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標題: | 視網膜失養症之基因流行病學及分子病理學暨其臨床應用 Genetic Epidemiology and Molecular Pathology of Retinal Dystrophies and the Clinical Applications |
作者: | Ta-Ching Chen 陳達慶 |
指導教授: | 胡芳蓉(Fung-Rong Hu) |
共同指導教授: | 陳沛隆(Pei-Lung Chen) |
關鍵字: | 視網膜失養症,遺傳性視網膜退化,次世代定序,基因流行病學,基因型-表型關聯性, Retinal dystrophy,Inherited retinal degeneration,Next-generation sequencing,Genetic epidemiology,Genotype-phenotype correlation, |
出版年 : | 2021 |
學位: | 博士 |
摘要: | 視網膜失養症,也被稱為遺傳性視網膜退化,是一群具有重大臨床與社經影響的疾病。它涵蓋了多種會造成進展性且不可逆視力喪失之視網膜退化疾病,據估計大約影響全台灣7000 – 8000位患者。過去由於複雜的疾病表現型及基因型,對此類疾病的精準分子診斷並不容易。近年隨著次世代基因定序技術的進步,可以提供快速準確且價格經濟的基因分析,在許多疾病領域都逐漸成為世界上基因診斷的主流,也大幅幫助了視網膜失養症的基因診斷。 為了對台灣本土視網膜失養症病患有更清楚的了解,與提升基因診斷的可近性與準確度,學生在指導教授胡芳蓉特聘教授與陳沛隆所長的支持下,從就讀博士班起,致力於建立以次世代基因定序為基礎之視網膜失養症診斷平台,並於2015年起開始「台灣視網膜失養症計畫」(Taiwan Inherited retinal degeneration Project, TIP)。在基因分析上,我們採取233個基因的次世代定序套組分析,可以達成57.1% 之診斷率,在312個家族中有178個家族得到基因確診。我們發現ABCA4 是台灣視網膜失養症最常見的致病基因,再來依序是EYS, USH2A, CYP4V2, RPGR, PRPF31, RP1L1, PROM1, CEP290, BEST1, RP1, 以及 GUCY2D。我們也從資料庫中發掘了數個台灣本土重要的致病熱點,包括ABCA4:c.1804C>T, CYP4V2:c.802-8_810delinsGC, 以及EYS:c6416G>A。並且在幾個本土常見的致病基因中,受ABCA4, RPGR, 以及PRPF31 基因變異而致病的家族傾向於有較早的發病年齡,而CYP4V2患者則傾向較晚發病。 有了上述「台灣視網膜失養症計畫」資料庫,我們得以繼續前行,探索台灣重要的視網膜失養症疾病,其基因型與表現型的關聯性,以及探索利用高效能病患專一性多能幹細胞培養,來體外重現病理機轉的相關研究。針對病患基因型與表現型的關聯性,我們發現「不同基因間的差異」以及「同一基因內不同變異位置之差異」都可能是病患預後評估的重要因子。以視網膜色素變性為例,我們發現典型視網膜色素變性的患者,大多有隨著年齡老化而視力喪失的趨勢;然而周邊血管型視網膜色素變性的患者,其中央視力隨著年齡變化的影響則較小;獨立性黃斑部病灶,無疑的,是重要的視力預後因子。而在幾個本土常見的視網膜色素變性致病基因中,受ABCA4 或 EYS 突變影響的患者,其視力預後比受 USH2A 或 PRPF31 突變影響的患者來的差。至於在ABCA4 突變相關的視網膜失養症中,我們發現,若將表現型視為一個連續的光譜,從最廣泛的視網膜色素變性,依序到錐狀-桿狀細胞失養症,散在型斯特格式症,以及影響範圍最侷限的中央型斯特格式症,當病患基因中包含越多的截斷變異或剪切位點突變時,病患會傾向產生較廣泛的表現型,反之則傾向產生較侷限的表現型。 為了體外模擬重現疾病模型,我們嘗試進行病患專一性多能幹細胞的培養,並以本土最常見的單一基因-單一表型視網膜失養症 – 由CYP4V2基因突變造成的Bietti 結晶性視網膜失養症為實驗目標。我們藉由實驗證實,從此疾病病患者體細胞培養分化出來的多能幹細胞與其衍生之色素上皮細胞,會表現細胞型態的不規則性,較差的細胞增殖能力,與較高的細胞凋亡比例。我們同時也利用組織工程的方式,透過以聚苯麩胺酸為基底的生物相容性支架,可以加速幹細胞分化成視網膜細胞之速率與神經成熟度,期望讓此培養系統更具效能,成為良好之體外實驗模型。 經過了數年的努力,我們對於視網膜失養症此類疾病,以及台灣本土的患者,在許多面向都有了更多的了解,包括: (1) 針對台灣本土患者的臨床表現、基因特徵與流行病學建立科學數據;(2) 針對台灣重要的疾病亞型,進一步了解基因型如何影響表現型,包括在視網膜色素變性患者中的視力預後變化,以及台灣最常見的致病基因 - ABCA4中,基因缺陷的程度如何影響臨床疾病的嚴重度;以及 (3) 成功建立本土 Bietti結晶性視網膜失養症患者之病患專一性多功能幹細胞株,並透過組織工程的協助,加速體外幹細胞培養視網膜相關組織的效率與神經分化成熟度,於體外重現其細胞病理生理學,提供後續研究與潛在治療的篩檢平台。 希望透過以上的結果,有助於在未來,對於視網膜失養症的患者,能更增進準確的臨床與基因分子診斷,更好的個人化治療區間評估,以及更健全的藥物開發研究平台。學生希望,這些我們所努力的目標,能確實幫助這些視網膜失養症患者達成精準醫療的目標,並提升患者醫療照護之品質。 Retinal dystrophies (inherited retinal degenerations, IRDs) are a group of important diseases with huge clinical and social impact. It consists of several severe retinal degenerative diseases which lead to a progressive, irreversible loss of vision. It is estimated that around 7,000 to 8,000 people are affected all around Taiwan. It was challenging to determine the specific disease-causing genes in individual patients because of their extensive genetic heterogeneity. In recent years, next-generation sequencing (NGS) technologies have been applied for genetic diagnosis. NGS allows for timely and cost-effective detection of disease-causing variants and has gradually become the mainstream technique for diagnosing patients with IRDs worldwide. To address a comprehensive genetic study for patients of IRDs in Taiwan, with the support of my mentors, Distinguished Professor Fung-Rong Hu and Associate Professor Pei-Lung Chen, my team members and I began to devote ourselves to launch Taiwan Inherited retinal degeneration Project (TIP) and setting up a probe capture-based NGS genetic test platform for IRDs. We designed a probe capture-based NGS approach targeting 233 IRD-related genes for genetic testing, which led to a diagnostic rate of 57.1% (178 of 312 families) for the cohort. ABCA4 was the most common disease-causing gene, followed by EYS, USH2A, CYP4V2, RPGR, PRPF31, RP1L1, PROM1, CEP290, BEST1, RP1, and GUCY2D. Several disease-causing hotspots were identified including ABCA4:c.1804C>T, CYP4V2:c.802-8_810delinsGC, and EYS:c6416G>A. Among the common disease-causing genes, patients affected by ABCA4, RPGR, and PRPF31 had an earlier age of onset, while patients affected by CYP4V2 had a relatively later age of onset. Accompanied with the establishment of TIP cohort, we can further step forward to explore the genotype-phenotype correlation of common retinal dystrophies in Taiwan, and to explore the pathophysiology via patient-derived iPSC with efficiently cultural platform. Regarding genotype-phenotype correlation, we found that inter-gene difference and intra-gene difference are both important in predicting prognosis of individual patients. For example, progressive loss of vision is often the rule of typical type retinitis pigmentosa (RP) while central vision was less influenced by aging in pericentral type RP. Isolated macular lesion was surely a negative parameter of VA. Among the four most common disease-causing genes of RP in Taiwan, patients with ABCA4 or EYS mutations had worse visual prognosis compared to patients with USH2A or PRPF31 mutations. As for the ABCA4-associated retinal dystrophies, those patients with more null/splicing variants tended to develop more extensive phenotype (eg. RP) while STGD1, especially central type STGD1, presented reverse trends when we hypothesized that the phenotypes are on a spectrum in the order of arRP, CRD, dispersed type STGD1, and central type STGD1. To simulate and explore the pathophysiology of genetically defective retinal cells, we tried to generate the patient-derived iPSCs from patients of CYP4V2-associated Bietti crystalline dystrophy (BCD), the most common single-gene IRD in Taiwan. BCD iPSC-derived RPE cells demonstrated cell irregularity, decreased proliferation, and increased apoptosis. Polybenzyl glutamate (PBG)-based biocompatible scaffolds can further promote the efficiency of retinal differentiation from iPSCs. The BCD iPSC-derived retinal cells could keep the original genotype and, potentially, a good disease model in vitro. With these efforts, we now get a better understanding of IRDs in Taiwan in several ways including (1) the clinical presentation, genetic characteristics and epidemiology of inherited retinal degeneration in Taiwan; (2) how genotypes may influence phenotypes and the genotype-phenotype correlation of common retinal dystrophies in Taiwan such as the preservation of vision in different morphological types of RP and ABCA4-assoiated retinal dystrophies; and (3) successfully simulating the pathophysiology via BCD patient-derived iPSCs and accelerating the maturation of iPSC-derived retinal neurons via bioengineering approaches. Hopefully, these results would facilitate the precise clinical and molecular diagnosis of the patients, a better prediction of the therapeutic windows for individual patients, and a better platform to the development of novel therapies. It should be important for achieving precision medicine of these patients. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8067 |
DOI: | 10.6342/NTU202100781 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2026-03-01 |
顯示於系所單位: | 臨床醫學研究所 |
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U0001-0903202119000700.pdf 此日期後於網路公開 2026-03-01 | 16.37 MB | Adobe PDF |
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