色素性視網膜炎患者黃斑部斷層攝影術與視覺功能間相關性之研究及人類視紫質基因之分析

Study of Macular Tomography and its Correlation with Visual Function and Analysis of Human Rhodopsin Gene in Retinitis Pigmentosa

陳裕芳(Yuh-Fang Chen)

指導教授 ： 陳慕師 ； 錢宗良

共同指導教授 ： 林隆光 ； 曹友平 ； 王一中

國立臺灣大學/醫學院/臨床醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02954

摘要

色素性視網膜炎(retinitis pigmentosa)是一群以影響視網膜桿狀感光細胞(rod photoreceptor)為主的疾病，而隨著疾病的進行與惡化，錐狀感光細胞(cone photoreceptor)也將受到影響。雖然至今色素性視網膜炎還沒有確切有效的治療，但由於病患的生活品質和視覺功能息息相關，因此對此疾病的監控與預測就顯得十分重要。而視網膜中央黃斑部的良窳決定了大部分視覺功能的優劣。是故雖然黃斑部在色素性視網膜炎的病程上較晚期才會受影響，如何早期評估及診斷黃斑部型態及功能，對預測色素性視網膜炎病患的生活品質而言便成為一個重要的課題。另一方面，由於色素性視網膜炎是一種和基因缺陷相關的疾病，因此進一步分析探討其基因與遺傳變異才能裨益於未來的基因治療。至今約有一百種視紫質(rhodopsin)的基因突變已被發現和色素性視網膜炎有關，而且絕大多數是造成體染色體顯性遺傳色素性視網膜炎(ADRP)，而這一類和視紫質突變相關的ADRP又大多導因於視紫質蛋白的錯誤摺疊(misfolding)或在內質網(endoplasmic reticulum, ER)中的阻滯與堆積。然而在對色素性視網膜炎的研究上，仍然有許多待釐清的問題。首先，色素性視網膜炎患者黃斑部型態的變化與年紀以及視覺功能的相關性研究並不完整；其次，視紫質中不同的蛋白質結構域(domain)如何交互作用以形成視紫質蛋白，以及不同的突變如何影響視紫質蛋白的生合成，都還需要進一步的探討。因此，我們將整個研究分成兩大部分：第一部分為臨床研究部分，重點在分析色素性視網膜炎患者黃斑部斷層掃瞄檢查之型態變化和視覺功能的相關性；第二部分為實驗室基礎研究的部分，主旨在分析色素性視網膜炎患者的視紫質基因。

第一部分：臨床研究
研究目的：以光學同調斷層掃描技術(optical coherence tomography, OCT)分析色素性視網膜炎患者黃斑部厚度及型態與其視覺功能之相關性。
研究方法：我們羅致了75位色素性視網膜炎患者為實驗組與75位無病變人員為對照組進行研究。這兩組人員皆依不同年齡層分成三個組，並接受光學同調斷層掃描儀檢查其黃斑部，再將檢查結果依照三個環狀區域、四個象限、以及九個區域等不同區分方式來分析。眼部則接受完整且詳細的眼科檢查，包括：眼底檢查、暗適應測試(dark adaptation test)、視力檢查(visual acuities, VA)、視野檢查(visual field examination, VF)、眼電圖記錄(electrooculography, EOG)、以及色覺區辨力檢查(color-sense discrimination test)等。
研究結果：黃斑部厚度在中年齡層(45-55歲) 色素性視網膜炎患者最薄，而在年齡最長(55歲以上)的患者群黃斑部相對較厚。以黃斑部不同區域的比較顯示：外側下方區域厚度在不同年齡層並無差異，而中央凹(fovea)的厚度在不同年齡層有最顯著的差異。在視覺功能的相關性上則發現色素性視網膜炎患者中央凹的厚度和眼電圖、視力、以及辨色力有顯著相關。
結論：色素性視網膜炎患者黃斑部厚度在不同年齡層有顯著不同，且厚度變化和特定視覺功能相關。

第二部分：基礎研究
研究目的：藉由對視紫質中不同獨立片段在COS-1細胞以及基因轉殖斑馬魚(zebrafish)的桿狀感光細胞之包內分布與表現，評估其對視紫質蛋白摺疊以及蛋白離出內質網的影響。
研究方法：我們合成人類視紫質的互補去氧核醣核酸(cDNA)，並將之分成氮端結構域(N-terminal domain)、羧基端結構域(Carboxyl-terminal domain)以及介於氮端與羧基端結構域之間的片段。接著觀察這些不同片段在COS-1細胞以及斑馬魚桿狀感光細胞中的表現。我們又分別置入F45L和G51V點突變於氮端與羧基端結構域之間的片段中，並觀察該點突變在不同細胞中對蛋白表現的影響。
研究結果：在COS-1細胞中，除羧基端結構域和完整視紫質蛋白被觀察到有和細胞膜相同的分布外，其餘截斷的視紫質片段皆堆積於內質網內或於其周圍形成聚合物。而在斑馬魚桿狀感光細胞中，氮端結構域、羧基端結構域、和完整視紫質蛋白都可傳輸到細胞的外側段(outer segment)。此外在這兩種細胞內，F45L和G51V的點突變都增加了不同程度和型態的聚合物，並影響蛋白在胞內的傳輸與表現。
結論：本研究觀察到視紫質不同結構域以及視紫質突變對蛋白的細胞生化之影響。

色素性視網膜炎是工作年齡層患者一大致盲原因，我們希望藉由本研究能提供對該疾病更多的瞭解，以期對未來的基因治療有所助益。

關鍵字

色素性視網膜炎 ； 光學同調斷層掃描 ； 視野檢查 ； 眼電圖檢查 ； 色覺區辨力檢查 ； 視紫質 ； 斑馬魚

並列摘要

Retinitis pigmentosa (RP) refers to a group of genetic disorders of the retina that primarily affect the rod photoreceptors, and the cone photoreceptors can also be affected as the disease progresses. It is important to predict and monitor the progression of this disease because a patient’s quality of life is closely related to visual function. Although the macula is usually less affected and well-preserved until late-stage RP, it determines a vast majority of visual functions; hence, evaluating its structure and function is important for determining the quality of life for RP patients. On the other hand, since RP is a genetic disorder, clarifying its genetics is important to gene therapy in the future. More than 100 mutations of rhodopsin have been identified to be associated with RP, and mostly autosomal dominant RP (ADRP). The majority of rhodopsin-associated ADRP is caused by protein misfolding and ER retention. However, several questions still remain unanswered. First of all, macular tomographic change with age and its correlation with visual function in RP are not comprehensive. Secondly, how different domains of rhodopsin work together to accomplish the assembly process and how mutations of rhodopsin interrupt with this process are still unclear. As a consequence, we divided the whole study into two parts: part I was a clinical study which focused on the macular tomography and its correlation with visual function in RP; part II was a laboratory study which focused on the analysis of human rhodopsin gene in RP.

Part I: the clinical tudy
Purpose: The present study was designed to analyze macular tomography in patients of different ages with RP and correlate their visual function with macular thickness, which was measured by optical coherence tomography (OCT).
Materials and methods: 75 RP patients and 75 controls were stratified into three age groups and the macular thickness was measured by OCT. The tomography was subdivided into three circular zones, four quadrants, and nine areas for analysis. Ophthalmic examinations, which involved ophthalmoscopic examinations, dark adaptation tests, visual acuities (VA), visual field (VF) examinations, electrooculography (EOG) and color-sense discrimination tests were performed.
Results: Macular thickness of the RP patients decreased in the middle age group (45-55-year-old), whereas the oldest group showed an increased thickness. The thickness of the outer inferior area remained virtually unchanged, whereas the thickness of the inner temporal area showed the most fluctuation with age. In terms of circular sections, the most dramatic changes in macular thickness were observed in the fovea, and the aging effect decreased outwards to the outer ring. Furthermore, the thickness of the fovea was more important than the thickness of the inner ring and the outer ring for EOG, VA and color sense discrimination in RP patients.
Conclusion: In middle age RP patients, the macular thickness decreased, whereas an increased thickness was observed in patients older than 55 years. In addition, the inner temporal area was the most fragile, and the outer inferior area was the least affected in patients with RP.
Part II: the laboratory study
Purpose: In this study, we aimed to evaluate rhodopsin folding, exiting the ER and intracellular localization through expression of the rhodopsin fragments in COS-1 cells as well as in the transgenic zebrafish.
Materials and methods: We cloned human rhodopsin cDNA, which was then divided into the N-terminal domain, the C-terminal domain, and the fragment between the N- and C-terminal domains, and examined their intracellular expression in vitro and in vivo. We introduced a point mutation, either F45L or G51V, into this fragment and observed the intracellular localization of these mutants in COS-1 cells and in the zebrafish.
Results: It revealed all of the truncated rhodopsin fragments except for the C-terminal domain and the full-length rhodopsin which had some plasma membrane localization, formed aggregates nearby or within the ER in COS-1 cells; however, the N-terminally truncated rhodopsin fragment, the C-terminal domain, and the full-length rhodopsin could traffic to the ROS in the zebrafish. Besides, the F45L mutation and the G51Vmutation in the rhodopsin fragment between the N- and C-terminal domains produced different effects on the aggresome formation and the intracellular distribution of the mutants both in vivo and in vitro.
Conclusion: This current study provided new information about the mutant rhodopsin as well as the treatment of the RP in humans in the future.
Since RP is one of the leading causes of blindness in the working age all over the world, we believe that this study will contribute to better understanding of links between RP, its visual function changes and the associated genes. And, this will further contribute to genetic counseling and gene therapy in the future.

並列關鍵字

retinitis pigmentosa ； optical coherence tomography ； visual field ； electrooculography ； Farnsworth Munsell 100-Hue test ； rhodopsin ； zebrafish

參考文獻

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