本文選題：高IgM綜合征 + CD40L��； 參考：《重慶醫(yī)科大學(xué)》2011年博士論文

【摘要】：第一部分:HIGM分子與T細(xì)胞亞群特征分析 目的:建立HIGM基因及流式細(xì)胞儀檢測(cè)方法,確診HIGM患兒,分析其臨床及分子特征。檢測(cè)HIGM患兒中Treg、Th17、Th1細(xì)胞亞群變化規(guī)律與自身免疫發(fā)生相關(guān)性。 方法:收集兩年來(lái)HIGM疑似患兒外周血,PCR擴(kuò)增CD40L、CD40、AID、UNG、NEMO等基因后測(cè)序比對(duì),并與健康對(duì)照比較,確定致病突變。流式細(xì)胞儀檢測(cè)突變患兒及移植患兒移植前后CD40L、CD40蛋白表達(dá)及Treg、Th1、Th17細(xì)胞亞群變化。 結(jié)果:通過(guò)臨床、免疫學(xué)篩查和基因分析,發(fā)現(xiàn)中國(guó)12例HIGM患兒,基因確診8例,均為CD40L突變。其中錯(cuò)義突變1例,無(wú)義突變3例,缺失突變4例。其P7發(fā)生染色體微缺失(缺失5369bp)。8例突變中發(fā)現(xiàn)新型突變6例。突變分布于各外顯子及啟動(dòng)子區(qū)域,主要集中在羧基末端胞外區(qū)。4例疑診患兒未發(fā)現(xiàn)上述基因突變。8例CD40L突變患兒其CD40L均無(wú)表達(dá)。同時(shí)發(fā)現(xiàn)XHIM患兒中Treg明顯下降(1.265±0.4801 N=6 VS 2.718±0.3963 N=12 P=0.04),Th17呈下降趨勢(shì)(0.4200±0.1525 N=6 VS 0.9600±0.2076 N=12 P=0.1)。同時(shí)Th1/Treg細(xì)胞比例在XHIM中亦呈上升趨勢(shì)(21.39±14.64 N=6 VS 10.50±2.596 N=12)。對(duì)移植前后2例XHIM患兒Treg、Th1、Th17檢測(cè)發(fā)現(xiàn),移植后Th17/Treg/Th1均較移植前呈上升趨勢(shì)。 結(jié)論:通過(guò)臨床及其基因蛋白篩查手段,確診中國(guó)較大宗HIGM患兒,發(fā)現(xiàn)6例CD40L新型突變。調(diào)節(jié)性T細(xì)胞降低與Th1/Treg比上升可能與XHIM患兒自身免疫發(fā)生相關(guān)。第二部分:IPEX臨床及其分子特征分析 目的:探討表現(xiàn)為頑固性腹瀉、有或無(wú)胰島素依賴性糖尿病以及皮疹的疑似IPEX患兒FOXP3基因變異及其蛋白表達(dá)水平。 方法:對(duì)近兩年來(lái)我院收治的5例表現(xiàn)為早發(fā)性頑固性腹瀉、有或無(wú)胰島素依賴性糖尿病、以及皮疹的疑似IPEX男性患兒進(jìn)行FOXP3基因擴(kuò)增及測(cè)序分析,將發(fā)現(xiàn)的可疑突變位點(diǎn)通過(guò)數(shù)據(jù)庫(kù)查詢及與100例健康兒童相同位點(diǎn)序列比較,采用流式細(xì)胞儀檢測(cè)CD4+CD25+FOXP3+調(diào)節(jié)性T細(xì)胞比例和FOXP3蛋白表達(dá)。 結(jié)果:5例疑似患兒中發(fā)現(xiàn)3例FOXP3突變,P1為FOXP3基因13098與13099位堿基之間插入堿基A(g.13098-13099 ins A),隨后立即形成終止密碼子。CD4+CD25+ FOXP3+調(diào)節(jié)性T細(xì)胞缺失。其臨床表現(xiàn)為典型IPEX三聯(lián)征。P2為13128位堿基錯(cuò)義突變(g.13128 GA),導(dǎo)致FOXP3蛋白370位氨基酸由甲硫氨酸替換為異亮氨酸(Met370Ile),CD4+CD25+ FOXP3+調(diào)節(jié)性T細(xì)胞比例升高,患兒母親為攜帶者�；純号R床表現(xiàn)為不完全癥狀。100例正常兒童FOXP3基因相同位點(diǎn)未見(jiàn)變異,故可排除該位點(diǎn)多態(tài)性可能。P3為此前已報(bào)道的錯(cuò)義突變(g.11628 TC;p.F324L),臨床表現(xiàn)為典型輕型癥狀。P1與P2為此前未見(jiàn)報(bào)道的新發(fā)突變。調(diào)節(jié)性T細(xì)胞比例降低。 結(jié)論:通過(guò)臨床、免疫學(xué)篩查和基因分析,首次發(fā)現(xiàn)中國(guó)3例IPEX患兒,其中兩例為新發(fā)突變。同時(shí)調(diào)節(jié)性T細(xì)胞數(shù)量與其臨床嚴(yán)重性呈正相關(guān)關(guān)系。對(duì)早發(fā)胰島素依賴性糖尿病、頑固性腹瀉及不明原因腎臟等多系統(tǒng)損害嬰幼兒,應(yīng)考慮IPEX可能并進(jìn)行FOXP3基因分析。第三部分:天然突變FOXP3蛋白抑制功能研究 目的:誘變及表達(dá)天然突變FOXP3蛋白,探索不同突變FOXP3蛋白抑制IL-2轉(zhuǎn)錄功能與IPEX臨床表型相關(guān)關(guān)系。 方法:常規(guī)由外周血cDNA擴(kuò)增FOXP3基因CDS區(qū),構(gòu)建表達(dá)載體,DpnI酶消化法在正常FOXP3表達(dá)載體基礎(chǔ)上定點(diǎn)誘變產(chǎn)生突變FOXP3蛋白(N326Kfs1X; V408M; A384T; R337Q; F324L; 251delE; L242P; R146W; P187L; T108M; M370I),與IL-2啟動(dòng)子熒光素酶報(bào)告基因載體共轉(zhuǎn)染Jurkat T細(xì)胞,PMA與離子霉素活化后雙熒光素酶報(bào)告體系檢測(cè)海腎熒光素與螢火蟲(chóng)熒光素。計(jì)算突變FOXP3對(duì)IL-2轉(zhuǎn)錄抑制活性變化。 結(jié)果:成功建立11種天然突變FOXP3蛋白表達(dá)載體,建立雙熒光素酶表達(dá)檢測(cè)系統(tǒng)。共轉(zhuǎn)染體系檢測(cè)發(fā)現(xiàn)N326Kfs1X; V408M; A384T; R337Q; F324L; 251delE; L242P; R146W; P187L; T108M; M370I突變體抑制IL-2轉(zhuǎn)錄功能均下降,其中嚴(yán)重突變類型251delE、N326Kfs1X和亮氨酸拉鏈區(qū)域突變L242P、R146W完全失去抑制功能。但僅251delE、N326Kfs1X等嚴(yán)重突變體與嚴(yán)重抑制功能障礙和IPEX臨床三聯(lián)征具有相關(guān)性。 結(jié)論:FOXP3嚴(yán)重突變和亮氨酸拉鏈區(qū)域突變常導(dǎo)致FOXP3抑制功能完全喪失,且僅嚴(yán)重突變體與嚴(yán)重抑制功能障礙及IPEX臨床三聯(lián)征具有相關(guān)性,因此基因診斷是IPEX最終確診手段。
[Abstract]:Part I: characteristics of HIGM and T cell subsets
Objective: to establish the HIGM gene and flow cytometry, to confirm the diagnosis of children with HIGM, to analyze the clinical and molecular characteristics, and to detect the correlation between the changes of Treg, Th17, Th1 cell subsets and the autoimmunity in children with HIGM.
Methods: the peripheral blood of HIGM suspected children was collected for two years. PCR amplification of CD40L, CD40, AID, UNG, NEMO and other genes was compared and compared with the health control, and the pathogenic mutation was determined. The flow cytometry was used to detect the CD40L, CD40 protein expression, Treg, Th1, and Th17 cell subsets before and after transplantation.
Results: through clinical, immunological screening and gene analysis, 12 cases of HIGM in China were found to have 8 cases of gene diagnosis, including 1 cases of missense mutation, 3 cases of nonsense mutation and 4 missing mutations. 6 cases of new mutation were found in P7 chromosome microdeletion (missing 5369bp) in.8 case, and the mutation was distributed in exons and promoter regions. At the end of the carboxyl group, the.4 cases of suspected children were not found to be expressed in CD40L, and the Treg decreased significantly (1.265 + 0.4801 N=6 VS 2.718 + 0.3963 N=12 P=0.04) in children with XHIM, and the Th17 decreased (0.4200 + 0.1525 N=6 0.9600 + 0.2076). The proportion of cell in XHIM also showed an upward trend (21.39 + 14.64 N=6 VS 10.50 + 2.596 N=12). Treg, Th1, Th17 of 2 children with XHIM before and after transplantation were detected, and the increase of Th17/Treg/Th1 was higher than that before transplantation.
Conclusion: through clinical and gene protein screening, 6 cases of HIGM children were diagnosed in China, and a new type of CD40L mutation was found. The rise of regulatory T cell reduction and Th1/Treg ratio may be related to the autoimmunity of children with XHIM. The second part: the analysis of the clinical and molecular characteristics of IPEX
Objective: To investigate the FOXP3 gene mutation and protein expression in children with suspected IPEX presenting with refractory diarrhea, with or without insulin dependent diabetes mellitus and rash.
Methods: 5 children with early onset intractable diarrhea, or without insulin dependent diabetes, and a suspected IPEX male child with skin rash in our hospital in the last two years were amplified and sequenced by FOXP3 gene. The suspicious mutation sites found by the database were querying and compared with the same sequence of the same site in 100 healthy children. The percentage of CD4+CD25+FOXP3+ regulatory T cells and the expression of FOXP3 protein were detected by cytometer.
Results: 3 cases of FOXP3 mutation were found in 5 cases of suspected children. P1 was FOXP3 gene 13098 and 13099 bases inserted into base A (g.13098-13099 ins A), and then immediately formed the termination codon.CD4+CD25+ FOXP3+ regulated T cell deletion. The clinical manifestation was that the typical IPEX triple sign was 13128 base missense mutations. 370 amino acids were replaced by methionine as isoleucine (Met370Ile), and the proportion of CD4+CD25+ FOXP3+ regulatory T cells increased and the mother was a carrier. The clinical manifestation of children with incomplete symptoms of.100 normal children had no variation in the same loci of FOXP3 gene. Therefore, the polymorphism of the loci could be excluded as the previously reported missense mutation (G.). 11628 TC; p.F324L). The clinical manifestation is a typical mild symptom..P1 and P2 are new mutations that have not been reported before. The proportion of regulatory T cells decreases.
Conclusion: by clinical, immunological screening and gene analysis, 3 children with IPEX were first found in China, of which two were new mutations. The number of regulatory T cells was positively correlated with its clinical severity. IPEX should be considered for early onset insulin dependent diabetes, refractory diarrhea and unexplained kidney and other multiple system damage to infants. FOXP3 gene analysis can be carried out. The third part: natural mutant FOXP3 protein inhibition function.
Objective: to mutagenesis and expression of natural mutant FOXP3 protein, and explore the correlation between the inhibition of IL-2 transcription function and IPEX phenotype by different mutant FOXP3 proteins.
Methods: the FOXP3 gene CDS region was amplified from peripheral blood by cDNA, and the expression vector was constructed. The mutation of FOXP3 protein (N326Kfs1X; V408M; A384T; R337Q; F324L) was produced on the basis of the normal FOXP3 expression vector by DpnI enzyme digestion. Rkat T cells, PMA and lincomycin activated double luciferase reporter system was used to detect fluorescein and fluorescein in the sea kidney. The changes of the transcriptional inhibitory activity of the mutant FOXP3 to IL-2 were calculated.
Results: 11 kinds of natural mutant FOXP3 protein expression vector were successfully established and the dual luciferase expression detection system was established. The co transfection system detected N326Kfs1X, V408M, A384T, R337Q, F324L, 251delE; L242P, R146W; P187L; T108M, and the M370I mutant inhibition, and bright ammonia The acid zipper region mutated L242P, R146W completely lost its inhibitory function. But only 251delE, N326Kfs1X and other severe mutants were associated with severe inhibition dysfunction and IPEX clinical triad.
Conclusion: severe FOXP3 mutation and leucine zipper regional mutation often lead to complete loss of FOXP3 inhibitory function, and only severe mutants are associated with severe dysfunction and IPEX clinical triad. Therefore, gene diagnosis is the final diagnosis of IPEX.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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相關(guān)期刊論文 前2條

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本文編號(hào)：2111827