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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NPHS2基因及突变特点。方法 研究对象为一个中国汉族人家族性SRNS家系中的先证者及其兄和父母 ,对照人群为 5 3例尿检正常成年人。取肾组织做常规光镜检查和 podocin、nephrin、α actinin、WT1表达的检查。提取外周血白细胞基因组DNA ,PCR扩增NPHS2基因 8个外显子 ;应用变性高效液相色谱(DHPLC)分析PCR产物 ,对DHPLC洗脱曲线异常者进行DNA序列测定。结果 先证者及其兄肾脏病理示 :局灶节段性肾小球硬化。先证者肾组织 podocin重组蛋白P35染色弱阳性 ,P2 1染色阴性 ;nephrin、α actinin和WT1染色的范围、定位、分布和荧光强度与对照组无差异。DHPLC示先证者及其父母洗脱曲线异常 ;DNA测序证实先证者为 4 6 7_4 6 8insT与 5 0 3G >A复合杂合突变 ,其父为 5 0 3G >A杂合突变 ,其母为 4 6 7_4 6 8insT杂合突变。结论 首次发现了一中国汉族人家族性SRNS家系中NPHS2基因突变——— 4 6 7_4 6 8insT与 5 0 3G >A复合杂合突变 ,且 5 0 3G >A突变为新发现的突变 ;同时还发现该复合杂合突变引起肾组织中抗 podocin重组蛋白P35的染色明显减弱 ,抗 podocin重组蛋白P2 1的染色阴性。
Objective To analyze the gene and mutation characteristics of NPHS2 in Chinese Han population with familial steroid resistant nephrotic syndrome (SRNS). Methods The subjects were probands and their relatives from a Chinese Han pedigree with SRNS pedigrees, and control subjects were 53 normal urine test adults. The kidneys were taken routine light microscopy and podocin, nephrin, α actinin, WT1 expression examination. The genomic DNA of peripheral blood leukocytes was extracted and 8 exons of NPHS2 gene were amplified by PCR. The PCR products were analyzed by denaturing high performance liquid chromatography (DHPLC), and the DNA sequence of DHPLC eluting curve was determined. Results proband and his brother kidney pathology: focal segmental glomerulosclerosis. The probands podocin recombinant protein P35 staining weakly positive, P2 1 negative staining; nephrin, α actinin and WT1 staining range, location, distribution and fluorescence intensity and the control group no difference. DHPLC showed probands and their parents eluting curve abnormalities; DNA sequencing confirmed that the proband was 4 6 7_4 6 8insT and 50 3G> A complex heterozygous mutation, the father of 50 3G> A heterozygous mutation, the mother As 4 6 7_4 6 8insT heterozygous mutation. Conclusions This study firstly found that the mutation of NPHS2 gene in Chinese Han familial SRNS pedigrees --- 467_4 6 8insT and 50 3G> A complex heterozygous mutations, and 50 3G> A mutations were found to be newly found mutations It was found that the hybrid heterozygous mutation caused a significant decrease in the staining of anti-podocin recombinant protein P35 in renal tissues and negative in the anti-podocin recombinant protein P2 1.