An Yun-fei, Zhao Xiao-dong, Xu Feng, Yang Xi-qiang
P2 Laboratory, Chongqing Children's Hospital, Chongqing Medical University, Chongqing 400014, China.
Zhonghua Er Ke Za Zhi. 2009 Nov;47(11):824-8.
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).
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.
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.
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.
研究伴有严重早发性肠病、皮疹且伴有或不伴有胰岛素依赖型糖尿病（IDDM）的男童中FOXP3的变异及其表达情况。
对4例伴有严重早发性肠病、皮疹且伴有或不伴有IDDM的男童，采用流式细胞术检测外周血单个核细胞（PBMC）上FOXP3的表达，并进行FOXP3基因分析。一旦发现FOXP3基因变异，随后进行母系基因分析。通过聚合酶链反应（PCR）从基因组DNA中扩增FOXP3基因的所有11个外显子和剪接位点。采用逆转录聚合酶链反应扩增FOXP3转录本。对大量PCR产物直接进行正向和反向序列分析。将候选突变位点与100名健康对照者的位点进行比较以排除多态性。采用流式细胞术测定CD4+CD25+T细胞上FOXP3的表达以及CD4+T细胞中调节性T细胞（Tregs）的频率。
4例患者中有1例在第11外显子出现G13128A基因变异，导致甲硫氨酸370被异亮氨酸替代。100名健康对照者在同一位置均未发现序列变异，表明甲硫氨酸370被异亮氨酸替代并非多态性，而是一种新的错义突变。该患者的母亲被确定为该突变的携带者。与对照组相比，该患者外周血中Tregs的频率未降低，且FOXP3蛋白表达正常。
通过免疫筛查和基因测序，在中国一名儿童中鉴定出一种导致免疫失调多内分泌病肠病X连锁综合征（IPEX）表型的新的FOXP3错义突变。对于早发性IDDM和持续性腹泻的婴儿应怀疑为IPEX，FOXP3基因分析将是IPEX可靠的诊断方法。
