研究背景：視網膜色素變性是遺傳性和異構性視網膜退化疾病的最普遍的群體之一，臨床常見表現為進行性視力減退，可在中青年階段即導致完全失明。全球大多數種族發病率在1：4000。視網膜色素變性的遺傳方式可以是常染色體顯性遺傳，常染色體隱性遺傳和性連鎖。此外，約有40的視網膜色素患者被列為散發性，因為他們未有明確的家族遺傳背景。到今天為止，已發現有63個與視網膜色素變性有關的染色體位點，其中23個常染色顯性遺傳視網膜色素變性基因，36個常染色隱性遺傳視網膜色素變性基因和2個性連鎖遺傳視網膜變性的基因被確定。然而在所有這些基因或位點中，沒有一個單一的基因或突變导致超过10的視網膜色素變性病例。 / 研究目的：在這項研究中，我們在所建立的視網膜色素變性致病基因網絡的基礎上，研究其中6個重要的基因，我們在中國漢族人群中進行此項研究的主要目標為：（1）描繪這些治病基因在中國漢族視網膜色素變性人群中的突變概況和貢獻; （2）狩獵新的功能性突變，揭示已知的視網膜色素變性基因中可能潛在的未知途徑;（3）測試一種新的假說，即某些基因變異可能是視網膜色素變性－經典傳統單基因疾病－易感性因素。此外，我們還對臨床評估的結果進行了定量分析，以探討這些特征性的臨床指標與疾病的嚴重程度和進展之間的關系。 / 研究對象：本研究包括了172名單純型中國漢族視網膜色素變性患者（平均年齡：44.9±18.1歲）這些患者中包含各種遺傳方式，他們的家庭成員如果有需要和願意參加也被邀請加入這項研究。對照組分別為180名彼此無任何血源關係的中國漢族受試者（平均年齡：73.1±10.1歲），除老年性白內障外未罹患其他任何主要的眼疾。所有研究對象均在香港眼科醫院或香港威爾斯親王醫院眼科及視覺科學系被招募。 / 研究方法：覆蓋RP1，RHO，NR2E3，NRL和BEST1 基因的所有外顯子部分和外顯子內含子邊界部位的基因序列由Sanger測序方法進行測序分析。同時被測序分析的還有SNRNP200基因包括兩個功能域部分的外顯子區域12-16，22-32和38-45。基於網絡分析的計算機程序：PolyPhen，SIFT，PMUT，SAP，PolyPhen-2和SIFT-Blink，被用來預測錯義變異對蛋白質功能的潛在影響。 ScanProsite，SMART和NetGene2等程序被用來研究蛋白質的功能片段和預測編碼外顯子和剪接位點。由CLUSTAL-W多重序列比對程序對進化保守性的變種進行關聯分析。SPSS 15.0平臺被用來檢測次要等位基因頻率（MAF）> 5的單核苷酸多態性的相關性分析 。Haploview的軟件測試被用來糾正在多重比較的p值。此外，我們收集了31例臨床資料完整的病例，使用計算機軟件後期處理視網膜圖像量化生成視網膜中央動脈和靜脈等值（視網膜中央動脈直徑及視網膜中央靜脈直徑）。使用ImageJ軟件數字化掃描分析自動視野計檢查結果，以作為臨床參數和評測標準對視網膜色素變性的嚴重程度進行定量分析。並對受試病例的視力，視野，視網膜中央動脈和靜脈等值進行了回顧性橫斷面研究分析。同時也對標準化和延時暗適應，視網膜電圖進行了描述性分析 。 / 結果：在6個基因中共有15例視網膜色素變性患者發現13個候選致病突變，占我們中國人群視網膜色素患者的8.2（15/172）。它們分別是：RP1的p.D984G p.R1652L，p.R1370E，p.R667X，p.S2RfxX16和p.P1648SfsX13; RHO的p.P327HfsX32和p.P347L; NR2E3 p.V118M和p.G56R; NRL的p.R202W ; SNRNP200 的p.C502R，和p.R1779H的。兩個RHO突變，p.P327HfsX32和p.P347L以及SNRNP200 p.C502R在功能上可能直接導致結構蛋白缺陷。而與之相反的， SNRNP200中發現的突變p.R1779H 和NR2E3 中的p.V118M，在發病機制中可能發揮間接作用。在這項研究中，首次確定的突變NRL p.R202W，是目前為止唯一位於該基因的TF-bZIP功能區域的突變，可能通過減少相關轉錄因子的活性而導致疾病而有異於其他所有NRL突變致病機理的功能假說。並且這個突變存在於常染色體隱性遺傳模式，也由此引導出與視網膜色素變性發病相關的潛在的致病新途徑。我們還進行了在這個六個基因中發現的20個的單核苷酸多態性與視網膜色素變性發病的關聯研究。其中四個：RP1的rs2293869（c.2953A>T）和ENSSNP13481334（c.6098G>A），BEST1的rs17156609（c.18151356 G> A） 和SNRNP200的rs772175（c.5317C> T），這種關聯關係表現為輕度統計學差異（p值分別為：0.033，0.02，0.032，0.02）。值得註意的是rs772175在顯性模式的矯正p值為0.025，比值比為2.05（95置信區間：1.31-3.21），使得這個的單核苷酸多態性的攜帶者在我們中國漢族人群中有增加視網膜色素變性易感性的傾向。同時我們發現在這組視網膜色素變性患者中，視力和視野的逐步下降呈現年齡依賴性。退化性黃斑病變，周邊退行性改變和視網膜炎和白點狀視網膜炎（RPA）在一些個例中也被觀察到。暗適應能力下降和視網膜電圖異常存在於所有年齡段。視網膜血管測量顯示視野每減少100平方厘米，視網膜中央動脈直徑和視網膜中央靜脈直徑分別降低12.2μm和23.7μm（p值均小於0.05）。 / 結論：在這項研究中，我們首先基於蛋白質相互作用的分析建立了一個視網膜色素變性基因網絡圖譜。這個基因網絡顯示在已知的視網膜色素變性基因有潛在的致病新途徑。六個基因中所發現的致病突變在我們中國漢族視網膜色素變性患者中所占的比例小於10， 表明視網膜色素變性在我們中國漢族人群中需要進一步的基因分析。首次發現的突變NRL p.R202W的表明在視網膜色素變性潛在可能的新致病途徑。本研究也同時提供了在SNRNP200和視網膜色素變性之間存在重要易感關聯的第一證據，提出一個新概念，即視網膜色素變性可能也存在遺傳易感基因而非僅僅只有傳統的致病基因。臨床數據的定量評估顯示，在所研究的患者的視野缺損的嚴重程度的視網膜中央動脈和視網膜中央靜脈管徑的縮小密切相關，他們是潛在有用的臨床指標以監測視網膜色素變性的進展和今後的治療反應。已知的視網膜色素變性基因的進一步研究應該有助於我們了解更全面的疾病的分子基礎，並進一步制訂治療的策略。 / Background: Retinitis pigmentosa (RP) is one of the most prevalent and heterogeneous groups of hereditary retinal degenerative diseases present with progressive vision loss and likely total blindness in the middle stage of life. It affects about 1 in 4000 people in most ethnic groups worldwide. Transmission of RP can be autosomal dominant (adRP), autosomal recessive (arRP) and X-linked (xlRP). Moreover, in most important studies about 40% of RP patients are classified as simplex or sporadic RP since they show uncertain familial background. To date, 63 chromosomal loci are mapped for RP, in which 23 adRP, 36 arRP and 2 xlRP genes have been identified. However, no single gene/mutation alone accounts for more than 10% of unrelated RP cases. / Purpose: In this study, we investigated six important genes selected on the basis of a RP gene network which we proposed in our Chinese cohort, our major objectives includes: depicting the mutation profiles and contributions of these genes in Chinese RP; hunting for novel functional mutations which may reveal potentially novel pathway in known RP genes; using SNP association studies to identify any SNP as bio-markers to the genes that are predisposing individuals to RP, which is conventionally regarded as a monogenic disease caused by single mutations. Moreover, we have performed quantitative clinical assessments to explore the presence of putative clinical markers and their respective relationships with the disease severity and progression of RP. / Study subjects: This study included a Chinese Han cohort of 172 non-syndromic RP patients (mean age: 44.9±18.1 years) with mixed inherited patterns and their family members who continued to participate in the study The control subjects were 180 unrelated Chinese subjects (mean age: 73.1±10.1 years) who are free of major eye diseases except for senile cataract. All study subjects were recruited at the Hong Kong Eye Hospital or the Department of Ophthalmology and Visual Sciences of the Prince of Wales Hospital, Hong Kong, China. / Methods: Genomic DNA was analyzed for the gene sequences covering all coding exons and exon-intron boundaries of the RP1, RHO, NR2E3, BEST1 and NRL genes by the Sanger sequencing strategy. Also sequenced were exons 12-16, 22-32, and 38-45, which cover the two functional domains, of the SNRNP200 gene. Web-based computer programs: PolyPhen, SIFT, PMUT, SAP, PolyPhen-2 and SIFT Blink, were used to predict the potential impact of the missense variants on protein functions. ScanProsite, SMART and NetGene2 were used to study the functional motifs sites and predict coding exons and splicing sites. The evolutionary conservation of the variants was analyzed by the CLUSTAL-W multiple sequence alignment program. Association analysis was performed, by using the SPSS 15.0 platform, among the detected SNPs with a minor allele frequency (MAF) > 5%. A permutation test in the Haploview software was used to correct the p-values in multiple comparisons. Moreover, we post-processed the retinal images using computer-based software to generate the central retinal artery and vein equivalents (CRAE and CRVE). The automated perimetry results were digitally scanned and quantitative analysis conducted using the ImageJ software as a clinical parameter to indicate the severity of RP. For the clinical characterizations 31 patients with complete clinical information were investigated by a retrospective cross-sectional study using parameters of visual acuity (VA), visual fields (VFs), CRAE and CRVE. Standardized dark adaptation and full-field electroretinograms (ERGs) were also analyzed and prolonged dark adaptometry were performed. / Results: A RP gene network was constructed. Five representative genes (RHO, NR2E3, NRL, SNRNP200 and BEST1) and the traditional important RP gene RP1 were selected for gene analysis. A total of 13 candidate disease-causing mutations in 15 patients was identified among the 6 genes, accounting for 8.2% (15/172) of overall RP in our Chinese cohort. They were p.D984G, p.R1652L, p.R1370E, p.R667X, p.S2RfxX16 and p.P1648SfsX13 of RP1; p.P327HfsX32 and p.P347L of RHO; p.V118M and p.G56R of NR2E3; p.R202W of NRL; p.C502R, and p.R1779H of SNRNP200. The two RHO mutations, p.P327HfsX32 and p.P347L, and SNRNP200 p.C502R were predicted to cause a direct structural protein defect. While p.V118M in NR2E3 and p.R1779H in SNRNP200, which were first identified in this study, on the contrast, may play an indirect role in the pathogenesis. The NRL p.R202W, also first detected in this study, is the only mutation so far located in the TF-bZIP module of this gene and predicted to have reduced transcriptional activity. It may contribute to recessive transmission and indicate a potentially new pathway associated with RP. We also performed an association study of RP with 20 SNPs of these genes. Four of them: rs2293869 (c.2953A>T) and ENSSNP13481334 (c.6098G>A) in RP1, rs17156609 (c.1815+1356G>A) in BEST1, and rs772175 (c.5317C>T) in SNRNP200 showed mild statistical difference (p-value: 0.033, 0.02, 0.032, 0.02, respectively). Notably, rs772175 showed a permutation p-value of 0.025, with an odds ratio of 2.05 (95% confidence interval: 1.31-3.21) in a dominant model, conferring an increased association to RP. Progressive decline of visual acuity and visual field were age-dependent in these patients. Degenerative maculopathy, peripheral degenerative changes and retinitis punctata albescens (RPA) were present. Reduced dark adaptation and affected ERGs were present in all ages. Retinal vessel measurement shows CRAE and CRVE decreased by -12.2μm and -23.7μm, respectively (both p<0.05) for each 100 cm² decrease in visual field. / Conclusions: Based on the proteinprotein interaction profiling a RP gene network was generated and it suggests potentially new pathways of known RP genes. The six genes selected altogether only account for <10% of Chinese RP, indicating the need of further gene analysis for this disease. The identification of NRL p.R202W may indicate a potentially new pathway in RP. The current study provided the first evidence of significant association between SNRNP200 and RP, hinting a new concept that RP may have association SNPs, apart from conventional disease-causing mutations. Quantitatively clinical data assessment revealed narrowing of CRAE and CRVE which were correlated with the severity of visual field loss in the studied patients. They are potentially useful clinical markers for monitoring progression of RP and future treatment response. Out finding throw light on the disease mechanism and susceptibility of RP. Continuing the intensive investigations of genes in the RP Gene Network highlighting our study strategies should lead us into deeper understanding for RP and providing information for therapies. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Xin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 173-193). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 中文摘要 --- p.iv / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xiii / List of Abbreviations (alphabetical order) --- p.xv / Publications --- p.xviii / ACKNOWLEDGEMENTS --- p.xix / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Retinitis Pigmentosa --- p.1 / Chapter 1.1.1 --- Clinical presentations, pathology and treatment --- p.1 / Chapter 1.1.2 --- Epidemiology --- p.4 / Chapter 1.1.3 --- Histopathologic changes in retinitis pigmentosa --- p.5 / Chapter 1.2 --- Molecular Genetics of Retinitis Pigmentosa --- p.6 / Chapter 1.2.1 --- Inherited patterns --- p.6 / Chapter 1.2.2 --- RP genes --- p.9 / Chapter 1.2.2.1 --- Causative genes and their relative contributions --- p.9 / Chapter 1.2.2.2 --- Ethnic and regional differentiation in heredity in gene contributions --- p.18 / Chapter 1.2.2.3 --- Classification of RP genes based on functional involvements --- p.24 / Chapter 1.3 --- Genetics Research Strategies --- p.30 / Chapter 1.3.1 --- Re-sequencing of known RP genes --- p.32 / Chapter 1.3.2 --- Identify new genes --- p.33 / Chapter 1.4 --- RP Genetic Studies in Chinese --- p.37 / Chapter 1.5 --- Genetic Background --- p.38 / Chapter 1.5.1 --- Strategies for sequence-tagged-gene selection --- p.38 / Chapter 1.5.2 --- rhodopsin (RHO) --- p.39 / Chapter 1.5.3 --- retinitis pigmentosa 1(RP1) --- p.43 / Chapter 1.5.4 --- nuclear receptor subfamily 2, group E, member 3 (NR2E3) --- p.47 / Chapter 1.5.5 --- neural retina leucine zipper (NRL) --- p.48 / Chapter 1.5.6 --- small nuclear ribonucleoprotein 200kDa (U5) (SNRNP200) --- p.50 / Chapter 1.5.7 --- bestrophin 1 (BEST1) --- p.51 / Chapter 1.6 --- Phenotypic Variabilities and Genotype-phenotype Correlations --- p.54 / Chapter 1.7 --- Objectives --- p.56 / Chapter Chapter 2 --- METHODS --- p.58 / Chapter 2.1 --- Study Subjects Recruitment --- p.58 / Chapter 2.2 --- Total Genomic DNA Extraction in Study Subjects --- p.60 / Chapter 2.3 --- Mutational Screening --- p.61 / Chapter 2.4 --- Statistical Methods --- p.68 / Chapter 2.4.1 --- Pairwise linkage disequilibrium test and Haplotype analysis --- p.68 / Chapter 2.4.2 --- Multivariable logistic regression analysis --- p.69 / Chapter 2.5 --- Variants Analysis Criteria --- p.70 / Chapter 2.6 --- Web-based in slico Bio-informatics Analysis Programs --- p.70 / Chapter 2.6.1 --- Web-based Gene Network Central[superscript TM] --- p.70 / Chapter 2.6.2 --- Polymorphism Phenotyping --- p.71 / Chapter 2.6.3 --- Sorting Intolerant From Tolerant --- p.72 / Chapter 2.6.4 --- The PMUT program --- p.73 / Chapter 2.6.5 --- Grantham Score --- p.74 / Chapter 2.6.6 --- PolyPhen-2 --- p.74 / Chapter 2.6.7 --- SIFT BLink --- p.75 / Chapter 2.6.8 --- Evolutionary conservation evaluation --- p.76 / Chapter 2.7 --- Retrospective Cross-sectional Clinical Investigations --- p.77 / Chapter 2.7.1 --- Patients and clinical materials --- p.77 / Chapter 2.7.2 --- Computer-assisted measurement of retinal vessel caliber --- p.77 / Chapter 2.7.3 --- Quantification assessment of visual fields --- p.78 / Chapter CHAPTER 3 --- RESULTS --- p.80 / Chapter 3.1 --- Study Subjects Demographics --- p.80 / Chapter 3.2 --- The RP Gene Network --- p.83 / Chapter 3.3 --- The RHO Gene --- p.85 / Chapter 3.3.1 --- Variants detected in the RHO gene --- p.85 / Chapter 3.3.3 --- Further investigations of those RHO mutations carrier families --- p.90 / Chapter 3.4 --- The RP1 Gene --- p.93 / Chapter 3.4.1 --- Variants detected in the RP1 gene --- p.93 / Chapter 3.4.2 --- Pathogenicity assessment --- p.95 / Chapter 3.4.3 --- The evolutionary conservation of RP1 --- p.98 / Chapter 3.4.4 --- Further investigations of those RP1 mutations carrier families --- p.98 / Chapter 3.4.4.1 --- Further investigations of p.R677X mutation family --- p.98 / Chapter 3.4.4.2 --- Further investigations of p.D984G mutation family --- p.101 / Chapter 3.4.4.3 --- Further investigations of those two novel frameshift mutations family --- p.104 / Chapter 3.4.4.3.1 --- Clinical manifestations of the individuals in the pedigree --- p.104 / Chapter 3.4.4.3.2 --- Mutation screening results of the individuals in the pedigree --- p.107 / Chapter 3.5 --- The NR2E3 Gene --- p.107 / Chapter 3.5.1 --- Variants detected in the NR2E3 gene --- p.107 / Chapter 3.5.2 --- Pathogenicity assessment --- p.109 / Chapter 3.5.3 --- The evolutionary conservation of NR2E3 --- p.112 / Chapter 3.5.4 --- Further investigations of novel p.V118M mutation family --- p.114 / Chapter 3.5.5 --- NR2E3 p.E121K in Chinese population --- p.114 / Chapter 3.6 --- The NRL Gene --- p.118 / Chapter 3.6.1 --- Variants detected in the NRL gene --- p.118 / Chapter 3.6.2 --- Pathogenicity assessment --- p.118 / Chapter 3.6.3 --- Further investigations of novel mutation p.R202W family --- p.122 / Chapter 3.7 --- The SNRNP200 Gene --- p.126 / Chapter 3.7.1 --- Variants detected in the SNRNP200 gene --- p.126 / Chapter 3.7.2 --- Pathogenicity assessment --- p.128 / Chapter 3.7.3 --- The evolutionary conservation of SNRNP200 --- p.131 / Chapter 3.8 --- The BEST1 Gene --- p.131 / Chapter 3.8.1 --- Variants detected in the BEST1 gene --- p.131 / Chapter 3.8.2 --- Pathogenicity assessment --- p.134 / Chapter 3.9 --- Association Study Between Selected SNPs and RP Among 6 Genes --- p.134 / Chapter 3.10 --- Clinical Features --- p.139 / Chapter 3.10.1 --- Clinical characteristics of study subjects --- p.139 / Chapter 3.10.2 --- Quantitative assessment of retinal arteriolar and venular calibers with visual field defect --- p.147 / Chapter CHAPTER 4 --- DISCUSSION --- p.152 / Chapter 4.1 --- RP in Hong Kong and Patient Recuritment --- p.152 / Chapter 4.2 --- Genes in the RP Gene Network --- p.152 / Chapter 4.3 --- RHO and RP --- p.154 / Chapter 4.4 --- RP1 and RP --- p.155 / Chapter 4.5 --- NR2E3 and RP --- p.157 / Chapter 4.6 --- NRL and RP --- p.160 / Chapter 4.7 --- SNRNP200 and RP --- p.161 / Chapter 4.8 --- BEST1 and RP --- p.164 / Chapter 4.9 --- Identifying Association SNPs vs Disease Causing Mutations in RP --- p.165 / Chapter 4.10 --- Perspectives Quantitative Clinical Assessment for Monitoring Progression of RP or Potential Response of RP therapy --- p.167 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.171 / References --- p.173