X连锁多内分泌腺病肠病伴免疫失调综合征一例基因及蛋白表达研究

A novel misseuse mutation of FOXP3 causes enteropathy, X-linked syndrome in a Chinese child

目的探讨表现为顽固性腹泻、皮疹以及有或无胰岛素依赖性糖尿病的疑似X连锁多内分泌腺病肠病伴免疫失调综合征（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4例表现为早发性顽固性腹泻、皮疹以及有或无胰岛素依赖性糖尿病的疑似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F0肼）3蛋白表达。结果4例疑似患儿中发现1例FOXP3突变，为11号外显子66位碱基错义突变（G〉A），导致FOXP3蛋白370位氨基酸由甲硫氨酸替换为异亮氨酸（Met370Ile），患儿母亲为携带者。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。该突变为此前未见报道的新突变，患儿CD4＋CD25＋FOXP3＋调节性T细胞比例升高而FOXP3表达量无减低。结论通过临床、免疫学筛查和基因分析，发现我国首例IPEX患儿（g：13128G〉Ac：1298G〉AMet370Ile），对早发胰岛素依赖性糖尿病、顽固性腹泻及不明肾脏损害婴幼儿，应考虑IPEX可能并进行FOXP3基因分析。

Objective To investigate variation of FOXP3 and it＇s expression in male children presented with severe and early-onset enteropathy, rash with or without insulin-dependent diabetes mellitus （IDDM）. Methods Four male children presented with severe and early-onset enteropathy, rash, with or without IDDM were subjected to detection of FOXP3 expression on the PBMC by flow cytometry and FOXP3 gene analysis. The maternal gene analysis was subsequently performed once the variant FOXP3 gene was found. All 11 exons and splice sites within FOXP3 gene were amplified by polymerase chain reaction （PCR） from genomic DNA. Reverse transcription polymerase chain reaction was used to amplify the FOXP3 transcripts. Sequence analysis was performed directly on the bulk PCR products forwardly and reversely. The candidate mutation site was compared with that of 100 healthy controls to exclude polymorphism. Flow cytometry was used to determine FOXP3 expression on CD4 ＋ CD25＋ T cells and the frequency of Tregs in CD4 ＋ T cells. Results One of the 4 patients showed a G13128A genetic variation in exon 11, which resulted in a Met370Ile substitution. No sequence variations were found at the same site in any of 100 healthy controls, indicating that the Met370Ile substitution is not a polymorphism but a novel missense mutation. The patient＇s mother was identified as a carrier for this mutation. There was no reduced frequency of Tregs in the peripheral blood of the patient and FOXP3 protein expression is normal as compared with controls. Conclusion A novel missense mutation of FOXP3 which causes IPEX phenotype was identified in a Chinese child according to immunologic screening and gene sequencing. Infants with early-onset IDDM and persistent diarrhea should be suspected as IPEX, FOXP3 gene analysis will be a reliable diagnostic approach to IPEX.