中国汉族人3个家族性激素耐药型肾病综合征家系WT1和PLCE1基因突变分析

Mutational analysis of WT1 and PLCE1 in three Chinese families with steroid-resistant nephrotic syndrome

目的分析中国汉族人家族性激素耐药型肾病综合征（SRNS）家系WT1和PLCE1基因突变及其特点。方法研究对象为A、B、C3个汉族人SRNS家系的先证者（已除外NPHS2基因突变）及其父母，A、B2个家系先证者的姐姐，50例尿检正常的汉族成年人作为对照人群。取所有研究对象外周静脉血3ml，提取基因组DNA，PCR扩增WT1基因全部10个外显子和PLCE1基因全部31个编码外显子及其周围的部分内含子，应用直接DNA序列测定法和限制性片段长度多态性PCR（RFLP—PCR）分析法检测WT1和PLCE1基因变异。结果未发现WT1和PLCE1基因的致病突变。但是，在3个SRNS家系的先证者检测到3个WT1基因多态性：126C〉T（P42P）、IVS5—64A〉G和903A〉G（R300R），其中IVS5—64A〉G为新发现的WT1基因多态性，126C〉T和903A〉G已见文献报道；还检测到13个PLCE1基因多态性一134A〉G、810T〉C（C270C）、960G〉A（E320E）、IVS11—28C〉G、IVS15＋26A〉C、4724G〉C（R1575P）、IVS20＋40C〉T、IVS21＋64G〉A、IVS22—26T〉A、5320C〉T（T1777I）、5780A〉G（H1927R）、IVS27＋24A〉G和IVS31＋48_49insT，其中IVS22—26T〉A为新发现的PLCE1基因多态性，其余12个PLCE1基因多态性已见公布。结论WT1和PLCE1基因突变不是本研究3个中国汉族人家族性SRNS家系的主要致病原因。

Objective To examine mutations in the WT1 and PLCE1 gene in three Chinese families with autosomal recessive steroid-resistant nephrotic syndrome （SRNS） once mutations in NPHS2 had been excluded. Methods Peripheral blood samples were collected for genetic analysis from three probands of three Chinese families and their parents, and two probands＇ siblings, and 50 adult volunteers with normal urinalysis. Genomic DNA was isolated from peripheral blood leucocytes. Ten exons and exon-intron boundaries of WT1, and 31 exons and exon-intron boundaries of PLCE1 were amplified by polymerase chain reaction （PCR）. Mutational analysis was performed by DNA sequencing directly and RFLP （restriction fragment length polymorphism） and/or PCR. Results No mutation in both WT1 and PLCE1 was identified in three probands from three Chinese families with autosomal recessive SRNS. However, three variants of WT1, 126C〉T, IVS5-64A〉G and 903A〉G, and 13 variants of PLCE1, -134A〉G, 810T〉C,960G〉A, IVS11-28C〉G, IVS15＋26A〉C, 4724G〉C, IVS20＋40C〉T, IVS21＋64G〉A, IVS22-26T〉 A, 5320C〉T, 5780A〉G, IVS27＋24A〉G and IVS31＋48_49insT, were detected in three probands and some controls, indicating that all these variants were gene polymorphisms. WT1 polymorphism IVS5-64A〉G, and PLCE1 polymorphism IVS22-26T〉A were novel. Conclusion All the encoding exons and exon-intron boundaries of both WT1 and PLCEI in three probands are examined, and no causative mutations in WT1 and PLCE1 are found, suggesting that mutation in WT1 and PLCE1 genes is not a major cause of the Chinese families with autosomal recessive SRNS.