|Molecular Vision 2007;
Received 13 May 2007 | Accepted 15 August 2007 | Published 15 August 2007
Pterygium and genetic polymorphism of DNA double strand break repair gene Ku70
Yi-Yu Tsai,1 Da-Tian
Bau,2,3 Chun-Chi Chiang,1,4
Ya-Wen Cheng,5 Sung-Huei Tseng,6 Fuu-Jen Tsai2,3,7
(The first two authors contributed equally to this publication)
1Departments of Ophthalmology, 2Medical Genetics, and 3Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan; 4Institute of Medical and Molecular Toxicology, and 5Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan; 6Department of Ophthalmology, National Cheng Kung University Hospital, Tainan, Taiwan; 7Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan
Correspondence to: Fuu-Jen Tsai, M.D., Ph.D., Department of Medical Genetics, Department of Ophthalmology, China Medical University Hospital, No. 2, Yuh Der Road, Taichung, Taiwan; Phone: 886-4-22052121-1141; FAX: 886-4-22052121-1139; email: email@example.com
Purpose: UV irradiation can produce a wide range of DNA damage, which will lead to gene mutation and uncontrolled cell proliferation. Of the many types of DNA damage, DNA double strand breaks (DSBs) are the most serious form, because of the intrinsic difficulty of their repair, inaccurate repair, or a lack of repair of DSBs can lead to mutations and large-scale genomic instability. DSBs are repaired by the DNA double strand break repair system. The DNA double strand break repair system consists of homologous recombination (HR) and nonhomologous end-joining (NHEJ). In humans, NHEJ is the predominant repair system and Ku70 protein plays an initial and important role in the NHEJ system. Genetic polymorphisms in NHEJ genes influence their DNA repair capacity and confer predisposition to UV-induced skin cancer. Because pterygium is an UV-related uncontrolled cell proliferation, it is logical to assume polymorphisms of Ku70 is associated with genetic predisposition to pterygium.
Methods: One hundred and twenty eight pterygium patients and 114 volunteers without pterygium were enrolled in this study. Polymerase chain reaction based analysis was used to resolve the Ku70 promoter G-57C (rs2267437) and T-991C (rs5751129) polymorphisms.
Results: There were significant differences between pterygium and control groups in the distribution of genotype (p=0.013) and allelic frequency (p=0.005) in the Ku70 promoter T-991C polymorphism. Individuals who carried at least one C allele (T/C and C/C) had a 2.83 fold increased risk of developing pterygium compared to those who carried the T/T wild type genotype (OR=2.83; 95% CI: 1.38-5.82). Moreover, individuals who carried at least one C allele (T/C and C/C) had a higher tendency to develop both sides of pterygium. In the Ku70 promoter C-57G polymorphism, there was no difference between both groups in the distribution of either genotype or allelic frequency.
Conclusions: The Ku70 promoter T-991C, but not the Ku70 promoter C-57G polymorphism, is correlated with pterygium. The Ku70 promoter T-991C polymorphism might become a potential marker for the prediction of pterygium susceptibility. It also provides a valuable insight into the pathogenesis of pterygium.
Although the pathogenesis of pterygia is still under-investigated, epidemiological evidence suggests that UV irradiation plays the most important role [1-3]. Moreover, after abnormal levels of p53 protein were found in the epithelium, more and more researchers feel that pterygium is a UV-related, uncontrolled, cell proliferation consistent with that of a tumor [4-8].
UV irradiation can produce a wide range of DNA damage, which leads to gene mutation and uncontrolled cell proliferation [9-11]. Of the many types of DNA damage, DNA double strand breaks (DSBs) are the most serious form because of the intrinsic difficulty of their repair as compared to other types of DNA damage. Inaccurate repair or a lack of repair of DSBs can lead to mutations and large-scale genomic instability .
DSBs are repaired by the DNA double strand break repair system [13,14]. The DNA double strand break repair system consists of homologous recombination (HR) and nonhomologous end-joining (NHEJ) . In humans, NHEJ is the predominant repair system. At present, five proteins involved in the NHEJ pathway have been identified; namely, the ligase IV and its associated protein XRCC1, and the three components of the DNA dependent protein kinase (DNA-PK) complex, Ku70, Ku80, and the catalytic subunit PKcs . Genetic polymorphisms in NHEJ genes influence DNA repair capacity and confer predisposition to UV-induced skin cancer .
Besides UV, there is evidence that genetic factors play a role in the development of pterygium . Several case reports have described clusters of family members with pterygium, and a hospital-based case-control study showed family history to be very significant, even suggesting a possible autosomal dominant pattern of occurrence [18-23].
Recently, researchers have begun to use single nucleotide polymorphism (SNP) to identify the genes associated with pterygium [24-27]. Single nucleotide polymorphisms are the most abundant types of DNA sequence variation in the human genome, and the SNP marker has provided a good method for identification of complex gene-associated diseases, and recognition of patients predisposing to the diseases [28,29]. Among the reported studies, glutathione S-transferase M1 (GSTM1) and human 8-oxoguanine DNA glycosylase 1 (hOGG1) polymorphisms were found to be associated with pterygium formation [24,25].
Because genetic polymorphisms in DSB repair genes influence DNA repair capacity and confer predisposition to UV-induced skin cancer , it is logical to assume that polymorphisms of DNA double strand break repair genes is also associated with genetic predisposition to pterygium.
In this study, we conducted a case control study and used SNPs in the NHEJ gene Ku70 to define its contribution to pterygium. After sequencing Ku70 from our volunteers, we found that not every reported polymorphism of Ku70 existed in our population but only promoter G-57C (rs2267437) and T-991C (rs5751129) showed high allele frequency. Hence, we evaluated the distribution of the Ku70 promoter G-57C and T-991C in our two study populations.
Patients and methods
A total of 128 pterygium patients (71 males and 57 females) treated at the Department of Ophthalmology, National Cheng-Kung University Hospital from January 2003 to June 2003 were enrolled in the study. Ages ranged from 35 to 90 years (mean, 64.6 years). Patients included in this study were primary pterygium with the apex of pterygium invading the cornea for more than 1 mm. One hundred and six (82.8%) patients' pterygium was on the nasal side, 7 (5.5%) patients' pterygium was temporal, and fifteen (11.7%) patients' pterygium was on both sides. Two patients had cancer. One was lung cancer and the other was breast cancer.
One hundred and fourteen volunteers aged 50 years or more without pterygium were enrolled as the control group. There were 64 males and 50 females in the control group (age range from 50 to 83 years with an average of 64.2 years). One female had lung cancer.
This study was carried out with approval from the Human Study Committee of the China Medical University Hospital and National Cheng Kung University Hospital. Informed consent was obtained from all individuals who participated in this study.
Genomic DNA was prepared from peripheral blood by use of a DNA Extractor WB kit (Wako, Osaka, Japan). Polymerase chain reactions (PCRs) were carried out in a total volume of 25 μl, containing genomic DNA; 2-6 pmol of each primer; IX Taq polymerase buffer (1.5 mM MgCl2); and 0.25 units of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA).
The primers used for Ku70 promoter G-57C were: forward 5'-AAC TCA TGG ACC CAC GGT TGT GA-3'; and backward 5'-CAA CTT AAA TAC AGG AAT GTC TTG-3', for Ku70 promoter C-991T were: forward 5'-AAC TCA TGG ACC CAC GGT TGT GA-3'; and backward 5'-CAA CTT AAA TAC AGG AAT GTC TTG-3'. PCR amplification was performed in a programmable thermal cycler, GeneAmp PCR System 2400 (Perkin Elmer Applied Biosystems, Foster City, CA). The primers and PCR conditions for Ku70 polymorphisms were designed and tuned by ourselves (Da-Tian Bau and Rou-Fen Wang). The cycling conditions for the Ku70 promoter G-57C and C-991T polymorphisms were both set as follows: one cycle at 94 °C for 8 min, 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and one final cycle of extension at 72 °C for 10 min. As for the Ku70 promoter G-57C, the resultant 298 bp PCR product was mixed with 2 U of HaeII. The restriction site was located at -57 with a C/G polymorphism, and the G form PCR products could be further digested while the C form could not. Two fragments measuring 103 bp and 195 bp were present if the product was digestible (G). The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (1) a G/G homozygote (digested), (2) C/C homozygote (undigested), or (3) C/G heterozygote. As for the Ku70 promoter C-991T, the resultant 301 bp PCR product was mixed with 2 U of DpnII. The restriction site was located at -991 with a C/T polymorphism, and the C form PCR products could be further digested while the T form could not. Two fragments measuring 101 bp and 200 bp were present if the product was digestible (C). The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (1) a C/C homozygote (digested), (2) T/T homozygote (undigested), or (3) C/T heterozygote.
For statistical analysis of the genotypes and allelic frequency distribution, both groups were compared using the χ2 test or Fisher's exact test.
There were no significant differences between both groups in age, sex, or the prevalence of cancers.
The frequency of the genotypes and alleles of the Ku70 promoter T-991C polymorphism in the pterygium and control groups was shown in Table 1. The distribution of the Ku70 promoter T-991C genotypes was in Hardy-Weinberg equilibrium. There were significant differences between both groups in the distribution of genotype (p=0.013) and allelic frequency (p=0.005). The odds ration of the T/C polymorphism was 2.90 (95% CI=1.38-6.10) and the C/C polymorphism was 2.13 (95% CI=0.19-23.82), compared to the T/T wild-type genotype. Hence, individuals who carried at least one C-allele (T/C and C/C) had a 2.83 fold increased risk of developing pterygium compared to those who carried the T/T wild type genotype (OR=2.83; 95% CI: 1.38-5.82). Allele C had a 2.53 fold increased risk of developing pterygium compared to allele T (OR=2.53; 95% CI: 1.30-4.93). Moreover, among the 128 pterygium patients, 15 patients' pterygium was both sides (nasal and temporal sides) and the other 113 was one side. There was a significant difference between T/T wild-type genotype and T/C and C/C genotypes in the distribution of two sides and one side pterygium. Individuals who carried at least one C-allele (T/C and C/C) had higher tendency to develop both sides of pterygium compared to those who carried the T/T wild-type genotype (Table 2).
In the Ku70 promoter C-57G polymorphism, the distribution of the Ku70 promoter C-57G polymorphism was in Hardy-Weinberg equilibrium and there was no difference between pterygium and control groups in the distribution of either genotype or allelic frequency (Table 3).
This is the first study concerned with the role of the DNA double-strand break repair system in pterygium. Our study revealed the Ku70 promoter T-991C polymorphism was associated with the susceptibility to pterygium, but the C-57G polymorphism was not. Although the Ku70 promoter T-991C genetic variation does not directly result in an amino acid coding change, it is plausible to suspect the alternative spicing, intervention, modification, determination, or involvement of this SNP influences the expression level or stability of the Ku70 protein. Moreover, Fu et al.  reported the Ku70 C-61G polymorphism was associated with breast cancer risk. Because this SNP also does not affect amino acid coding and therefore probably does not affect protein function, they suggested the observed association between breast cancer risk and the Ku70 C-61G polymorphism should be interpreted as the presence of linkage disequilibrium (LD) between the SNP and other SNPs in exons, resulting in functional polymorphism. Our result may also be in the same situation. There may be LD between the Ku70 promoter T-991C polymorphism and other SNPs in exons, resulting in functional polymorphism and predisposing to pterygium formation.
UV is capable of causing DNA DSBs. Although UVB exposure does not directly produce DNA DSBs, it has been suggested that UV-induced photoproducts cause blockage of DNA replication, which can lead to the formation of DSBs, chromosomal aberrations, and recombination during the course of replication arrest . Reactive oxygen species generated after the absorption of UVA by cellular chromophores can cause oxidative DNA damage that can lead to single and double strand breaks . HR and NHEJ are two distinct mechanisms in the repair of DSBs in mammalian cells. In the NHEJ pathway, after bound by the complex of the Ku 70 and 80 heterodimer and DNAdependent protein kinase, the DNA double strand break is repaired by the complex of LigaseIV and XRCC4 . Hence, Ku70 protein plays an initial and important role in DNA double-strand break repair. The association between pterygium and Ku70 polymorphism suggests that DNA double-strand break repair plays a role in pterygium formation and provides an indirect evidence that there may be insufficient DNA repair related genetic mutation in pterygium.
The most common alteration in human tumor is a mutation in the p53 gene . Though more and more researchers feel that pterygium is a UV-related uncontrolled cell proliferation or tumor, whether there is p53 genetic mutation in pterygium is controversial. To date, there have been 4 studies in Medline regarding p53 gene mutations in pterygium [35-38]. Schneider et al.  found the p53 gene in pterygia is not mutated in their 11 Caucasian patients. Reisman et al.  found that the p53 gene had undergone a mono-allelic deletion, and the remaining allele remained wild-type in 9 American patients, and Shimmura et al.  reported that no mutation was found in exons 5 to 8 in 6 Japanese. In our hands, mutations within the p53 gene were detected in 8 of 51 Taiwanese pterygial samples (15.7%) . Though our result of a p53 gene mutation in pterygium is different from others, the result of this Ku70 study further supports our previous report.
UV irradiation can produce a wide range of DNA damage and most DNA damage is repaired by the DNA repair system. In humans, more than 70 genes are involved in five major DNA repair pathways: direct repair, base excision repair (BER), nucleotide excision repair (NER), mismatch repair and double strand break repair [13,14]. HOGG1 belongs to the BER system and Ku70 belongs to the double strand break repair system. Polymorphisms of both are reported to be associated with pterygium . In our unpublished study, polymorphism of X-ray repair cross complementary 1 (XRCC1), a major gene in BER system, is also associated with pterygium. However, polymorphisms of xeroderma pigmentosum group A (XPA) and xeroderma pigmentosum group D (XPD), two important genes in NER system, are not. We suggest different DNA repair systems may play different roles in pterygium. These could be the basis of future surveys. Further study on polymorphisms of the genes in other repair systems are necessary for clearly figuring out the molecular mechanism of pterygium formation by UV.
In conclusion, Ku70 promoter T-991C, but not Ku70 promoter C-57G polymorphism, is correlated with pterygium. Ku70 promoter T-991C polymorphism might become a potential marker for the prediction of pterygium susceptibility. It also provides a valuable insight into the pathogenesis of pterygium.
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