OBJECTIVEAutosomal 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.

METHODSRenal 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.

RESULTSThe 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.

CONCLUSIONThe 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.