高度近視的基因及表徵基因學研究

Genetic and Epigenetic Study of High Myopia

眭致遠(Edward Hsi)

指導教授 ： 卓夙航

共同指導教授 ： 余明隆(Ming-Lung Yu)

高雄醫學大學/醫學院/醫學研究所/博士(2014年)

摘要

背景：
近視是全球最常見的眼睛疾病，亞洲國家的盛行率高於世界各國，台灣高中三年級的學生有85%有近視。基因遺傳以及後天環境都是造成近視的成因。儘管在基因關連性研究中已發現許多近視易感基因，但缺乏後續的功能研究且在不同種族間的再現性問題，目前並沒有一個公認的近視基因。因此，我們整合基因關聯性研究、基因功能測定及動物模型來評估潛在的近視基因，FGF10。另一部份，表徵遺傳學是在不改變DNA序列下調節基因表現的一些基因修飾，這些修飾是會因應環境壓力改變且可遺傳給子代。我們探討DNA甲基化的程度，表徵遺傳學的其中一種修飾，與近視的關連性。
方法：
在第一項研究中，我們以引發近視小鼠當中引發近視的眼睛，與其對側眼和沒有引發近視的正常實驗小鼠眼睛三類眼睛中比較FGF10基因的表現量。並收集1,020個高度近視（近視度數大於六百度）和960個對照組（近視度數小於150度）受試者進行基因分型實驗，研究8個FGF10的tagSNPs與近視的關連性。並接續分別建立包含兩個不同allele的luciferase reporter，評估allele間基因表現量的差異。
在第二項研究中，長重複序列1（LINE-1）的甲基化程度被視為全域DNA甲基化的一個替代指標。本研究以300個高度近視和300個性別、年齡匹配的對照組受試者，尋找高度近視和LINE-1甲基化的關連性。並探討血中白細胞，視網膜和鞏膜組織中LINE-1甲基化程度在引發近視小鼠與正常小鼠兩組間的差異。此外，同型半胱氨酸和蛋氨酸是體內DNA甲基化過程的重要氨基酸，我們在FDM小鼠的眼睛觀察這兩個氨基酸的表現程度與LINE-1甲基化程度是否相關。最後，我們在培養的視網膜細胞外加多巴胺，以模擬戶外明亮環境當中的生理現象，觀察能否改變細胞中的甲基化程度。
結果：
在第一個研究中，引發近視小鼠眼睛的鞏膜組織內，FGF10的mRNA比對側眼高2.57倍（P = 0.018）。在關連研究中，SNP rs339501與極端近視有顯著關連（P = 0.008），帶有G allele的人罹患極端近視的機會是A allele人的1.58倍。同時在luciferase reporter assay中也顯示，高風險G allele的基因表現量顯著比A allele的基因表現量高（P = 0.011）。
在第二項研究中，若將受試者的LINE-1甲基化分為高中低三個程度，高度及中度甲基化的受試者，近視的風險是低度甲基化的2倍（P≤0.002）。在引發近視的小鼠中，白細胞、視網膜和鞏膜的LINE-1甲基化程度比沒有引發近視的小鼠高。且在免疫組織染色顯示，引發近視小鼠的眼睛中有較高的半胱氨酸和蛋氨酸含量。最後，全域甲基化程度在多巴胺刺激的在24小時後顯著降低2％。
結論：
FGF10基因是近視的危險因子。近視眼睛的鞏膜中有較高的FGF10表現。 FGF10 SNP rs339501的危險G allele與超過1,000度的極端近視有關連，且會造成較高的FGF10表現量。
LINE-1的甲基化程度增加與高度近視有關連。在動物實驗中，誘發近視後眼睛組織內全域甲基化及累積的同型半胱氨酸都提高。最後在細胞接受多巴胺後減少了全域甲基化程度，顯示與引發近視造成相反的改變。

關鍵字

近視 ； SNP ； 表徵基因學 ； FGF10 ； LINE-1

並列摘要

Purpose:
Myopia is a common eye condition worldwide, and its prevalence varies widely among populations and ages. Both genetic and environmental factors are important for the development of myopia. Although several susceptibility genes to myopia were identified by genetic association study, no gene was concluded as myopia gene. Therefore, we conducted a study integrated genetic association, genetic functional assay and animal model to evaluate a potential myopia gene, FGF10. In the second part, we conducted a global DNA methylation study. DNA methylation is an epigenetic process that modulates gene expression without changing DNA sequence and is known to be altered by environmental factors. We aimed to discover the association and mechanism between global DNA methylation and high myopia.
Methods:
In the first study, the expression levels of FGF10 gene were compared among the form deprivation myopic (FDM) eyes, the fellow eyes of the FDM group and the normal eyes of experimental mice. In the present study 1,020 cases (≤-6.0 D) and 960 controls (≥-1.5 D) were enrolled from a Chinese population. Eight tagging SNPs were genotyped to test for an association between genotypes and myopia. The luciferase reporter assay was conducted for the particular SNP to assess the allelic effect on gene expression.
In the second study, the methylated levels of long interspersed nucleotide elements (LINE-1) are regarded as a surrogate marker of global DNA methylation. The present study included 300 highly myopic and 300 sex- and age-matched control subjects to find the association between high myopia and LINE-1 methylation in leukocytes. The form deprivation myopia (FDM) mice were used to further explore LINE-1 methylation among leukocytes, retina and sclera tissues. Homocysteine and methionine that are related to methylation process were also measured in animal eyes. We use dopamine to mimic brighter environment to rescue the myopia state.
Results:
In the first study, the sclera of FDM eyes had a 2.57-fold higher level of FGF10 mRNA (p=0.018) than the fellow eyes. Although no SNP was associated with high myopia, SNP rs339501 was significantly associated with extreme myopia (≤-10D, p=0.008) and the OR was 1.58 for G allele carriers. The luciferase assay showed that the risk G allele significantly caused a higher expression level than the A allele (P = 0.011).
In the second study, subjects with high and middle tertile of LINE-1 methylation had approximately 2-fold increase of myopia risk (p≤0.002). In the mouse model, a significant increase of LINE-1 methylated level was found in leukocytes, retina and sclera in the myopic animals. Immunohistochemical staining indicates elevated homocysteine and methionine levels in the eyes of myopic animals. Finally, the global methylated level was reduced 2% significantly after 24 hours of dopamine stimulation.
Conclusions:
The evidence suggested FGF10 to be a risk factor for myopia. The sclera of myopic eyes had higher FGF10 levels. The risk G allele of SNP rs339501 was associated with extreme myopia in human and caused a higher gene expression in the luciferase assay. It is concluded that the FGF10 could have been involved in the development of myopia.
The second study demonstrates that LINE-1 DNA hypermethylation is associated with high myopia. Global hypermethylation and accumulated homocysteine indicate a constitutional change which is induced by FDM. Reduced global methylated level is observed after treatment of dopamine may elucidate increased outdoor activity reduced the development of myopia.

並列關鍵字

myopia ； SNP ； epigenetic ； FGF10 ； LINE-1

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