摘要:葛瑞夫玆氏病(Gravesdisease,GD,MIM%275000)是造成甲狀腺機能亢進以及甲狀腺眼病變的最重要原因,是一種在臨床上以及基礎研究上都非常重要的疾病。本研究團隊長期目標是找出葛瑞夫茲氏病的致病基因,並進一步研究出為何這些基因的致病機轉。利用人類檢體,本團隊已建立了葛瑞夫玆氏病很深厚的研究基礎。在本計劃中,除了將我們在人類領域的研究做更深入研究外,我們將更進一步地製造出擬人化的主要組織相容性基因(MHC)轉殖嵌入小鼠,然後更進一步做出葛瑞夫茲氏症之小鼠模式。在未來三年內,我們團隊將致力於以下二大目標。首先,本團隊將進行葛瑞夫玆氏症在HLA區域的最完整研究。本團隊過去已發表葛瑞夫玆氏症在HLA區域裡新的以及主要的相關對偶基因型,但是最近我們又發現新證據顯示在HLA區域還有其他的相關基因是獨立於這些我們已經研究過的經典HLA基因的。本團隊將利用次世代定序技術針對HLA特定區域進行再定序,以找出最有可能的疾病相關基因變異點,然後將針對這樣的變異點利用大規模的家族性以及族群性檢體進行相關研究。因為HLA區域實在是太複雜而且又有很長的連鎖不平衡(linkagedisequilibrium)構造,除非說像我們這樣的深入投資HLA研究(包含經典HLA基因以及全基因體規模相關研究,GWAS)否則其他團隊是沒有辦法完整的找出真正致病基因的。本團隊在此一領域也是佔有很大的研究優勢。第二,我們要建立擬人化的第二型主要組織相容性(MHC)小鼠,然後更進一步建立出好的葛瑞夫茲氏症小鼠模式。本團隊將會把C57BL/6純種品系小鼠的第二型主要組織相容性基因移除,然後置入單一劑量的人類第二型HLA基因,而且這個植入的基因會像是『卡夾』的形式,在未來如果我們要置換入新的基因,只需要透過方便的轉植實驗模式(transgenic)而不需進行耗時耗錢的基因剔除並植入(knock-out/knock-in)模式之實驗。本團對設計的基因轉殖嵌入鼠之所以會有突破性之重要份量主因在於我們利用了許多精巧的竅門,包括PITT,recominbeering,C57BL/6N幹細胞,以及TALENs等尖端技術。這樣的人類化的第二型主要組織相容性小鼠將會是許多人類疾病的重要研究工具,當然也包括了本團隊最有興趣的葛瑞夫玆氏症。過去其他研究人員試圖建立的葛瑞夫玆氏症小鼠模型,由於只著重於外在刺激因素的找尋,當然就無法做出很好的模式。本團隊計畫同時考量基因以及環境因素以建立最好的葛瑞夫玆氏症小鼠模式,這對未來整個疾病的臨床以及研究會有非常重要貢獻。
Abstract: Graves disease (GD, MIM%275000) is the leading cause of hyperthyroidism andthyroid eye disease with both clinical and research importance. Our long-term goals are tofind the susceptibility genes of GD, and to further figure out the detailed mechanisms howthese genes cause susceptibility or protection to GD. Our team has established solid GDresearch foundation using valuable human samples. In this proposal, in addition to the“extension of our human study”, we will go one step further to generate humanized majorhistocompatibility complex (MHC) mice, and to establish a good mouse model for GD. Forthe upcoming 3-year period, we will work on the following 2 aims. First, to perform themost comprehensive study in the HLA regions for GD. Our team previously reportedmajor and novel classical HLA alleles associated with GD. Now we have shown newevidence that there are additional association signals in the HLA region beyond the classicalHLA alleles. We will apply NGS to do targeted re-sequencing in the HLA region, and, afteridentification of candidate genetic variants, will then perform large-scale association studyusing both family samples and unrelated samples. Since the HLA region is probably the mostcomplicated genomic region with high gene density and extended linkage disequilibrium, webelieve that the best way to find the genuine susceptibility/protective genetic variants is tocomprehensively investigate the classical HLA loci together with other variants, such as SNPs,at the same time. We are probably the only GD team worldwide to heavily invest in bothgenome-wide association study (GWAS) and classical HLA loci. Our team has huge researchadvantage in this field. Second, to generate humanized class II major compatibilitycomplex (MHC) mice, and then to further generate a good GD mouse model. Our teamwill replace the mouse MHC class II genes with an “exchangeable” single copy of humanclass II HLA allele into the homogeneous C57BL/6 inbred mice background. Thecassette-like nature of our design makes it possible to exchange the old allele with any newallele of interest using the convenient “transgenic” approach (instead of the time-consuming“knock-in” approach). We have several clever state-of-the-art tricks to make our mousemodels groundbreaking, including using pronuclear injection-based mouse targetedtransgenesis (PITT), recombineering, C57BL/6N embryonic stem cell lines, and transcriptionactivator-like effector nuclease (TALENs) technologies, etc. Furthermore, our team is aversatile combination with the expertise to handle mouse targeting experiments, mousephenotyping and immunological studies. These humanized class II MHC mice can be verypowerful for studies of various human (autoimmune) diseases, including our favorite, GD.Previous attempts by other researchers to make GD mouse models only focused on externaltriggers (by introducing antigens) but failed to address genetic susceptibility, which, with nosurprise, ended up with no satisfactory GD model. We propose to combine both genetic andenvironmental factors to generate a good GD mouse model, which will have fundamentalimpact on the whole GD research and clinical fields.