IPEX综合征及XLP-1临床与分子特点分析

发布时间：2018-05-31 09:16

本文选题：原发性免疫缺陷病 + 调节性T细胞　；参考：《重庆医科大学》2013年硕士论文

【摘要】：第一部分：IPEX新发突变一例分子与临床特征研究目的：探讨1例X连锁多内分泌腺病、肠病伴免疫失调综合征患儿FOXP3基因变异、蛋白表达水平、免疫表型及临床特征。方法：对我院收治的1例表现为早发性顽固性腹泻、自身免疫现象、内分泌功能异常、生长发育落后、IgE增高的疑似IPEX男性患儿外周血单个核细胞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，PCR法进行外周血FOXP3基因扩增及测序、比对分析，并与100例正常儿童FOXP3基因序列比较除外多态性以确定致病突变。结果：该患儿CD4+CD25+FOXP3+调节性T细胞比例较正常对照明显减低，FOXP3蛋白表达量与正常对照无显著差异。经基因分析确诊为IPEX，其FOXP3基因第8号外显子末位碱基错义突变(GA)，导致FOXP3蛋白323位氨基酸由谷氨酸替换为赖氨酸(Glu323Lys)，突变基因的转录产物中除了一部分为错义突变外，还形成了多种异常拼接体，部分为缺失第8号外显子，部分同时缺失7、8号外显子，另有部分完全或部分缺失5号外显子及部分11号外显子。患儿母亲为该突变的携带者。100例正常儿童FOXP3基因相同位点未见变异。结论：通过临床、免疫学筛查、基因分析及流式细胞术，确诊一例发生FOXP3多种不同异常拼接及错义突变的IPEX患儿，为此前未报到的新发突变，，对早发顽固性腹泻、湿疹、内分泌功能异常伴自身免疫现象及不明肾脏损害婴幼儿，应考虑IPEX可能并进行Treg流式细胞术快速筛查及FOXP3基因分析以最终确诊。第二部分：XLP-1型患者临床、基因和蛋白表达分析目的：探讨SH2D1A基因突变所致XLP-1型患儿的临床及分子特征，为临床甄别和诊断此病提供信息。方法：对15例临床疑诊为XLP-1型的男性患儿，收集归纳临床资料和相关实验室检查结果，采取患儿及其亲属新鲜外周血进行流式细胞术检测PBMC内SAP蛋白的表达，提取外周血DNA和RNA，PCR法扩增SH2D1A基因cDNA序列直接进行测序。在NCBI基因库比对目的序列，发现突变位点后扩增相应外显子及或进行TA克隆进一步分析确定突变类型，同时对相关亲属进行基因分析。结果：通过对15例疑诊XLP-1型患儿的基因和蛋白检测，发现3例来自无血缘关系的不同家族的患儿存在SH2D1A基因突变，除外12例。3例突变均除外了基因多态性可能，其中2例为新发突变。患儿1的母亲、外祖母及患儿2的母亲为致病突变的携带者。行流式细胞术检测的患儿1和2，SAP蛋白表达均明显降低。患者1的母亲SAP蛋白表达为双峰，患者2的母亲蛋白表达正常。结论：本研究通过对患儿进行临床表现和免疫学初筛，同时借助基因序列分析和流式蛋白检测，确诊了3例SH2D1A基因不同突变的患者。对于符合XLP-1型临床表现的患儿应尽快进行SAP蛋白检测和SH2D1A基因分析。
[Abstract]:Part one: molecular and clinical characteristics of a new mutation of IPEX Objective: to investigate the FOXP3 gene variation, protein expression, immunophenotype and clinical features in a case of X-linked polyendocrine adenopathy and enteropathy with immune maladjustment syndrome. Methods: one patient with early onset refractory diarrhea, autoimmune phenomenon and abnormal endocrine function was treated in our hospital. Peripheral blood mononuclear cells (PBMC) of suspected male children with increased IPEX were amplified and sequenced by FOXP3 gene amplification and sequencing using flow cytometry to detect the proportion of CD4 CD25 FOXP3 regulatory T cells and the expression of FOXP3 protein. In order to identify the pathogenicity mutation, the FOXP3 gene sequence was compared with that of 100 normal children. Results: the percentage of regulatory T cells in CD4 CD25 FOXP3 was significantly lower than that in normal controls. There was no significant difference in the expression of FOXP3 protein between the two groups. It was confirmed by gene analysis that the missense mutation at the end of exon 8 of FOXP3 gene resulted in the amino acid substitution of FOXP3 protein 323 from glutamic acid to Glu323LysN. The transcriptional product of the mutant gene was missense mutation except for part of the transcriptional product. A variety of abnormal splicing bodies were formed, some of them were missing exon 8, some of them were deletion of exon 7,8 at the same time, and some of them were completely or partially deleted exon 5 and exon 11. The mother was the carrier of the mutation. There was no mutation in the same locus of the FOXP3 gene in the normal children. Conclusion: through clinical, immunological screening, gene analysis and flow cytometry, a case of IPEX with multiple abnormal splicing and missense mutation of FOXP3 was diagnosed. For early onset refractory diarrhea eczema abnormal endocrine function with autoimmune phenomenon and unknown kidney damage infants should consider the possibility of IPEX and carry out rapid screening of Treg flow cytometry and FOXP3 gene analysis to determine the final diagnosis. Part two: clinical, gene and protein expression in patients with type XLP-1 Objective: to investigate the clinical and molecular characteristics of XLP-1 type in children with SH2D1A gene mutation, and to provide information for clinical identification and diagnosis. Methods: 15 male children with suspected XLP-1 type were collected and summarized. The expression of SAP protein in PBMC was detected by flow cytometry in the fresh peripheral blood of the children and their relatives. The cDNA sequence of SH2D1A gene was amplified by DNA from peripheral blood and sequenced directly. After the target sequence of NCBI gene bank alignment was found, the corresponding exons were amplified and TA cloning was carried out to determine the mutation type, and the related relatives were analyzed. Results: by detecting the gene and protein of 15 children with suspected XLP-1 type, it was found that 3 children from different families without blood relationship had SH2D1A gene mutation, except 12 cases with SH2D1A gene mutation, all of them excluded the possibility of gene polymorphism. Two of them were new mutations. The mother of child 1, grandmother and mother of child 2 were carriers of the disease mutation. The expression of 1 and 2 SAP protein was significantly decreased by flow cytometry. The expression of SAP protein in the mother of patient 1 was bimodal, while the expression of protein in the mother of patient 2 was normal. Conclusion: in this study, three patients with different mutations of SH2D1A gene were diagnosed by screening their clinical manifestations, immunology, gene sequence analysis and flow protein detection. SAP protein detection and SH2D1A gene analysis should be carried out as soon as possible for children with XLP-1 type clinical manifestations.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R725.9

【参考文献】