臺灣博碩士論文加值系統

DNA錯配修復機制(MMR)在維持基因體穩定性扮演重要的角色，當此機制缺失時會使突變累積進而造成癌症的發生。這些MMR缺失相關癌症被發現有微衛星不穩定(MSI)的特徵，微衛星序列為一至六個核苷酸重複所組成，分布於基因體編碼及非編碼區。這些序列在複製過程時很容易發生錯誤，因此相當依賴MMR機制，然而MMR功能喪失時便會使微衛星序列產生改變。如果分布於編碼區的微衛星序列不穩定時就有可能造成移碼突變，提早產生終止密碼子，進而影響基因的功能。近年來許多研究指出編碼區具有單核苷酸重複序列(cMNR)突變的基因參與在人類MMR缺失癌症進程中，然而在人類較常被報導的cMNR在小鼠內並不一定保守，因此參與MMR缺失癌症的cMNR突變基因在MMR缺失的小鼠模式和人類是否相同仍不完全完全清楚。在這個研究中，我以ICE CRIM系統針對Trp53、Mlh1、Msh2進行破壞，成功建立MMR缺失小鼠模式。另外我設計一個可藉雜交反應來富集相對應基因體序列的探針擷取模組，以次世代定序的方式在我們建立的MMR缺失小鼠模式中偵測MSI靶基因保守性。在我們建立的MMR缺失小鼠模式所產生的MSI-H腫瘤中，我發現無論是保守cMNR區域或是編碼區域的非重複序列皆有出現部分與人類MMR缺失癌症相同的突變基因。另外，我也在一些致癌基因中觀察到出現和人類癌症一樣的突變，例如Kras G12D以及G12V。在小鼠MSI-H腫瘤中出現與人類MSI靶基因保守的小鼠直系同源基因突變顯示使用小鼠研究人類MSI-H腫瘤的可行性。我們研究結果對於參與小鼠MMR缺失癌症的MSI靶基因提供更深入的觀點並有助於我們能更好的了解導致MSI-H腫瘤發生的突變路徑中MMR突變特徵。

DNA mismatch repair mechanism (MMR) plays a critical role in maintaining the stability of the genome. Loss of MMR function leads to the accumulation of mutations and promotes tumorigenesis. Microsatellite instability (MSI) is the hallmark of MMR-deficient cancers. Microsatellites are one to six nucleotide repeats, located in either coding or non-coding regions. These repeats are prone to errors and frequently change in size during DNA replication. Normally, the errors are repaired through MMR. In other words, the lengths of microsatellites can be altered when the MMR pathway is defected. If an alteration of the microsatellite length occurred in the coding region, it may cause a frameshift mutation and result in a premature stop codon. Recent studies indicated that some genetic loci carrying coding mononucleotide repeat (cMNR) mutants are involved in the MSI-H carcinogenesis. However, the sequences of these frequently often reported cMNRs in human are not all conserved in mice. So far, whether those cMNR mutations generally seen in human MSI-H tumors are conserved in the MMR-deficient mouse model is unclear. In this study, I disrupted Trp53, Mlh1, and Msh2 by the ICE CRIM system and successfully generated the MMR-deficient mouse model. In addition, I designed a probe capture panel to enrich the conserved MSI target genes in mice for next-generation sequencing analysis. My result identified some conserved MSI target genes in human patient samples and our mouse MSI-H tumors, including both of those mutated in cMNR regions and non-repetitive sequences of coding regions. Oncogenic mutations, identical to those found in human, such as Kras G12D and G12V were observed in our mouse tumorous tissue. The significant enrichment for mouse orthologous variants of the human predicted MSI target genes involved in tumorigenesis reinforce the adequacy of using mice to study human MSI-H tumors. Our study provided an insight into the MSI target genes in MMR-deficient mice and allowed us to better understand the MMR signatures of possible mutational steps leading to MSI-H tumorigenesis.

口試委員會審定書………………………………………………………………i
致謝………………………………………………………………………………ii
中文摘要……………………………………………………………………………iv
Abstract………………………………………………………………………………v
Contents……………………………………………………………………………vii
List of figures………………………………………………………………………ix
List of tables……………………………………………………………………xi
List of supplementary tables………………………………………………………xii
Supplemental Information…………………………………………………………xiii
1. Introduction………………………………………………………………….…1
1.1 DNA mismatch repair (MMR) mechanism and diseases…….………….…1
1.2 cMNR as a potential target in MSI-H tumors…….………………..……… 3
1.3 Different approaches to detect MSI……………..……...…….…………..…4
1.4 Mouse models to study MMR-associated tumorigenesis…….……….…… 6
1.5 Conserved cMNR genes in human and mouse MSI-H tumors………..……7
2. Materials and methods……………………….……………………………………9
2.1 Animals……………………………………………………………….…. 9
2.2 Genotyping………….…………………………………………………..… 9
2.3 Genomic DNA extraction…………………………………………………10
2.4 Amplicon-based deep sequencing…………………….………………. 10
2.5 Microsatellite analysis……………………….……………………………11
2.6 The DNA capture probe design for MSI target genes panel………………12
2.7 Hybridization-based deep sequencing….…………………………………13
2.8 Sanger Sequencing……………………………………….…………….…15
3. Results……………………………………………………………………….…16
3.1 Generation of MMR-deficient mice by the ICE CRIM system………….16
3.2 Identification of mutations in conserved coding regions of mouse MSI-H tumors……………………………………………………………………18
3.3 Characterization of non-repetitive coding region of disease-related genes in mouse MSI-H tumors…………………………………………………20
4. Discussion……………………………………………………………………….23
5. References………………………………………………………………………30

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