高IgM综合征及IPEX临床与分子特征研究

发布时间：2018-07-10 03:18

本文选题：高IgM综合征 + CD40L　；参考：《重庆医科大学》2011年博士论文

【摘要】：第一部分:HIGM分子与T细胞亚群特征分析目的:建立HIGM基因及流式细胞仪检测方法,确诊HIGM患儿,分析其临床及分子特征。检测HIGM患儿中Treg、Th17、Th1细胞亚群变化规律与自身免疫发生相关性。方法:收集两年来HIGM疑似患儿外周血,PCR扩增CD40L、CD40、AID、UNG、NEMO等基因后测序比对,并与健康对照比较,确定致病突变。流式细胞仪检测突变患儿及移植患儿移植前后CD40L、CD40蛋白表达及Treg、Th1、Th17细胞亚群变化。结果:通过临床、免疫学筛查和基因分析,发现中国12例HIGM患儿,基因确诊8例,均为CD40L突变。其中错义突变1例,无义突变3例,缺失突变4例。其P7发生染色体微缺失(缺失5369bp)。8例突变中发现新型突变6例。突变分布于各外显子及启动子区域,主要集中在羧基末端胞外区。4例疑诊患儿未发现上述基因突变。8例CD40L突变患儿其CD40L均无表达。同时发现XHIM患儿中Treg明显下降(1.265±0.4801 N=6 VS 2.718±0.3963 N=12 P=0.04),Th17呈下降趋势(0.4200±0.1525 N=6 VS 0.9600±0.2076 N=12 P=0.1)。同时Th1/Treg细胞比例在XHIM中亦呈上升趋势(21.39±14.64 N=6 VS 10.50±2.596 N=12)。对移植前后2例XHIM患儿Treg、Th1、Th17检测发现,移植后Th17/Treg/Th1均较移植前呈上升趋势。结论:通过临床及其基因蛋白筛查手段,确诊中国较大宗HIGM患儿,发现6例CD40L新型突变。调节性T细胞降低与Th1/Treg比上升可能与XHIM患儿自身免疫发生相关。第二部分:IPEX临床及其分子特征分析目的:探讨表现为顽固性腹泻、有或无胰岛素依赖性糖尿病以及皮疹的疑似IPEX患儿FOXP3基因变异及其蛋白表达水平。方法:对近两年来我院收治的5例表现为早发性顽固性腹泻、有或无胰岛素依赖性糖尿病、以及皮疹的疑似IPEX男性患儿进行FOXP3基因扩增及测序分析,将发现的可疑突变位点通过数据库查询及与100例健康儿童相同位点序列比较,采用流式细胞仪检测CD4+CD25+FOXP3+调节性T细胞比例和FOXP3蛋白表达。结果:5例疑似患儿中发现3例FOXP3突变,P1为FOXP3基因13098与13099位碱基之间插入碱基A(g.13098-13099 ins A),随后立即形成终止密码子。CD4+CD25+ FOXP3+调节性T细胞缺失。其临床表现为典型IPEX三联征。P2为13128位碱基错义突变(g.13128 GA),导致FOXP3蛋白370位氨基酸由甲硫氨酸替换为异亮氨酸(Met370Ile),CD4+CD25+ FOXP3+调节性T细胞比例升高,患儿母亲为携带者。患儿临床表现为不完全症状。100例正常儿童FOXP3基因相同位点未见变异,故可排除该位点多态性可能。P3为此前已报道的错义突变(g.11628 TC;p.F324L),临床表现为典型轻型症状。P1与P2为此前未见报道的新发突变。调节性T细胞比例降低。结论:通过临床、免疫学筛查和基因分析,首次发现中国3例IPEX患儿,其中两例为新发突变。同时调节性T细胞数量与其临床严重性呈正相关关系。对早发胰岛素依赖性糖尿病、顽固性腹泻及不明原因肾脏等多系统损害婴幼儿,应考虑IPEX可能并进行FOXP3基因分析。第三部分:天然突变FOXP3蛋白抑制功能研究目的:诱变及表达天然突变FOXP3蛋白,探索不同突变FOXP3蛋白抑制IL-2转录功能与IPEX临床表型相关关系。方法:常规由外周血cDNA扩增FOXP3基因CDS区,构建表达载体,DpnI酶消化法在正常FOXP3表达载体基础上定点诱变产生突变FOXP3蛋白(N326Kfs1X; V408M; A384T; R337Q; F324L; 251delE; L242P; R146W; P187L; T108M; M370I),与IL-2启动子荧光素酶报告基因载体共转染Jurkat T细胞,PMA与离子霉素活化后双荧光素酶报告体系检测海肾荧光素与萤火虫荧光素。计算突变FOXP3对IL-2转录抑制活性变化。结果:成功建立11种天然突变FOXP3蛋白表达载体,建立双荧光素酶表达检测系统。共转染体系检测发现N326Kfs1X; V408M; A384T; R337Q; F324L; 251delE; L242P; R146W; P187L; T108M; M370I突变体抑制IL-2转录功能均下降,其中严重突变类型251delE、N326Kfs1X和亮氨酸拉链区域突变L242P、R146W完全失去抑制功能。但仅251delE、N326Kfs1X等严重突变体与严重抑制功能障碍和IPEX临床三联征具有相关性。结论:FOXP3严重突变和亮氨酸拉链区域突变常导致FOXP3抑制功能完全丧失,且仅严重突变体与严重抑制功能障碍及IPEX临床三联征具有相关性,因此基因诊断是IPEX最终确诊手段。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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