腦及肌腱黃瘤病(cerebrotendinous xanthomatosis )簡稱CTX，是一種極為罕見的體細胞隱性脂類堆積家族遺傳疾病；常伴隨永久性神經症狀。最早於1937年由van Bogaert等人發現並提出病例報告。導致CTX的主要原因為人類第二對染色體上的CYP27基因出現突變，此基因產物是粒線體的一個水解酵素；可將膽固醇等脂類支鏈第27個碳加上一個氫基並進行代謝與分解。一旦此酵素喪失功能將使膽固醇無法順利轉化成膽酸排出反而形成大量膽固醇(cholesterol)與代謝中間產物膽醇(cholestanol)堆積在體內各種器官；特別是肌腱與神經系統因而造成CTX病人的臨床症狀，包括癡呆、幼年型白內障、黃瘤、小腦病變症候群、動脈硬化及各種神經系統障礙等等。在臨床診斷上主要可從血清中膽醇上升及肌腱黃瘤兩方面確認。CTX特別好發在某些族群；如日本人、葡萄牙或西班牙裔猶太人及義大利族裔。
透過高雄長庚神經內科張文能主任的幫助，本研究中收集到一個台灣CTX家族的樣本；其中包含三位具典型臨床症狀之CTX患者及其未有病徵應為突變基因攜帶者的父親。經病人及家長同意並由醫院相關單位核准後進行CTX疾病的基因與蛋白質體研究。本實驗計畫的第一部分即需釐清台灣CTX病患是否也有CYP27基因突變現象；希望能明確鑑定出此疾病的基因關連性並與其他地區或國家之CTX患者基因突變作比較，試圖尋找更多的基因線索。首先，CYP27基因上三端與五端臨接處(3’ and 5’-flanking region)與所有的八個Intron及九個Exon都以多聚合酶連鎖反應(PCR)自基因組去氧核醣核酸(genomic DNA)大量增幅後；再以最佳條件進行單股構型多樣性(SSCP)實驗篩選可能產生突變之片段。結果顯示CTX病患、突變基因攜帶者與正常人對照組除了第二個Exon的單股構型多樣性型式有明顯不同外，其他部份呈現完全相同的情形；由此可知突變極可能存在第二個Exon上。接下來，為了更進一步確認突變所在，利用直接去氧核醣核酸定序分析(direct DNA sequencer analysis)定出突變位置。果然在CTX病人第二個Exon上 326處的密碼子發現一個同質接合體突變(homozygous mutation)；一個cytosine的缺失在三個CTX患者樣本中皆存在。這個點突變導致原本第102個氨基酸從原本的proline變成leucine；形成所謂框架位移(frameshift)的情形因而造成第106個氨基酸valine被早發性終結密碼(premature termination codon)TGA取代。雖然只是一個點突變卻造成極為嚴重的後果，整個CYP27基因功能喪失而造成CTX症狀。
然而，因為各種後轉譯修飾作用(post-translational modification) ，基因分析有時無法忠實地呈現出複雜生物系統中相對應的功能性蛋白質表現。於是，蛋白質體學成為基因體學在表現蛋白質這部份的互補成份。近來蛋白質體分析已被廣泛應用在生命科學研究的各個領域，以基因及蛋白質角度同時分析較能完整顯現CTX的成因或可能的生理途徑。
本研究中的第二個部分，即以血清與白血球各部份的蛋白質圖譜檢視造成CTX症狀的可能病理機制。蛋白質體分析主要包含兩大技術：二維電泳(2-DE)及質譜分析；從CTX病人白血球蛋白質圖譜發現某些細胞骨架蛋白如：vinculin、talin、vimentin和ABP-280表現量皆明顯高出突變基因攜帶者與正常人對照組樣本很多；以上結果反應出大量膽醇堆積影響細胞膜動態改變因而造成細胞失常或死亡。將蛋白質圖譜分析鎖定的目標蛋白質以專一性抗體西方點墨法再次確認其表現量顯示與二維電泳結論相符。
本研究首次結合基因及蛋白質體學觀念分析CTX疾病可能的機制，希望可提供未來相關實驗更多有用的資訊。

Cerebrotendinous xanthomatosis (CTX), an autosomal recessive lipid-storage disorder with prominent neurological features, was first described by van Bogaert et al. in 1937. A deficiency of the mitochondrial sterol 27-hydroxylase due to mutations in the CYP27 gene (CYP27) blocks the oxidization of cholesterol side chain at the first step in the formation of bile acids. The accumulation of great amount of cholesterol and cholestanol in various tissues, especially in tendons and neural system, leads to the clinical symptoms including dementia, juvenile cataracts, xanthoma, cerebellar syndrome, atherosclerosis and a variety of neurological dysfunctions in CTX subjects. The diagnosis can be made by demonstrating elevated level of cholestanol in the serum and apprearance of xanthoma in tendon. There is a high prevalence of CTX in the Japanese, Sephardim Jewish and Italian populations.
Here in this investigation, a one-reported pedigree of three affected individuals with typical characteristics of CTX and a heterozygous paternal carrier in Taiwan were assembled. The first part of the project was to clarify the genetic causes of these CTX patients and to design a series of analytical tests for achieving rapid and correct confirmation of the diagnosis. First, 3’ and 5’-flanking region as well as all 8 introns and 9 exons fragments of CYP27 were amplified from genomic DNA by polymerase chain reaction (PCR) and followed by single strand conformation polymorphism (SSCP) under optimal conditions. The SSCP patterns were identical among CTX subjects, the carrier, and normal controls for all exons except exon 2, implying some kind of mutation may exist on it. Then, direct DNA sequencer analysis was performed on the suspected PCR fragment of exon 2. A new homozygous mutation of one base-pair deletion of cytosine at codon 326 on exon 2 was found in all three CTX subjects in this family. This novel point deletion of cytosine at Pro102 (CCT) would cause a frameshift in mRNA (Pro102 →Leu) and result in the appearance of a premature termination condon (TGA) to substitute for Val106(GTG). This severe mistake would cause the breakdown in the normal function of sterol 27-hydroxylase and lead to CTX.
However, gene analysis could not represent the corresponding functional proteins under various post-translational modifications in complex biological systems. Proteome is the set of expressed protein complement of a genome and proteomic analysis has been widely used in studies of life sciences.
The second part of this study is to characterize the pathological mechanism of CTX patients with serum protein profiles and leukocytes isolated from CTX subjects by means of proteomic technologies, including two-dimensional electrophoresis (2-DE) and MALDI-TOF analysis. The results showed that the amount of vinculin, ABP-280, talin and vimentin in leukocytes of CTX patients increased significantly, reflecting the changes in membrane dynamics concerning cholestanol accumulation. The expression of target proteins in CTX patients and control was further confirmed by Western blotting with specific antisera which indicated the same tendency as 2-DE data.
This is the first report to integrate both genomic and proteomic concepts for analyzing the possible mechanisms of CTX and information provided by this report should be very helpful for the future studies on CTX.

Table of Contents
序…………………………………………………………………………………1
Abstract……………………………………………………………………………2
中文摘要…………………………………………………………………………5
Table of Contents…………………………………………………………………8
List of Tables……………………………………………………………………10
List of Figures……………………………………………………………………11
Chapter 1. Introduction and Literature Review…………………………………12
1-1 Cerebrotendinous xanthomatosis: clinical
and biochemical features……………………………………………12
1-2 Sterol 27-hydroxylase (CYP27) gene………………………………15
1-3 Metablism of cholesterol and cholestanol…………………………18
1-4 Biomedical applications of proteomic………………………………21
1-5 Bioinformatics………………………………………………………23
Chapter 2. Motive and Research Objective……………………………………25
2-1 Motive…………………………………………………………………25
2-2 Research objectives……………………………………………………27
Chapter 3. Materials and Methods………………………………………………28
3-1 Patients…………………………………………………………………28
3-2 Biochemical analysis……………………………………………………29
3-3 Genomic DNA preparation and PCR amplification……………………29
3-4 Single strand conformation polymorphism (SSCP) analysis…………30
3-5 DNA sequence analysis…………………………………………………30
3-6 Detection of point mutation by RFLP…………………………………31
3-7 Structural modeling……………………………………………………31
3-8 Albumin depletion and SDS-PAGE analysis of seral samples…………32
3-9 Two-dimensional electrophoresis (2-DE ) ……………………………32
3-10 Tryptic in-gel digestion of 2-D PAGE-resolved proteins and MALDI-TOF mass spectrometry………………………………………………………………33
3-11 Separation of the leukocyte compartments and Western blotting……34
Chapter 4. Genomic Studies on Patients with Cerebrotendinous Xanthomatosis in Taiwan………………………………………………………………37
4-1 Introduction……………………………………………………………37
4-2 Materials and methods…………………………………………………39
4-3 Results…………………………………………………………………39
4-4 Discussion………………………………………………………………44
Chapter 5. New Insights into the Pathological Mechanisms of Cerebrotendinous Xanthomatosis in the Taiwanese Using Proteomic Tools……………46
5-1 Introduction……………………………………………………………46
5-2 Materials and methods…………………………………………………46
5-3 Results…………………………………………………………………48
5-4 Discussion………………………………………………………………52
Chapter 6.Conclusion and Future Work…………………………………………55
6-1 Conclusion………………………………………………………………55
6-2 Future work……………………………………………………………57
References………………………………………………………………………58
Appendixes………………………………………………………………………92

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