台灣眼翳致病機轉之研究：(I)甲基轉移酵素3b與p16INK4a轉錄起始區過度甲基化的關聯性(II)去氧核醣核酸修補基因 APE1、hOGG1及XRCC1 之基因多型性之分析

A study on the pathogenesis of Taiwanese pterygium: (i) The association between DNA Methyltransferase 3b (DNMT3b) and p16INK4a promoter hypermethylation in pterygium. (ii) Relationship between pterygium and genetic polymorphism of DNA repair gene APE1, hOGG1, XRCC1 in Taiwan

陳培亮(Pei-Liang Chen)

指導教授 ： 李輝 ； 鄭雅文

中山醫學大學/醫學院/醫學研究所/博士(2010年)

https://doi.org/10.6834/CSMU.2010.00144

摘要

Part I
眼翳（pterygium）長久以來認為是一種慢性退化性疾病，不過隨著有些研究發現在眼翳之上皮細胞有 p53 蛋白表現異常，認為眼翳可能是一種腫瘤，不過仍需更多的證據來證實。在許多的癌症中 p16INK4a 基因啟動區的不正常甲基化所引起的基因靜默（gene silence）扮演一個重要的角色。本研究目的在研究 p16INK4a 基因啟動區的過度甲基化與 p16INK4a 蛋白、DNMT3b蛋白表現與眼翳的相關性。本研究共收集 129個眼翳及 16位正常結膜組織，各別以Methylation specific PCR（MS-PCR）及免疫組織化學染色法來分析p16INK4a基因啟動區過度甲基化及 p16INK4a、DNMT3b 蛋白表現。結果發現有 16.3%（21 of 129）的眼翳有 p16INK4a基因啟動區過度甲基化現象，但在對照組中並未發現此現象。129個眼翳中有35.7%（45 of 129）p16INK4a 蛋白表現，p16蛋白染色均局限在上皮細胞層的細胞核中。我們觀察到 p16INK4a 過度甲基化與p16INK4a 蛋白的表現呈顯著負相關（p=0.006）。以上結果顯示 p16INK4a 基因啟動區過度甲基化的確會造成 p16INK4a 蛋白的不表現。本研究進一步分析 p16INK4a 基因啟動區過度甲基化與 DNMT3b 蛋白表現的相關性，有29.5%（38 of 129）DNMT3b 蛋白表現，且 DNMT3b 蛋白染色均局限在上皮細胞層的細胞核中。我們發現 p16INK4a 基因啟動區過度甲基化與 DNMT3b 蛋白的表現呈顯著相關（p=0.001）。結論：在眼翳的 p16INK4a基因啟動區發生過度甲基化的現象，而這過度甲基化的現象緊密與甲基轉移酶 DNMT3b蛋白表現聯繫及抑制 p16INK4a 蛋白的表現。這些分子學上的證據證實甲基化現象存在於眼翳及在眼翳生長可能扮演的角色。
Part II
眼翳(pterygium)長久以來認為是一種慢性退化性疾病，不過隨著有些研究發現眼翳之上皮細胞有p53 蛋白表現異常，而認為眼翳可能是一種腫瘤，不過仍需更多的證據來證實。流行病學的研究認為紫外線照射是眼翳發生的最主要因子，紫外線幅射引起的毒害經由光毒性或活性氧造成細胞的 DNA、蛋白質及脂質的損壞而傷害細胞。鹼基切除修補（base excision repair，BER）在修補輻射造成的 DNA 損傷途徑中扮演極重要的角色。根據先前的研究指出鹼基切除修補基因的AP-endonuclease-1 (APE1)、8-oxoguanine glycosylase-1 (OGG1) 及 X-ray repair cross-complementing group 1 (XRCC1)之單一鹼基多型性（single-nucleotide polymorphisms，SNPs）會影響到對輻射照射引起 DNA 傷害的敏感性，因此假設這些修補基因的多型性增加眼翳發生的可能性。本研究利用螢光標記的TaqMan® 探針對 83個眼翳檢體和 206位健康對照組，分析 APE1、hOGG1、XRCC1 之基因多型性。研究結果顯示 XRCC1 codon 399 的基因多型性與眼翳的形成顯著相關（p=0.038），但在 hOGG1 codon 326（p=0.383）及 APE1 codon 148（p=0.898）則無相關性。此外，在 XRCC1 (Arg399Gln) 的基因型 Arg/Gln 及 Gln/Gln 相較於 Asp/Asp 野生型基因的 OR分別為2.592（95% CI：1.225-5.484）及1.212（95% CI：0.914-1.607）。更進一步來說，若至少帶有1個C-對偶基因（C-allele，A/G、G/G）的個體將比帶有 A/A 野生型基因增加1.7倍的風險，而hOGG1 及 APE1 基因多形型並不會增加眼翳的風險。因此XRCC1（Arg399Gln）基因多型性可能提高罹患眼翳的風險，並且可能成為預測眼翳感受性的一個重要指標。

關鍵字

眼翳 ； 基因多型性 ； 甲基化

並列摘要

Part I
A pterygium has long been considered as a degenerative condition. After p53 protein was found to be abnormally expressed in the epithelium, researchers suggested that a pterygium may be a tumor, but additional evidence is required to support this hypothesis. Aberrant methylation of the p16INK4a gene promoter and resultant gene silencing play important roles in the pathogenesis of many types of human cancers. The purpose of this study was to investigate hypermethylation of the p16INK4a promoter in pterygium and the relationship between this hypermethylation and the expression of p16CDKN2A and DNA methyltransferase 3b (DNMT3b) proteins. We studied the methylation status of p16INK4a and the expression of p16INK4a and DNMT3b proteins by performing methylation-specific polymerase chain reaction（MS-PCR） and immunohistochemistry, respectively, in specimens of 129 pterygium and 16 normal conjunctivas. The results were statistically analyzed. Hypermethylation of the p16INK4a gene promoter was detected in 21 (16.3%) of 129 pterygial specimens. Among them, 46 (35.7%) were positive for p16INK4a protein expression. Staining for p16 was limited to the nuclei of the epithelial layer. We observed a significant reverse correlation between hypermethylation of the p16INK4a promoter and the expression of p16INK4a protein (p=0.006). Thirty-eight (29.5%) pterygial specimens were positive for DNMT3b protein expression. DNMT3b staining was limited to the nuclei of the epithelial layer. A significant correlation was found between hypermethylation of the p16INK4a promoter and the expression of DNMT3b protein (p=0.001). CONCLUSIONS: The p16INK4a gene promoter was hypermethylated in pterygium, and this hypermethylation was strongly linked to expression of the positive expression of DNMT3b protein and to the suppression of p16INK4a protein. These data provided molecular evidence that methylation occurs in pterygium and that it may play a role in their development.
Part II
A pterygium has long been considered as a degenerative condition. After p53 protein was found to be abnormally expressed in the epithelium, researchers suggested that a pterygium may be a tumor, but additional evidence is required to support this hypothesis. Epidemiological evidence suggests that UV irradiation plays an important role in pterygium pathogenesis. UV irradiation can produce a wide range of DNA damage. The base excision repair (BER) pathway is considered the most important pathway involved in the repair of radiation-induced DNA damage. Based on previous studies, single-nucleotide polymorphisms (SNPs) in 8-oxoguanine glycosylase-1 (OGG1), X-ray repair cross-complementing group-1 (XRCC1) and AP-endonuclease-1 (APE1) genes in the BER pathway have been found to affect the individual sensitivity to radiation exposure and induction of DNA damage. Therefore, we hypothesize that the genetic polymorphisms of these repair genes increase the risk of pterygium.
XRCC1, APE1 and hOGG1 polymorphisms were studied using fluorescence-labeled TaqMan® probes on 83 pterygium specimens and 206 normal controls. There was a significant difference between the case and control groups in the XRCC1 genotype (p=0.038) but not in hOGG1 (p=0.383) and APE1 (p=0.898). The odds ratio of the XRCC1 A/G polymorphism was 2.592 (95% CI=1.225-5.484, p=0.013) and the G/G polymorphism was 1.212 (95% CI=0.914-1.607), compared to the Arg/Arg wild-type genotype. Moreover, individuals who carried at least one C-allele (Arg/Gln and Gln/Gln) had a 1.710 fold increased risk of developing pterygium compared to those who carried the Arg/Arg wild type genotype (OR=1.710; 95% CI: 1.015-2.882, p=0.044). The hOGG1 and APE1 polymorphisms did not have an increased odds ratio compared with the wild type. In conclusion, XRCC1 (Arg399Gln) is correlated with pterygium and might become a potential marker for the prediction of pterygium susceptibility.

並列關鍵字

pterygium ； SNP ； p16 ； XRCC1 ； DNMT3b

參考文獻

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