NPHS2 mutations in late-onset focal segmental glomerulosclerosis: R229Q is a common disease-associated allele

Abstract

Mutations in NPHS2, encoding podocin, have been identified in childhood onset focal and segmental glomerulosclerosis (FSGS). The role of NPHS2 in adult disease is less well defined. We studied 30 families with FSGS and apparent autosomal recessive inheritance and 91 individuals with primary FSGS. We screened family members for NPHS2 mutations. NPHS2 mutations appeared to be responsible for disease in nine of these families. In six families, the affected individuals were compound heterozygotes for a nonconservative R229Q amino acid substitution. This R229Q variant has an allele frequency of 3.6% in a control population. In these families, R229Q was the only mutation identified on one of the two disease-associated NPHS2 alleles. We used in vitro-translated podocin and purified nephrin to investigate the effect of R229Q on their interaction and found decreased nephrin binding to the R229Q podocin. These data suggest that this common polymorphism contributes to the development of FSGS. Chromosomes bearing the R229Q mutation share a common haplotype defining an approximately 0.2-Mb region. R229Q appears to enhance susceptibility to FSGS in association with a second mutant NPHS2 allele. Identification of R229Q mutations may be of clinical importance, as NPHS2-associated disease appears to define a subgroup of FSGS patients unresponsive to corticosteroids.

Figure 1

NPHS2 missense mutations in family FS-W. (a) DNA sequence chromatograms. Left: DNA sequence analysis of exon 5 amplified from an affected individual in family FS-W. A G→A transition predicts an Arg→Gln substitution. Right: DNA sequence analysis of exon 7 shows a C→T transition resulting in an Arg→Trp substitution. (b) Cosegregation of mutations with disease. Affected individuals are showed by filled circles (female) and squares (male). Agarose gel electrophoresis demonstrates the presence or absence of mutations within family FS-W. Top: R229Q mutations detected by ClaI digestion. The point mutation G755A results in a loss of ClaI site, and mutant R229Q alleles are shown by the top bands (545 bp), representing PCR products missing a ClaI site. Bottom: R291W mutations identified by PflMI digestion. A C941T mutation creates a PflMI restriction site and is visualized as two bands of 155 bp and 128 bp. Affected individuals are compound heterozygotes for R229Q and R291W. (c) Alignment of species orthologs of podocin. Amino acid sequences encoded by human NPHS2 (positions 221–240, GenBank NP_055440), rat NPHS2 (GenBank AF309631), mouse EST (GenBank AI552534), Drosophila melanogaster EST (GenBank AA141235), and Globodera rostochiensis EST (GenBank AW505792) were aligned by the PILEUP program (Wisconsin package version 10.0 [Unix]; Genetic Computer Group, Accelrys, San Diego, California, USA). Identical amino acid residues are indicated by shading. The arginine residue at position 229 is highlighted by an arrow.

Figure 2

Haplotype analysis for 1q25 markers surrounding the NPHS2 gene. Haplotypes of chromosomes bearing the R229Q-encoding mutations are shown. (a) The genomic context of the markers used. (b) Portions of the haplotypes that are totally conserved (dark gray) and partially shared (light gray) are indicated. The orders and intervals of the frame markers are determined based on Genemap ’99 (www.ncbi.nlm.nih.gov/genemap), the Sanger Center chromosome database. The markers CA126, CA1419, and D1S3760 are derived from the BACs harboring NPHS2 and two neighboring genes SOAT1 (sterol-O-acetyltransferase) and KIAA0475. Cent., centromere; Tel., telomere.

Figure 3

Nephrin binding assay. (a) The bottom autoradiograph shows in vitro–translated and labeled mutant and wild-type (WT) podocin after incubation with purified nephrin, immunoprecipitation with an anti-nephrin antibody, transfer to nitrocellulose, and exposure to radiographic film (as described in detail in Methods). Shown above is the result of Western analysis of the same blots using the anti-nephrin antibody for immunodetection. (b) Densitometry of the R229Q and R291W bands from repeated paired experiments. Intensity (with error bars indicating SEM) is given as a percentage of wild-type intensity, which is set by definition at 100%.