【摘要】：播散性卡介苗感染(Mendelian susceptibility to mycobacterial disease,MSMD)疾病于1951年首次報(bào)道,該病的臨床表型是患者對(duì)一些弱毒性分枝桿菌(例如卡介苗、非結(jié)核性的環(huán)境分枝桿菌)易感,屬于一種罕見的原發(fā)性免疫缺陷疾病。本研究對(duì)一個(gè)播散性卡介苗感染家系進(jìn)行了分子遺傳學(xué)分析,找到了其致病基因,并通過分子生物學(xué)和生物化學(xué)等實(shí)驗(yàn)方法研究了該致病基因的突變是如何影響其編碼蛋白質(zhì)的功能,初步闡明其致病機(jī)制。家系的先證者臨床診斷為播散性卡介苗感染疾病(MSMD),先癥者的母親也有類似的表型,對(duì)符合遺傳規(guī)律的已知基因進(jìn)行篩查,最終發(fā)現(xiàn)致病基因STAT1外顯子15,第1228位堿基從A突變成G(c.1228AG),導(dǎo)致該基因編碼蛋白質(zhì)的第410位氨基酸由賴氨酸突變?yōu)楣劝彼?p.K410E),先癥者的母親攜帶同樣的突變,為了進(jìn)一步確定該突變?yōu)橹虏⊥蛔?對(duì)家系內(nèi)成員以及家系外100個(gè)正常人進(jìn)行了限制性片段長度多態(tài)性分析驗(yàn)證,證實(shí)只有患者攜帶該突變,正常人都不攜帶該突變,說明該突變不是單核苷酸多態(tài)位點(diǎn)。進(jìn)一步通過對(duì)STAT1基因突變位點(diǎn)保守性及其蛋白的編碼結(jié)構(gòu)區(qū)域分析,發(fā)現(xiàn)突變的氨基酸位點(diǎn)在物種進(jìn)化上高度保守,而且突變位點(diǎn)位于STAT1蛋白的DNA結(jié)合區(qū)。將野生型和突變型的STAT1基因分別構(gòu)建到pRc/CMV表達(dá)載體上,轉(zhuǎn)染到HEK293細(xì)胞,經(jīng)干擾素IFN-γ刺激后,進(jìn)行免疫熒光定位并提取細(xì)胞核內(nèi)總蛋白做凝膠遷移率實(shí)驗(yàn)(Electrophoretic Mobility Shift Assay,EMSA)實(shí)驗(yàn),結(jié)果顯示突變型的STAT1蛋白入核并未受到影響,但與野生型相比,STAT1蛋白組裝的gamma激活因子(gamma-activating factor,GAF)與gamma激活序列(gamma-activating sequence,GAS)結(jié)合能力顯著減弱,因此STAT1基因的突變影響了其生化功能。本研究通過對(duì)一個(gè)MSMD家系進(jìn)行遺傳學(xué)研究發(fā)現(xiàn)STAT1基因的一個(gè)新的致病突變(c.1228AG,p.K410E),通過相關(guān)分子生物學(xué)和生化實(shí)驗(yàn)初步闡明了STAT1基因K410E突變導(dǎo)致MSMD疾病的生化機(jī)制,進(jìn)一步加深了對(duì)STAT1基因?qū)е翸SMD疾病的認(rèn)識(shí),為該疾病的遺傳診斷與治療提供了理論支持。
[Abstract]:Disseminated BCG infection with (Mendelian susceptibility to mycobacterial disease,MSMD was first reported in 1951. The clinical phenotype of the disease is the susceptibility of patients to some weakly toxic mycobacteria, such as Bacillus Calmette-Guerin (BCG), non-tuberculous environmental mycobacterium. It is a rare primary immunodeficiency disease. In this study, molecular genetic analysis was carried out on a family with disseminated BCG infection, and its pathogenic gene was found. Molecular biology and biochemistry were used to study how the mutation of the pathogenicity gene affected the function of the encoded protein and the pathogenicity mechanism was preliminarily elucidated. The mothers of the probands who were clinically diagnosed with disseminated BCG infection (MSMD), had similar phenotypes and screened known genes according to genetic rules, and finally found the pathogenic gene STAT1 exon 15. The 1228 base mutated from A to G (c.1228AG), causing the 410th amino acid of the protein encoded by the gene to mutate from lysine to glutamic acid (p.K410E), and the mother of the first patient carried the same mutation. In order to further identify the mutation as a pathogenic mutation, the restriction fragment length polymorphism (RFLP) analysis was carried out among family members and 100 normal persons outside the family. It was confirmed that only the patient carried the mutation, and none of the normal persons carried the mutation. The mutation is not a single nucleotide polymorphic site. By analyzing the conserved mutation sites of STAT1 gene and the coding structure of its protein, it was found that the amino acid sites of the mutations were highly conserved in species evolution, and the mutation sites were located in the DNA binding region of STAT1 protein. The wild-type and mutant STAT1 genes were constructed into pRc/CMV expression vectors and transfected into HEK293 cells. After stimulation with interferon IFN- 緯, immunofluorescence localization was carried out and the total protein in the nucleus was extracted for gel mobility experiment (Electrophoretic Mobility Shift Assay,. The results of EMSA showed that the entry of mutant STAT1 protein was not affected, but the binding ability of gamma activator (gamma-activating factor,GAF) assembled by STAT1 protein to gamma activation sequence (gamma-activating sequence,GAS) was significantly decreased compared with wild type. Therefore, the mutation of STAT1 gene affects its biochemical function. In this study, a new pathogenic mutation of STAT1 gene (c. 1228 AGp.K410E) was found in a MSMD pedigree. The biochemical mechanism of MSMD disease caused by K410E mutation of STAT1 gene was preliminarily clarified by molecular biology and biochemical experiments. The understanding of MSMD disease caused by STAT1 gene was further deepened, which provided theoretical support for the genetic diagnosis and treatment of the disease.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.2;R394

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