2015-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/708336葛瑞夫茲氏病(MIM%275000)是全世界造成甲狀腺功能亢進及甲狀腺眼病變的最重要原因，而具 有臨床及研究的重要性。抗甲狀腺藥物導致之無白血球症(TiA, 定義為服用抗甲狀腺藥物時絕對 顆粒球數目在每立方毫米空間小於五百顆), 是使用抗甲狀腺藥物治療時最叫人害怕的副作用，其 發生率大約為0.1-0.37%。本團隊終極目標是要找尋葛瑞夫茲氏病及其相關臨床表現（如TiA） 的致病基因並進一步釐清其致病機轉。我們長久以來已經利用珍貴臨床病人檢體及資料建立了堅 實的葛瑞夫茲氏病研究根基，而最近我們發現的TiA 的幾個主要致病基因位址更可能會改變整個 葛瑞夫茲氏病領域。在本計劃中，除了繼續延伸我們的人類研究外，我們更進一步來設計產生擬 人化主要組織相容性複合體小鼠，這不只對於我們自己有興趣的疾病（如葛瑞夫茲氏病及TiA） 之研究是非常有力的工具之外，對其他數不盡的免疫相關疾病都會很重要。在未來三年計劃期 間，我們有以下兩個主要目標。首先，我們要確實找出TiA 的致病基因。本團隊最近進行了 一個正統兩階段全基因體相關研究，共納入42 個TiA 病人以及1,208 位葛瑞夫茲氏病對照組， 在研究中我們發現了一個 HLA-B 對偶基因型 (Armitage trend Pcombined = 6.75 × 10-32), 一個 HLA-DRB1 對偶基因型 (Pcombined = 1.83 × 10-9) 以及一個在第三對染色體3q13 (Pcombined = 7.75 × 10-8) 的位置是TiA 的主要基因區域（本研究之論文正在Nature Genetics 期刊進行 revision）。在 此新的計劃期間，我們預計要倍增我們的檢體數目（再額外納入約40 個TiA 病人以及約1,000 葛瑞夫茲氏病對照組）來大幅繼續提高我們的統計檢力以利全面性地找出致病相關『區域』，然 後利用次世代定序方法來確實找出真正致病基因及其致病變異點。我們將利用本計劃要做出的擬 人化主要組織相容性複合體基因轉殖小鼠來作為功能研究的利器，而對於我們全基因體相關研究 發現之『非』人類白血球表面抗原之致病基因，我們未來也將利用其他研究經費來進行功能研究。 第二，我們將建立擬人化主要組織相容性複合體基因轉殖小鼠以利各種免疫相關疾病之 研究。在過去一年半的時間當中，本團隊對於建立擬人化主要組織相容性複合體『第二型』基 因轉殖小鼠已經有相當不錯的進展，這個實驗設計是要將單一基因背景小鼠的第二型主要組織相 容性複合體基因，改由單一人類白血球第二型表面抗原基因來替代，而這個替代基因未來又可以 簡便地利用快速轉基因(transgenic)（而非複雜的基因敲入, knock-in）的方法由其他人類白血球第 二型表面抗原基因來置換。要達到這樣劃時代的目標我們結合了許多精巧的設計，包括使用核注 射專一性轉殖基因小鼠(pronuclear injection-based mouse targeted transgenesis (PITT))，使用 C57BL/6N 純種幹細胞，以及使用TALENs 技術等等。在這次的三年新計劃中，我們要將這些經 驗與成果，延伸到人類白血球『第一型』表面抗原基因。許多免疫相關的疾病，包括我們最感興 趣的葛瑞夫茲氏病以及 TiA 都被發現『同時』具有第一型以及第二型人類白血球表面抗原基因 的相關性，因此如果能夠將兩型的基因都能夠來轉殖到小鼠，才會是完整的工具。我們的終極目 標更希望能夠將第一型以及第二型人類白血球表面抗原基因轉殖到小鼠的『同一條染色體』上， 這樣子的轉殖鼠將會是對於未來研究非常有力的幫助。Graves disease (GD, MIM%275000) is the leading cause of hyperthyroidism and thyroid eye disease with both clinical and research importance. Anti-thyroid drug (ATD)-induced agranulocytosis, (TiA, defined as an absolute granulocyte count < 500 per cubic millimeter while taking ATDs), is the most feared adverse effect of ATDs and can occur in 0.1-0.37% of individuals who take these medications. Our long-term goals are to find the susceptibility genes of GD and its related phenotypes (such as TiA), and to further figure out the detailed mechanisms. We have established solid GD research foundation using valuable human samples, and our recent identification of major TiA susceptibility loci is potentially paradigm-shifting. In this proposal, in addition to the “extension of our human study”, we will go one step further to generate humanized major histocompatibility complex (MHC) mice, which will be a powerful tool not only for the diseases we are interested in (such as GD and TiA) but also for numerous other immune-related diseases. For the upcoming 3-year period, we will work on the following 2 aims. First, identification of the susceptibility genes of TiA. Our team recently conducted a two-stage genome-wide association study (GWAS)(in total 42 TiA cases and 1,208 GD controls) and demonstrated a HLA-B allele (Armitage trend Pcombined = 6.75 × 10-32), a HLA-DRB1 allele (Pcombined = 1.83 × 10-9) and a locus at chromosome 3q13 (Pcombined = 7.75 × 10-8) as major independent susceptibility loci of TiA (manuscript under revision at Nature Genetics). For this proposed study, we plan to double our sample size (additional ~40 TiA cases and ~1,000 GD controls) to drastically increase our statistical power and comprehensively identify major TiA association loci. Then we will use next-generation sequencing (NGS)-based approach to narrow down and identify the genuine susceptibility/protective genes/variants. We will take advantage of the humanized MHC mouse model (covered by this grant) for studying TiA pathophysiology. We will also perform other functional study (covered by other grants) on other susceptibility genes identified through GWAS. Second, establishment of humanized MHC mice for study of immune-related diseases. During the past one and half years, our team has made substantial progress in making the humanized MHC class II mice. The overall study design is to replace the mouse MHC class II genes with an “exchangeable” single copy of human class II HLA allele into the homogeneous C57BL/6 inbred mice background. The cassette-like nature of our design makes it possible to exchange the old allele with any new allele 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 mouse models groundbreaking, including using pronuclear injection-based mouse targeted transgenesis (PITT), recombineering, C57BL/6N embryonic stem cell lines, and transcription activator-like effector nuclease (TALENs) technologies, etc. For the new proposal, in addition to accomplishing the class II mouse project, we will extend the design to also make humanized class I MHC mouse models. Many immune-related diseases or drug adverse effects (including GD and TiA) have been shown to have susceptibility/protective alleles in “both” class I and class II HLA genes. Our ultimate goal is to put both human class I and class II HLA risk alleles together on the same mouse chromosome/haplotype, which can be extremely powerful for future research.