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研究生： 李嘉怡
Li, Chia-Yi
論文名稱： 粒線體蛋白質輸入複合體中的錯義變異導致小頭畸形骨發育不全侏儒症伴隨毛毛樣疾病
A missense variant in mitochondrial protein import complex leads to microcephalic osteodysplastic dwarfism with moyamoya disease
指導教授： 陳芃潔
Cheng, Peng-Chieh
學位類別： 博士
Doctor
系所名稱： 醫學院 - 臨床醫學研究所
Institute of Clinical Medicine
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 169
中文關鍵詞： TOM 複合體 、iPSC 衍生的內皮細胞 、腦血管疾病 、粒線體蛋白質輸入 、代謝重編程
外文關鍵詞： TOM complex, Moyamoya disease, iPSC-derived endothelial cells, Mitochondrial protein import, Metabolic reprogramming
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    • 幾乎所有的粒線體蛋白質在細胞質中合成後，需要透過粒線體外膜上的蛋白質輸入複合體（TOM complex）進入到粒線體，其中 TOM7 為此複合體的重要調控蛋白。粒線體蛋白質運輸系統的缺陷則會導致表型異質性高度多樣的粒線體疾病。
    利用全外顯子定序，我們在來自七個無血緣關係的家庭中，共發現九名患者皆攜帶TOMM7 p.P29L變異。所有患者均表現出小頭症、身材矮小、面部畸形以及黃斑部病變。值得注意的是，其中有五位個案出現腦血管病變。目前已知內皮細胞中的粒線體功能缺失與毛毛樣腦血管病變（moyamoya disease）相關。然而，在臨床上還沒有研究提出TOMM7 p.P29L變異會導致腦血管病變，因此我們在此研究要確立TOMM7 變異與毛毛樣腦血管疾病的關聯及其在內皮細胞中對粒線體功能的影響。
    我們利用CRISPR/Cas9建立tomm7缺失斑馬魚，觀察到其呈現腦較小、顱面異常與腦血管缺失，與患者表徵相似。為探討TOMM7 p.P29L變異的致病機制，我們利用CRISPR/Cas9技術編輯在iPSCs中敲入TOMM7變異，並將其分化為血管內皮細胞（endothelial cells）。於CRISPR/Cas9編輯的TOMM7 p.P29L同型合子內皮細胞中，我們觀察到TOM7蛋白量上升、細胞衰老活性上升，以及血管形成效率減弱。粒線體呈現異常增大且趨於圓形型態。粒線體蛋白質體分析顯示，與ATP合成及代謝相關的粒線體蛋白質顯著減少。儘管粒線體呼吸功能略有下降，來自糖解作用的ATP產生顯著增加，轉錄體分析亦顯示糖解相關基因明顯上調，顯示出代謝重編程（metabolic reprogramming）的現象。為進一步驗證其致病性，我們將患者的外周血單核細胞（PBMCs）重編程為iPSCs，並分化為血管內皮細胞。在患者來源的血管內皮細胞中，我們觀察到粒線體異常增大、皺褶（cristae）結構缺失及粒線體活性降低，並伴隨糖解作用活性上升與HK2基因及蛋白質表現增加。透過CRISPR/Cas9編輯將TOMM7 p.P29L變異校正回野生型（wild-type），可恢復部分粒線體型態與皺褶結構，並提升線粒體呼吸功能及降低糖解作用活性。總結本研究結果，我們首次將 TOMM7 p.P29L 變異與人類腦血管病變建立關聯，並證實該變異會損害內皮細胞的粒線體功能，是導致小頭骨發育不良伴隨毛毛樣腦血管病的致病基因。

    The majority of mitochondrial proteins are produced in the cytosol and transported into mitochondria through the translocase of the outer membrane (TOM) complex, where TOM7 serves a regulatory role. Disruption of this import machinery can cause phenotypically heterogeneous mitochondrial disorders. In endothelial cells, mitochondrial dysfunction has been associated with the development of moyamoya disease.
    Whole-exome sequencing revealed a recurrent TOMM7 p.P29L variant in nine patients from seven unrelated Taiwanese families. These patients consistently exhibited microcephaly, growth retardation, craniofacial dysmorphism, and macular scarring. Notably, five patients developed moyamoya disease. However, the impact of TOMM7 p.P29L variant on mitochondrial function in endothelial cells remained unknown.
    Phenotypic analysis of tomm7 knockout zebrafish revealed microcephaly, craniofacial anomalities, and absent cerebral arteries, phenocopying some of patient’s clinical symptoms. To evaluate the causality of TOMM7, we introduced the TOMM7 p.P29L variant into induced pluripotent stem cells via CRISPR/Cas9 gene editing and differentiate them into endothelial cells (iEndos). CRISPR-edited TOMM7PL/PL iEndos exhibited elevated TOM7 protein levels, increased senescence, impaired angiogenic capacity and increased enlarged, rounded mitochondria. Mitochondrial proteomes revealed reductions in proteins involved in ETC complexes and metabolic pathways. Despite modest decreased of mitochondrial respiration, glycolytic ATP production and glycolysis-related gene expression were elevated, indicating metabolic reprogramming. Patient-derived endothelial cells (patient iEndos) showed the abnormally enlarged mitochondria with disrupted cristae, reduced respiration and elevated glycolytic activity. Importantly, CRISPR/Cas9-mediated correction of the TOMM7 p.P29L variant in patient iEndos restored mitochondrial morphology, improved cristae architecture, enhanced respiratory function, and normalized glycolytic flux.
    Together, our study is the first to link the TOMM7 variant to moyamoya disease and provides strong evidence that TOMM7 p.P29L is a pathogenic variant that impairs mitochondrial function in endothelial cells, contributing to microcephalic osteodysplastic dwarfism with moyamoya disease.

    中文摘要 i Abstract ii Acknowledgement iii Table of Contents v Lists of Appendices x Abbreviations xi Chapter 1. Introduction 1 1.1 Microcephalic osteodysplastic dwarfism with moyamoya disease 1 1.1.1 Clinical features and genetics of microcephalic primordial dwarfism 1 1.1.2 Moyamoya disease is linked to mitochondrial dysfunction in endothelial cells 2 1.2 Role of TOM7 in development and mitochondrial function 4 1.2.1 Mitochondrial protein import machinery 4 1.2.2 Mitochondrial protein import defects in human diseases 5 1.2.3 Mitochondrial quality control 6 1.2.4 Previous in vivo and in vitro studies of TOM7 8 Chapter 2. Material and Methods 10 Chapter 3. Results 25 3.1 A novel TOMM7 p.P29L variant in humans 25 3.1.1 Clinical presentations of four patients from three unrelated families 25 3.1.2 TOMM7 p.P29L variant identified by whole exome sequencing 26 3.1.3 Cerebrovascular phenotypes in humans with TOMM7 p.P29L variant 29 3.2 Phenotypic studies of tomm7∆/∆ zebrafish 30 3.2.1 Generating tomm7 knockout zebrafish by CRISPR/Cas9 genome editing 30 3.2.2 tomm7∆/∆ zebrafish recapitulate patient clinical features 30 3.2.3 Mitochondrial and transcriptomic profiles in tomm7∆/∆ zebrafish 31 3.3 Functional studies of TOMM7 p.P29L variant in CRISPR-edited endothelial cells 33 3.3.1 Gene editing of iPSCs and in vitro endothelial differentiation 33 3.3.2 Deleterious effects of TOM7P29L on endothelial and mitochondrial functions 34 3.3.3 Defective protein import and metabolic imbalance in TOMM7PL/PL iEndos 36 3.3.4 Bioenergetic and transcriptomic profiles in TOMM7PL/PL iPSCs 38 3.4. Phenotypic rescue by gene correction in TOMM7 p.P29L variant 41 3.4.1 Generation of isogenic controls from patient-derived iPSCs 41 3.4.2 Normalization of vascular networks in variant-corrected patient iEndos 42 3.4.3 Mitochondrial and metabolic recovery in variant-corrected patient iEndos 43 3.4.4 No cell-type specific effects of TOMM7 p.P29L on mitochondrial respiration 44 3.5 Summary of the study 46 Chapter 4. Discussion 48 4.1 Phenotypic variability in TOMM7-variant cells 48 4.2 Metabolic alterations in TOMM7-variant cells 49 4.3 Molecular functions of TOM7P29L protein 51 4.4 Clinical insights and future prospects 52 Chapter 5. References 55 Chapter 6. Figures 61 Chapter 7. Tables 144 Chapter 8. Appendices 150

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