Nephrin mutations can cause childhood-onset steroid-resistant nephrotic syndrome

Abstract

Classically, infants with mutations in NPHS1, which encodes nephrin, present with nephrotic syndrome within the first 3 mo of life (congenital nephrotic syndrome of the Finnish-type), and children with mutations in NPHS2, which encodes podocin, present later with steroid-resistant nephrotic syndrome. Recently, however, NPHS2 mutations have been identified in children with congenital nephrotic syndrome. Whether NPHS1 mutations similarly account for some cases of childhood steroid-resistant nephrotic syndrome is unknown. In this study, 160 patients who belonged to 142 unrelated families and presented with nephrotic syndrome at least 3 mo after birth were screened for NPHS1 variants once mutations in NPHS2 had been excluded. Compound heterozygous NPHS1 mutations were identified in one familial case and nine sporadic cases. Mutations included protein-truncating nonsense and frameshift mutations, as well as splice-site and missense variants. Mutations were classified as "severe" or "mild" using prediction algorithms and functional assays. Most missense variants trafficked normally to the plasma membrane and maintained the ability to form nephrin homodimers and to heterodimerize with NEPH1, suggesting retained function. The presence of at least one "mild" mutation in these patients likely explains the later onset and milder course of disease. These results broaden the spectrum of renal disease related to nephrin mutations.

Figure 1.

Schematic diagram of the NPHS1 mutations detected in a cohort of patients with SRNS and their localization with respect to exons and protein functional domains. Nephrin consists of Ig-like domains, a fibronectin III (FNIII)-like domain, a transmembrane (TM) region, and a cytosolic C-terminal tail. Mutations included nonsense and frameshift, splice-site, and missense mutations and were classified as severe or mild on the basis of prediction algorithms and functional analyses.

Figure 2.

Effects of missense mutations on nephrin intracellular trafficking. (A) Schematic representation of full-length V5- and Flag-tagged and ec.Fc-tagged nephrin constructs and the locations of missense variants. (B) The p.L96V and p.R460Q mutants co-localized with epithelial membrane antigen (green) at the plasma membrane, similar to wild-type nephrin, when transiently transfected into HeLa cells. Nephrin was detected using an anti-V5 antibody (red), under nonpermeabilized conditions. Conversely, the p.L832P mutant failed to traffic to the membrane. (C) Similar to the p.S366R variant used as a positive control, the p.L832P mutant co-localized with calnexin (green) in transiently transfected HeLa cells, suggesting retention in the endoplasmic reticulum. Cells were permeabilized with saponin before immunolabeling. Magnification, ×1000.

Figure 3.

Effects of missense mutations on nephrin homodimerization and heterodimerization with NEPH1. (A) HEK 293T cells were transfected with wild-type or mutant full-length nephrin-ec.Fc and V5-nephrin-Flag constructs. Nephrin-ec.Fc was immunoprecipitated using Protein A-Sepharose beads. Immunoblotting using anti-Flag antibody revealed intact dimerization between the two nephrin constructs. These data are representative of three experiments. (B) Similarly, cells were transfected with wild-type or mutant full-length nephrin-ec.Fc and full-length V5-NEPH1 plasmid constructs. Protein A-Sepharose beads were used to immunoprecipitate nephrin. Immunoblotting using anti-V5 antibody revealed intact heterodimerization between missense nephrin mutants and NEPH1. These data represent five replicate experiments.