Yu Zi-hua, Ding Jie, Guan Na, Shi Yan, Zhang Jing-jing, Huang Jian-ping, Yao Yong, Yang Ji-yun
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Zhonghua Er Ke Za Zhi. 2004 Feb;42(2):108-12.
Autosomal recessive steroid-resistant nephrotic syndrome (SRNS) is a subgroup of familial nephrotic syndrome. A causative gene has been identified, that is NPHS2, in chromosome 1q25-31, which encodes podocin. This study aimed to detect NPHS2 mutation in a Chinese family with SRNS.
Renal biopsy was performed on the proband and her sibling for routine histologic and immunohistochemical investigation and electron microscopic examination. The expressions of podocin, nephrin, alpha-actinin and WT1 in glomeruli of the proband were detected by indirect immunofluorescence. Peripheral blood samples were collected for genetic analysis from the proband and her parents, and 53 adults with normal urinalysis. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons of NPHS2 were amplified by polymerase chain reaction. Mutational analysis was performed using denaturing high-performance liquid chromatography (DHPLC) and DNA fragments with aberrant elution profiles of both strands revealed by DHPLC were re-amplified and sequenced directly.
The histologic findings on kidney biopsies were focal segmental glomerulosclerosis. In controls, the distribution of staining with P35, rabbit against a human podocin recombinant protein (amino acids 135 - 383 = all the C-terminal part of the protein downstream the transmembrane domain), and P21, rabbit against a human podocin recombinant protein (amino acids 15 - 89 = all the N-terminal part of the protein upstream the transmembrane domain) showed a linear pattern along glomerular capillary walls on glomeruli, and the fluorescent intensity of the staining with P35 was intensely positive. The fluorescent intensity of the staining with P21 was positive. In the proband, the distribution of the staining with P35 showed uneven and nonlinear, and the fluorescent intensity of the staining with P35 was weakly positive. The staining with P21 was negative. The area, location, distribution and fluorescent intensity of the staining with nephrin, alpha-actinin and WT1 on glomeruli of the proband were the same as those in the controls. The DHPLC elution profiles of exon 4 of NPHS2 from the proband and her parent were aberrant. The chromatograms by sequencing detected in the exon 4 of NPHS2 showed a composite heterozygous mutation of both 467_468insT and 503G > A in the proband, a heterozygous mutation of 503G > A in her father, and a heterozygous mutation of 467_468insT in her mother, respectively.
The study demonstrated for the first time a novel mutation, 503G > A, of NPHS2 in Chinese kindred with autosomal recessive SRNS. A significantly decreased or negative expression was also revealed in glomeruli of the proband stained with two kinds of anti-podocin antibodies.
常染色体隐性遗传性类固醇抵抗型肾病综合征（SRNS）是家族性肾病综合征的一个亚组。已在1q25 - 31染色体上鉴定出致病基因NPHS2，其编码足突蛋白。本研究旨在检测一个中国SRNS家系中的NPHS2突变。
对先证者及其同胞进行肾活检，进行常规组织学、免疫组织化学检查及电子显微镜检查。通过间接免疫荧光检测先证者肾小球中足突蛋白、nephrin、α - 辅肌动蛋白和WT1的表达。采集先证者及其父母以及53名尿检正常的成年人的外周血样本进行基因分析。从外周血白细胞中分离基因组DNA。通过聚合酶链反应扩增NPHS2的8个外显子。使用变性高效液相色谱（DHPLC）进行突变分析，对DHPLC显示两条链洗脱图谱异常的DNA片段进行重新扩增并直接测序。
肾活检的组织学结果为局灶节段性肾小球硬化。在对照组中，用P35（兔抗人足突蛋白重组蛋白，氨基酸135 - 383 = 该蛋白跨膜结构域下游的整个C末端部分）和P21（兔抗人足突蛋白重组蛋白，氨基酸15 - 89 = 该蛋白跨膜结构域上游的整个N末端部分）染色的分布在肾小球的肾小球毛细血管壁上呈线性模式，且P35染色的荧光强度为强阳性。P21染色的荧光强度为阳性。在先证者中，P35染色的分布不均匀且非线性，P35染色的荧光强度为弱阳性。P21染色为阴性。先证者肾小球上nephrin（nephrin）、α - 辅肌动蛋白和WT1染色的面积、位置、分布及荧光强度与对照组相同。先证者及其父母的NPHS2第4外显子的DHPLC洗脱图谱异常。对NPHS2第4外显子测序的色谱图显示，先证者存在467_468insT和503G > A的复合杂合突变，其父亲存在503G > A的杂合突变，母亲存在467_468insT的杂合突变。
本研究首次在中国常染色体隐性遗传性SRNS家系中证实了NPHS2的一种新突变503G > A。在先证者肾小球中用两种抗足突蛋白抗体染色也显示表达显著降低或阴性。
