Nephrin Mutations Can Cause Childhood-Onset Steroid-Resistant Nephrotic Syndrome

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Aure´lie Philippe,*† Fabien Nevo,*† Ernie L. Esquivel,*† Dalia Reklaityte,*† ʈ Olivier Gribouval,*† Marie-Jose`phe Teˆte,*‡ Chantal Loirat,§ Jacques Dantal, Michel Fischbach,¶ Claire Pouteil-Noble,** Ste´phane Decramer,†† Martin Hoehne,‡‡ Thomas Benzing,‡‡ Marina Charbit,‡ Patrick Niaudet,*†‡ and Corinne Antignac*†§§

† *Inserm U574, Hoˆpital Necker-Enfants Malades, Universite´ Paris Descartes, Faculte´deMe´ decine Rene´ Descartes, BRIEF COMMUNICATION ‡Pediatric Nephrology and §§Department of Genetics, Hoˆpital Necker-Enfants Malades, Assistance Publique-Hoˆpitaux de Paris, and §Pediatric Nephrology Department, Universite´ Paris VII, Assistance Publique-Hoˆpitaux de Paris, Hoˆpital ʈ Robert Debre´, Paris, ITERT, Department of Nephrology and Clinical Immunology, CHU Nantes, Nantes, ¶Nephrology Dialysis Transplantation Children’s Unit, Hoˆpital de Hautepierre, Strasbourg, **Transplantation and Nephrology Unit, Centre Hospitalier Lyon-Sud, Pierre-Be´nite, and ††Department of Pediatric Nephrology, Hoˆpital des Enfants, and Inserm, U858/I2MR, Department of Renal and Cardiac Remodeling, Toulouse, France; and ‡‡Department of Medicine IV, University of Cologne, Cologne, Germany

ABSTRACT Classically, infants with mutations in NPHS1, which encodes nephrin, present with but has since been described in other nephrotic syndrome within the first 3 mo of life (congenital nephrotic syndrome of populations.3–5 Nephrin is a single-pass the Finnish-type), and children with mutations in NPHS2, which encodes podocin, transmembrane protein consisting of present later with steroid-resistant nephrotic syndrome. Recently, however, eight extracellular Ig-like modules, a fi- NPHS2 mutations have been identified in children with congenital nephrotic syn- bronectin type III–like motif, and a cy- drome. Whether NPHS1 mutations similarly account for some cases of childhood tosolic C-terminal tail. Homodimers of steroid-resistant nephrotic syndrome is unknown. In this study, 160 patients who nephrin and heterodimers with the glo- belonged to 142 unrelated families and presented with nephrotic syndrome at merular protein NEPH1 constitute the least 3 mo after birth were screened for NPHS1 variants once mutations in NPHS2 structural basis of the slit diaphragm.6,7 had been excluded. Compound heterozygous NPHS1 mutations were identified in In addition to its structural function, one familial case and nine sporadic cases. Mutations included protein-truncating nephrin is involved in podocyte signal- nonsense and frameshift mutations, as well as splice-site and missense variants. ing events.8 Mutations were classified as “severe” or “mild” using prediction algorithms and Mutations in the NPHS2 gene en- functional assays. Most missense variants trafficked normally to the plasma mem- coding podocin were, thereafter, de- brane and maintained the ability to form nephrin homodimers and to heterodimer- scribed in patients presenting with au- ize with NEPH1, suggesting retained function. The presence of at least one “mild” tosomal recessive SRNS with onset mutation in these patients likely explains the later onset and milder course of typically between 3 mo and 5 yr of age.9 disease. These results broaden the spectrum of renal disease related to nephrin NPHS2 mutations account for 42% of mutations. Received January 17, 2008. Accepted May 14, J Am Soc Nephrol 19: 1871–1878, 2008. doi: 10.1681/ASN.2008010059 2008.

Published online ahead of print. Publication date available at www.jasn.org.

Idiopathic nephrotic syndrome (NS) improved understanding of the heredi- A.P., F.N., and E.L.E. contributed equally to this represents a heterogeneous group of tary basis of NS.1 Mutations in the work. glomerular disorders occurring mainly NPHS1 gene, encoding the podocyte- Correspondence: Dr. Corinne Antignac, Inserm in children and may be classified as ste- expressed protein nephrin, lead to the U574, 6e`mee´ tage, Tour Lavoisier, Hoˆpital Necker- Enfants Malades, 147, rue de Se`vres, 75015 Paris, roid-sensitive (SSNS) or steroid-resis- congenital NS of the Finnish-type France. Phone: ϩ33-1-44-49-50-98; Fax: ϩ33-1- tant (SRNS) on the basis of response to (CNF), which is inherited in an autoso- 44-49-02-90; E-mail: [email protected] corticosteroid therapy. Gene discovery mal recessive manner. It affects approx- Copyright ᮊ 2008 by the American Society of efforts in the past decade have led to an imately 1:10,000 newborns in Finland2 Nephrology

J Am Soc Nephrol 19: 1871–1878, 2008 ISSN : 1046-6673/1910-1871 1871 BRIEF COMMUNICATION www.jasn.org familial and 10% of sporadic cases of and who presented with noncongenital volvement was reported. At the end of SRNS.10 Proper assembly of nephrin and onset and a more protracted course of follow-up, only five patients had reached other slit diaphragm constituents and renal disease. ESRD, at a mean age of 13.6 yr (range 6 to trafficking to the plasma membrane and Mutation screening was performed in 25 yr), whereas six patients had normal to lipid rafts require interaction with a cohort of familial and sporadic cases of serum creatinine (Table 1). Four patients podocin.11,12 SRNS, for which NPHS2 mutations had successfully received a renal allograft, Classically, mutations in the NPHS1 been excluded by sequencing the exonic with no disease recurrence. and NPHS2 genes have been distin- regions and intronic junctions. Nephrin In total, 14 mutations, 12 of which are guished by their implications in familial mutations were found in one family with novel, were identified in 10 unrelated pa- congenital (onset at birth to 3 mo) and in two affected siblings, among 44 families tients. These included six nonsense and childhood-onset (later than 3 mo) cases, with familial SRNS, using a combination frameshift, two splice-site, and six mis- respectively. Hinkes et al.13 recently con- of linkage analysis and NPHS1 sequencing sense mutations, uniformly distributed firmed the findings of others that pa- (Table 1). Of 98 patients with sporadic throughout the NPHS1 gene (Figure 1). tients with NPHS1 mutations present SRNS, nine individuals were compound Segregation of mutations confirmed re- with NS exclusively during the first 3 mo heterozygotes for NPHS1 mutations (Ta- cessive inheritance. All nonsense and of life.3–5,14 They also identified NPHS2 ble 1). The mean age of onset of NS in these frameshift mutations are predicted to re- mutations in 39% of children with con- 11 patients was 3.0 yr (range 6 mo to 8 yr); sult in a truncated protein. In addition, genital onset of NS,13 as described previ- hence, later than previously described for the c.3720_3735delC (p.L1240fs1286X) ously,15 therein broadening the spec- patients with NPHS1 mutations. The NS mutation is unusual because it involves a trum of NPHS2-associated renal disease. was resistant to corticosteroids in all cases, deletion of the last seven nucleotides of We therefore sought to determine as well as to cyclosporine and cyclophos- the NPHS1 coding and of 9 bp of the 3Ј- whether mutations in the nephrin gene phamide, when additional treatments untranslated regions. The resulting pro- may similarly account for a wider range were attempted. Renal biopsy performed tein lacks the usual terminal valine resi- of disease presentations to include child- at the time of presentation revealed me- due and instead bears an additional 45 hood-onset SRNS. Our studies revealed sangioproliferative lesions in one pa- amino acids with no known sequence that compound heterozygous mutations tient, minimal-change disease in six pa- homology. in the NPHS1 gene were responsible for tients, and FSGS in three patients (Table Six missense mutations were identi- SRNS in a cohort of patients in whom 1). Characteristic tubular lesions of CNF fied in this cohort of patients, and all NPHS2 mutations had been excluded were not observed. No extrarenal in- were ruled out as polymorphisms by se-

Table 1. Clinical data of patients who had SRNS and in whom NPHS1 mutations were identifieda Age of Onset Patient Gender Biopsy Therapy Evolution Mutation 1 Severe Mutation 2 Mild Pu (NS) (yr) 1420 F 0.25 (3.00) MCNS CS, CP Normal Cr at 14 yr c.609–2A3C (M) c.319G3A p.A107T (P) 446 F 0.80 (0.80) MCNS CS ESRF at 9 yr c.3720_3735del16 c.1724C3A p.L1240fs1286Xb (P) p.P575Q (M) 1075 F 0.50 (0.50) FSGS Unknown ESRF at 13 yr c.1379G3A c.2928G3T p.R460Qb (?) p.R976S (?) 841 F 3.80 (3.80) FSGS CS, CsA Normal Cr at 6 yr c.468C3G c.2928G3T p.Y156X (P) p.R976S (M) 466 F 0.25 (0.75) MCNS CS, CP Normal Cr at 10 yr c.2479C3T c.2928G3T p.R827X (M) p.R976S (P) 1167 F 3.10 (3.10) FSGS CS, CP ESRF at 15 yr c.516delC c.2928G3T p.T712fs175X (M) p.R976S (P) 693 M 8.00 (8.00) MCNS CS Normal Cr at 16 yr c.1134–1135delGC c.286C3G p.R379fs417X (M) p.L96V (P) 1407 F 5.00 (5.00) MCNS CS, CP ESRF at 25 yr c.516delC c.2928G3T p.T712fs175X (M) p.R976S (P) 634 M 3.00 (3.00) MP Unknown ESRF at 6 yr c.1491delC c.2072–6C3G (P) p.S494fs547X (M) 771c F 2.20 (2.80) MCNS CS, CsA Normal Cr at 9 yr c.2495T3C c.2928G3T 1462c M 2.50 (2.50) Not performed No treatment Normal Cr at 6 yr p.L832P (P) p.R976S (M) aCP, cyclophosphamide; Cr, creatinine; CS, corticosteroids; CsA, cyclosporin A; ESRF, end-stage renal failure; MCNS, minimal-change glomerulonephritis; MP, mesangioproliferative glomerulonephritis; Pu, proteinuria. bThis mutation may potentially be a “mild” one. Mode of transmission of mutant alleles are indicated in parentheses: M, maternal; P, paternal; ?, unknown. cSiblings with the same parents.

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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. quencing at least 176 control chromo- c.609–2A3C mutation affects the in- membrane antigen, a plasma mem- somes, and 352 control chromosomes variant A of the splice acceptor site, re- brane marker, was observed for the for the most commonly detected sulting in a decrease in the score from p.L96V, p.A107T, p.P575Q, p.R460Q, p.R976S mutation. These missense 0.88 to 0.00 and hence would be expected and p.R976S mutants, just as with changes were evaluated for pathogenicity to lead to absence of correct splicing (Ta- wild-type nephrin, under nonperme- using two widely used prediction meth- ble 2). Conversely, the c.2072–6C3G abilized conditions (Figure 2B, Supple- ods: SIFT (Sorting Intolerant From Tol- mutation only moderately decreases the mental Figure S2). Furthermore, im- erant)16 and PolyPhen (Polymorphism score of the normal splice junction se- munolabeling under permeabilized Phenotyping).17 SIFT predicts whether quence from 0.88 to 0.26. Normally conditions revealed co-localization of an amino acid substitution will affect spliced nephrin transcripts are, there- the p.L832P variant with calnexin, sim- protein function on the basis of the de- fore, likely to exist, although in reduced ilar to the p.S366R mutant,19 suggest- gree to which the amino acid residue is amounts (Table 2). In addition, the ing retention in the endoplasmic retic- conserved during evolution, thereafter c.2928G3T (p.R976S) mutation, which ulum (Figure 2C). designating changes as either tolerated or leads to a probably damaging substitu- Thereafter, the effects on nephrin deleterious.16 Of the six missense mutation in the fibronectin-like domain (ac- homodimerization and NEPH1 hetero- tions, five were classified as deleterious, cording to the PolyPhen program)17 af- dimerization of the missense p.L96V, with the exception of the p.R460Q muta- fects the first base of exon 22. The Genie p.A107T, p.R460Q, and p.P575Q mutants, tion (Table 2). PolyPhen designates vari- program predicted a slight decrease in which affect the nephrin Ig domains, ants as either probably or possibly dam- the splicing score from 0.96 to 0.79. De- were tested. Upon co-transfection of aging or benign on the basis of sequence spite this modest score change, an in vitro mutagenized nephrin-ec.Fc and Flag- annotation, sequence alignment, and exontrap system demonstrated an alter- tagged nephrin constructs in HEK293 structural parameters.17 PolyPhen pre- ation in the splicing around exon 22 cells, co-immunoprecipitation experi- dicted only three of the missense changes (Supplemental Figure S1). Unfortu- ments demonstrated maintenance of to be damaging: p.P575A, p.L832P, and nately, verification in vivo was not possi- nephrin–nephrin interaction, despite p.R976S mutations (Table 2). ble given the absence of available RNA the missense changes (Figure 3A). Sim- Two splice-site mutations were iden- from these patients. ilarly, variants heterodimerized with tified. Potential splice sites were addi- To determine the putative effects of full-length V5-tagged NEPH1 in tran- tionally evaluated using the neural net- missense mutations, we additionally siently transfected cells (Figure 3B). works program Genie, and scores performed functional studies. The in- Our assays were not, however, suffi- ranging from 0 to 1 (where 1 is indicative tracellular trafficking of V5- and Flag- ciently quantitative to assess whether of the presence of an ideal splice site) tagged missense nephrin variants (Fig- the strengths of these interactions were were assigned and compared between ure 2A) was studied upon transient altered. wild-type and mutant sequences to de- transfection in HeLa cells. Except for Our analysis of a cohort of patients termine the extent to which the mutation the p.L832P mutant, co-localization presenting with familial and sporadic is predicted to alter a splice site.18 The with fluorescently labeled epithelial SRNS after 3 mo of life broadens the phe-

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notypic spectrum of renal disease associated with mutations in the NPHS1 gene encoding nephrin. Previously, NS caused by nephrin mutations had been de- Intact Intact Intact Intact NEPH1 scribed exclusively in children presenting at the time of birth or within the first Heterodimerization 3 mo of life. Our study identified patients bearing NPHS1 mutations in which the renal disease presented later in childhood, was resistant to steroids, and had a

Intact Intact Intact Intact spectrum of renal histologic findings Nephrin atypical for CNF,20 ranging from mini-

Homodimerization mal-change disease to FSGS. These fea- tures are reminiscent of SRNS caused by NPHS2 mutations. A protracted course was also observed, averaging 13.6 yr from Cellular membrane membrane membrane membrane reticulum membrane disease onset to end-stage renal failure in Localization five of 11 patients. We attempted to classify these mutations on the basis of prediction algorithms and functional studies as either SIFT “severe” or “mild.” Nephrin mutations Prediction

Not tolerated Plasma Not tolerated Endoplasmic Not tolerated Plasma were classified as severe when they result in truncated, aberrantly spliced, or non- functional proteins. This includes all nonsense and frameshifting, the c.609–

Score 2A3C splice, and the endoplasmic re- PolyPhen damaging) damaging) damaging) ticulum–retained p.L832P missense mu- 1.40 (benign) Tolerated1.80 (possibly 2.20 Plasma (probably 1.34 (benign) Not tolerated1.50 (benign) Plasma Not tolerated Plasma 2.20 (probably tations. Greater than 75% of missense nephrin variants leading to CNF have previously been shown to be retained in the endoplasmic reticulum.21 Mutations Control were designated mild when partial func- Chromosomes 0/176 0/190 0/176 0/182 0/188 0/188 0/352 0/186 tion of nephrin was likely maintained. The five other missense mutations were shown to retain the abilities to traffic in the cell properly and to homo- and heterodimerize with NEPH1 and thus were Mutant:0.79 Mutant:0.00 Mutant:0.26

Acceptor Site classified as mild. The c.2072–6C3G Score of Donor/ splice mutant likely allows for some cor- 3 4 3 rect splicing and therefore is included as 11 13 18 22 Normal:0.95 18 IVS5 Normal:0.88

Exon a mild mutation.

Involved The mutation detection rate in familial cases was lower than might normally be expected, because most of these pa- mutations according to prediction algorithms and functional studies tients were offsprings of consanguineous

Coding relationships; thus, compound heterozy- Sequence Alteration

NPHS1 gosity is less likely. Compound heterozy- p.T172fs175X 4 p.R460Q p.Y156X Splice site (?) gosity for at least one mild mutation in all cases may explain the lesser severity of disease in our cohort of patients. Indeed, Effects of 22 22 less severe phenotypes of cystinosis, Pier- son syndrome, and autosomal recessive Alteration Nucleotide polycystic kidney disease have been ob- c.319G 3 Ac.379G 3 A p.A107T c.1724C 3 Ac. 2495T 3 C p.P575Q c.2928G 3 T p.L832P p.R976S c.516delC c.1134–1135delGC p.R379fs417Xc.1491delCc.2479C 3 Tc. 3720–3735del16 p.L1240fs1286X p.S494fs547X 9 p.R827X 29 12 c.609–2A 3 C Splice site c.468C 3 G c.2072–6C 3 G Splice site IVS15 Normal:0.88 c.286C 3 G p.L96V Table 2. served in patients bearing missense mu-

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tance of screening for NPHS1 mutations in patients with SRNS for disease prognostication. Our method of classifying mutations as either severe or mild was based on ap- plying the best available prediction algorithms and coupling these with estab- lished functional assays of known nephrin functions. Despite this added rigor, there were, however, discrepancies in the results of functional assays, of prediction algorithms, and in clinical correlation in the case of previously described mutations. For instance, although the p.R460Q mutant is predicted to be a tolerated variant and was shown to traffic normally in the cell and to homodimer- ize and to heterodimerize with NEPH1, it had previously been identified in cases of CNF,3,5,25 suggesting that the mutation may disrupt other structural or functional properties of nephrin. It is likewise possible that this mutation leads to a decrease in the ability to homo- or heterodimerize, a feature that we were unable to quantify with precision in our assays. Future studies will additionally need to address whether missense variants that traffic to the membrane affect nephrin phosphorylation, actin reorga- nization in the cytoskeleton of podocytes,26 or downstream signaling events involved in transcriptional regulation27 and apoptosis.28 Conversely, the protein- truncating p.L1240fs1286X mutation might be considered a mild mutation because it involves the very C-terminal end of the protein. This is reminiscent of the p.R1160X mutant, which results in a Figure 2. Effects of missense mutations on nephrin intracellular trafficking. (A) Schematic truncation of the nephrin protein down- representation of full-length V5- and Flag-tagged and ec.Fc-tagged nephrin constructs stream of the last amino acid residue of and the locations of missense variants. (B) The p.L96V and p.R460Q mutants co-localized exon 27 and which has been shown to be with epithelial membrane antigen (green) at the plasma membrane, similar to wild-type expressed in the kidney.4 This mutation nephrin, when transiently transfected into HeLa cells. Nephrin was detected using an results in renal disease of lesser severity in anti-V5 antibody (red), under nonpermeabilized conditions. Conversely, the p.L832P 50% of female patients.4 mutant failed to traffic to the membrane. (C) Similar to the p.S366R variant used as a Mutations in the NPHS2 gene and in positive control, the p.L832P mutant co-localized with calnexin (green) in transiently exons 8 and 9 of the WT1 gene were ex- transfected HeLa cells, suggesting retention in the endoplasmic reticulum. Cells were cluded in our cohort of patients after se- ϫ permeabilized with saponin before immunolabeling. Magnification, 1000. quencing the exonic and intronic splice junctional regions. We are, therefore, tations in the CTNS,22 LAMB2,23 and mutations, as demonstrated by patient confident that the renal disease in these PKHD124 genes, respectively, which 446, in whom two potentially mild mu- patients may be attributed to the patho- maintained residual protein function. At tations were detected. Additional work genic NPHS1 mutations we have identi- present, however, it is difficult to be cer- is required, but such genotype–pheno- fied, rather than to the triallelic inheri- tain of the functional effects of these type correlations exemplify the impor- tance of NPHS1 and NPHS2 mutations

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them, 62 patients belonging to 44 families (31 consanguineous) were classified as familial cases, defined previously10 either as families in which two or more children were affected or families in which one affected child was the product of a consanguineous relationship. Cases were defined as sporadic when only one child, in families with no history of consan- guinity, was affected. Mean age at discovery (proteinuria and/or NS) was 67 Ϯ 49 mo in AR SRNS (n ϭ 57), whereas it was lower in sporadic cases (54 Ϯ 39 mo; n ϭ 98). In- formed consent was obtained from patients or their parents. Experiments were performed in accordance with French ethical committee recommendations.

Mutation Analysis Genomic DNA was extracted from peripheral blood by standard methods. Linkage analyses were carried out using four polymorphic mi- Figure 3. Effects of missense mutations on nephrin homodimerization and heterodimer- crosatellite markers (D19S224, D19S225, ization with NEPH1. (A) HEK 293T cells were transfected with wild-type or mutant D19S425, and D19S608), spanning a 0.5-Mb full-length nephrin-ec.Fc and V5-nephrin-Flag constructs. Nephrin-ec.Fc was immuno- region containing the NPHS1 locus. Pedi- precipitated using Protein A-Sepharose beads. Immunoblotting using anti-Flag antibody grees were constructed with CYRILLIC 2.1 revealed intact dimerization between the two nephrin constructs. These data are repre- (Cherwell Scientific, Oxford, UK). Mutations sentative of three experiments. (B) Similarly, cells were transfected with wild-type or in the NPHS2 gene encoding podocin were mutant full-length nephrin-ec.Fc and full-length V5-NEPH1 plasmid constructs. Protein excluded in all cases by sequencing exonic A-Sepharose beads were used to immunoprecipitate nephrin. Immunoblotting using and intronic junctional regions, as were mu- anti-V5 antibody revealed intact heterodimerization between missense nephrin mutants tations in exons 8 and 9 of the WT1 gene in and NEPH1. These data represent five replicate experiments. phenotypically female patients. Mutation screening of NPHS1 was performed by direct in a few rare cases, as had been reported patients carrying similar NPHS1 muta- sequencing of the 29 coding exons and the by others.4,29 It is conceivable that vari- tions should be spared immunosuppres- adjacent intronic junctions. All variants were ants in the promoter and intronic re- sive therapies. Furthermore, therapies screened in at least 88 unrelated controls us- gions of the NPHS2 gene may have been aimed at correcting mis-trafficking of ing direct sequence analysis and/or the missed, but no pathogenic mutations in misfolded mutant proteins in the cell SNAPshot method.10 In silico analyses of mis- the promoter region of the gene have may provide hope for retarding the pro- sense mutations was carried out using the previously been associated with SRNS; gression of SRNS by preserving residual SIFT (http://blocks.fhcrc.org/sift/SIFT.html)16 however, variants and haplotypes in the nephrin function.21,31 and PolyPhen (http://genetics.bwh.harvard. promoter region of the podocin gene edu/pph/)17 software. The evaluation of splice have been associated with variations in sites and potential splice sites was performed us- the levels of proteinuria in patients with CONCISE METHODS ing the Genie program (http://www.fruitfly. IgA nephropathy but not in FSGS.30 It org/seq_tools/splice.html).32 will be of great interest in the future to Patients explore the possibility of genetic modifi- A total of 160 patients belonging to 142 fam- Plasmid Constructs and Antibodies cation as a basis for phenotypic variabil- ilies were included in this study. They origi- The generation of full-length Nephrin-Flag, ity in patients with familial forms of NS. nated from a large number of countries Nephrin-ec.Fc, and V5-NEPH1 constructs These studies, however, will require worldwide, but most are from Europe has previously been described.7,24 For sub- larger numbers of patients and should (mainly from various regions in France) and cloning of the NPHS1-Flag cDNA into the V5 involve genome-wide approaches. from North Africa. All patients presented be- plasmid, a unique MluI restriction site was Finally, our findings bear significant tween 3 mo and 18 yr of age with SRNS, de- inserted in position 57 by site-directed mu- implications on the therapy of NS. As fined as a lack of response to three bolus in- tagenesis (Quick-Change Kit; Stratagene, La with patients bearing NPHS2 muta- fusions of methylprednisolone, followed by 4 Jolla, CA), digested with MluI and NotI and tions,15 our limited cohort revealed that wk of treatment with prednisone. Among ligated into corresponding sites in the plas-

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mid. Site-directed mutagenesis was similarly washed extensively with lysis buffer, and keri U, Tryggvason K, Scambler P: Geno- used to generate all missense variants, fol- bound proteins were split in two and resolved type/phenotype correlations of NPHS1 and lowed by verification by automated sequenc- on a 6% SDS-PAGE. Western blot analysis NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in ing. Commercial antibodies used for this was performed with anti-IgG (diluted glomerular filtration. Hum Mol Genet 11: study are detailed in Supplemental Table S1. 1:5000), anti-Flag (diluted 1:2000), or mouse 379–388, 2002 Affinity-purified rabbit anti-podocin anti- anti-V5 (diluted 1:2000) antibodies followed 5. Sako M, Nakanishi K, Obana M, Yata N, bodies were generated in the laboratory and by incubation with appropriate horseradish Hoshii S, Takahashi S, Wada N, Takahashi Y, have previously been described.33 peroxidase–coupled secondary antibodies. Kaku Y, Satomura K, Ikeda M, Honda M, Iijima K, Yoshikawa N: Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese pa- Cellular Immunolocalization Studies tients with congenital nephrotic syndrome. 5 HeLa cells (7 ϫ 10 ) were plated on poly-L- ACKNOWLEDGMENTS Kidney Int 67: 1248–1255, 2005 lysine–coated glass coverslips (Sigma, St. 6. Liu G, Kaw B, Kurfis J, Rahmanuddin S, Kan- war YS, Chugh SS: Neph1 and nephrin inter- Louis, MO) and grown to 90% confluence in We acknowledge financial support for this work action in the slit diaphragm is an important DMEM supplemented with 10% FBS. Cells from the Association pour l’Information et la determinant of glomerular permeability. were transiently transfected 24 h later with 0.5 Recherche sur les Maladies Re´nales Ge´ne´tiques J Clin Invest 112: 209–221, 2003 ␮g of plasmid DNA, using Lipofectamine ac- (AIRG), the Programme Hospitalier de Re- 7. Gerke P, Huber TB, Sellin L, Benzing T, Walz G: Homodimerization and heterodimeriza- cording to the manufacturer’s instructions cherche Clinique (AOM02123), and the GIS- tion of the glomerular podocyte proteins (Invitrogen, Carlsbad, CA), thereby achiev- Institute des Maladies Rares (4MR02F). A.P. nephrin and NEPH1. J Am Soc Nephrol 14: ing 80% transfection efficiency. After 48 h of was the recipient of a PhD grant from the Min- 918–926, 2003 incubation, HeLa cells were rinsed in PBS and iste`re de l’Enseignement Supe´rieur et de la Re- 8. Benzing T: Signaling at the slit diaphragm. incubated with rabbit anti-V5 (diluted 1:1300 cherche. E.L.E. was supported by a Ruth Kirch- J Am Soc Nephrol 15: 1382–1391, 2004 9. Boute N, Gribouval O, Roselli S, Benessy F, in PBS) and/or mouse anti–epithelial mem- stein National Research Service Award Lee H, Fuchshuber A, Dahan K, Gubler MC, brane antigen (diluted 1:50) antibodies for 15 (DK065409) from the National Institutes of Niaudet P, Antignac C: NPHS2, encoding min at 4°C. Cells were fixed in 4% parafor- Health, National Institute of Diabetes and Di- the glomerular protein podocin, is mutated maldehyde for 20 min and subsequently gestive and Kidney Diseases, and by the EuRe- in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24: 349–354, treated with 50 mM NH4Cl. Alternatively, Gene Project, an integrated project (5085) of 2000 cells were incubated for 1 h with mouse an- the 6th Framework Program of the European 10. Weber S, Gribouval O, Esquivel EL, ti-V5 (diluted 1:200) or anti-calnexin (di- Commission. Moriniere V, Tete MJ, Legendre C, Niaudet luted 1:100) in PBS/BSA/saponin for perme- P, Antignac C: NPHS2 mutation analysis abilization. Coverslips were then incubated shows genetic heterogeneity of steroid-re- with fluorescently labeled secondary antibod- sistant nephrotic syndrome and low post- ies for 1 h, before mounting using Fluoprep DISCLOSURES transplant recurrence. Kidney Int 66: 571– C.A. and E.L.E. are recipients of a Renal Innova- 579, 2004 (Biome´rieux, Lyon, France). Images were ob- 11. Schwarz K, Simons M, Reiser J, Saleem MA, tions Program grant from Genzyme Corp. tained using a Zeiss Pascal confocal laser scan- Faul C, Kriz W, Shaw AS, Holzman LB, Mun- ning microscope (Carl Zeiss, Jena, Germany) del P: Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 108: Homo- and Heterodimerization 1621–1629, 2001 Studies REFERENCES 12. 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J Am Soc Nephrol 19: 1871–1878, 2008 Nephrin and SRNS 1877 BRIEF COMMUNICATION www.jasn.org

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1878 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 1871–1878, 2008