054 and 0.2834). When analyzed under a dominant model, carriers of either CT or CC genotypes exhibited a 1.83-fold increased risk of pterygium (95%CI=1.21-2.77, p=0.0052) (Table III, middle section). In contrast, no significant differences in genotype distributions for MMP-11 rs738791, rs2267029, and rs28382575 were observed between the pterygium and non-pterygium groups (all p>0.05) (Table III, top and bottom sections).
Physical map of MMP-11 rs738791, rs2267029, rs738792, and rs28382575 polymorphic sites.
Genotypic frequency distributions of matrix metalloproteinase-11 genotypes among the pterygium cases and the non-pterygium controls.
Association of MMP-11 allelic frequencies and pterygium risk. The analysis of allelic frequencies revealed a potential association between the C allele at MMP-11 rs738792 and an increased risk of pterygium (34.6% vs. 26.4%, p=0.0169) (Table IV, middle section). However, for MMP-11 rs738791, rs2267029, and rs28382575, the presence of variant alleles did not show a significant correlation with pterygium risk (OR=1.05, 1.07, and 0.99, respectively; 95%CI=0.77-1.42, 0.77-1.48, and 0.44-2.24; p=0.8152, 0.7574, and 0.9859, Table IV, top and bottom sections).
Allelic frequencies of matrix metalloproteinas-11 polymorphisms among the pterygium cases and healthy controls.
Discussion
There is ongoing debate among ophthalmologists regarding the precise mechanisms underlying the etiology of pterygium. The pathophysiological characteristics of pterygium share notable similarities with those of ocular surface squamous neoplasia and skin cancers, particularly in their response to ultraviolet (UV) damage and subsequent cellular adaptations (24, 25). MMPs have been implicated in the pathology of both pterygium and various types of cancer (26-29). Despite this, the specific roles of MMPs in pterygium have not been fully established. Previous studies have documented over-expression of several MMPs in pterygium, including MMP-1 (7, 11, 30-32), MMP-2 (9, 31), MMP-3 (7, 30, 31), MMP-9 (9, 10), MMP-10 (10), and MMP-14 (12). However, studies focusing on other MMPs are rare, and previous research often involves small sample sizes. From a genomic perspective, investigations into MMPs have been limited. Recently, our team has studied the genotypic distributions of MMP-1 (33), MMP-2 (34), MMP-7 (35), MMP-8 (36) and MMP-9 (37) in a representative Taiwanese pterygium cohort. A key finding is that the MMP-1-1607 (rs1799705) 1G/2G and 1G/1G genotypes could serve as protective biomarkers for pterygium (33).
To date, the genetic or proteomic roles of MMP-11 in pterygium risk have not been elucidated. In this study, we explored the potential impact of MMP-11 genotypes rs738791, rs2267029, rs738792, and rs28382575 on the susceptibility to pterygium within a representative Taiwanese cohort of 140 pterygium cases and 280 non-pterygium controls (Table I). Notably, our analysis revealed that the heterozygous CT genotype of MMP-11 rs738792 is significantly associated with an increased risk of pterygium (Table III). In contrast, the homozygous CC genotype of MMP-11 rs738792 did not show a significant correlation with pterygium risk (Table III). The dominant model analysis indicated that the combined CT+CC genotypes had a similar odds ratio to the CT genotype alone (1.83 versus 1.88), and allelic frequency analysis demonstrated a significant association between the C allele and pterygium risk (Table IV). These findings suggest that individuals carrying at least one variant C allele at MMP-11 rs738792 are at a higher risk of developing pterygium.
This discovery is noteworthy and suggests that further examination of MMP-11 genetic profiles, particularly the MMP-11 rs738792 genotypes, in a larger pterygium cohort could be more informative. Our study appears to be the first to reveal the potential role of MMP-11 rs738792 genotypes in pterygium susceptibility on a global scale. The C allele frequencies of MMP-11 rs738792 observed in our study are consistent with those reported for East Asian populations on the NCBI website, which report minor allele frequencies of 30.3% among 1,170 individuals and 29.2% among 660 individuals (38). While the C allele frequency of 29.8% in our study aligns with global averages, it shows notable variation among different populations, such as 52.2% in Africans (n=1786), 7.8% in Europeans (n=1266), and 15.2% in Americans (n=980) as reported in the 1000 Genomes Project. This variation underscores the importance of collecting and analyzing pterygium samples from diverse populations to validate our findings regarding the MMP-11 rs738792 C allele as a potential risk factor for pterygium. Furthermore, a deeper investigation into the mechanisms by which MMP-11 contributes to pterygium development is warranted.
MMP-11 rs738792 has been identified as a novel biomarker in various human diseases. For example, individuals with CT+CC genotypes at the MMP-11 rs738792 have been linked to advanced stages of hepatocellular carcinoma (39). Patients with urothelial cell carcinoma carrying the MMP-11 rs738792 CC homozygous variant exhibit improved relapse-free survival, longer disease-free intervals, and overall survival compared to those with the CT+CC genotypes (40). In 2022, Huang et al. demonstrated that MMP-11 rs738792 CC and CT genotypes are associated with higher levels of MMP-11 expression compared to the wild-type TT genotype (41). Additionally, these variant genotypes were significantly linked to perineural invasion in colon cancer patients (41). Moreover, MMP-11 rs738792 variant genotypes CC and CT have been correlated with an increased risk of Kawasaki disease (42). These findings highlight the need for further research to elucidate the underlying mechanisms of MMP-11 in disease pathogenesis and to explore the variations across different ethnic groups.
In summary, this investigation represents the first comprehensive analysis of MMP-11 genotype patterns within a Taiwanese cohort, revealing a significant association between the MMP-11 rs738792 C allele and an increased susceptibility to pterygium. The findings highlight the C allele as a notable genetic factor contributing to pterygium risk. This novel insight into the genetic underpinnings of pterygium underscores the importance of further research into the specific mechanisms by which MMP-11 influences the pathogenesis of pterygium. Understanding these mechanisms is crucial for advancing the development of targeted MMP-11-based therapeutic strategies for pterygium. Future studies should focus on elucidating the precise biological processes involved and exploring potential interventions that could mitigate the risk or progression of pterygium through modulation of MMP-11 activity.
Acknowledgements
The Authors are grateful to Yu-Cheng Luo and Yu-Hsin Yen for their excellent technical assistance. All the participants in this study are appreciated. This study is supported with grants from Show Chwan Memorial Hospital (SRD-113009), China Medical University Hospital (DMR-113-085) and Taichung Veterans General Hospital (TCVGH-1128901B). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.
Footnotes
Authors’ Contributions
Conceptualization: NYH, HCC and HWT; Data curation: NYH, HWT and TCH; Formal analysis: DCC, HCC and YCW; Funding acquisition: NYH, HCC, and HWT; Investigation: HYS, CWT and WSC; Methodology: YCW, HYS, CWT, WSC and DTB; Project administration: CWT and DTB; Resources: NYH, HCC, PSH and TCH; Supervision: DTB; Validation: TCH, CWT and DTB; Writing – original draft: NYH, CWT and DTB; Writing – review & editing: CWT and DTB.
Conflicts of Interest
All the Authors declare no conflicts of interest regarding this study.
- Received August 20, 2024.
- Revision received September 5, 2024.
- Accepted September 6, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
