Article

Clinical Value of NPHS2 Analysis in Early- and Adult-Onset Steroid-Resistant Nephrotic Syndrome

Sheila Santín,* Ba´rbara Tazo´n-Vega,* Irene Silva,* María A´ ngeles Cobo,† Isabel Gime´nez,‡ Patricia Ruíz,* Rafael García-Maset,‡ Jose´Ballarín,‡ Roser Torra,‡ and Elisabet Ars,* on behalf of the FSGS Spanish Study Group

Summary Background and objectives To date, very few cases with adult-onset focal segmental glomerulosclerosis *Molecular Biology (FSGS) carrying NPHS2 variants have been described, all of them being compound heterozygous for the Laboratory, Fundacio´ p.R229Q variant and one pathogenic mutation. Puigvert, Universitat Auto`noma de Design, setting, participants, & measurements Mutation analysis was performed in 148 unrelated Spanish Barcelona, REDinREN, Instituto de patients, of whom 50 presented with FSGS after 18 years of age. Pathogenicity of amino acid substitutions Investigacio´n Carlos III, was evaluated through an in silico scoring system. Haplotype analysis was carried out using NPHS2 single Barcelona, Spain; nucleotide polymorphism and microsatellite markers. †Nephrology Department, Hospital Universitario de Results Compound heterozygous or homozygous NPHS2 pathogenic mutations were identified in seven Canarias, Tenerife, childhood-onset steroid-resistant nephrotic syndrome (SRNS) cases. Six additional cases with late child- Spain; and ‡Nephrology hood- and adult-onset SRNS were compound heterozygotes for p.R229Q and one pathogenic mutation, Department, Fundacio´ mostly p.A284V. p.R229Q was more frequent among SRNS cases relative to controls (odds ratio ϭ 2.65; Puigvert, Universitat ϭ Auto`noma de P 0.02). Significantly higher age at onset of the disease and slower progression to ESRD were found in Barcelona, REDinREN, patients with one pathogenic mutation plus the p.R229Q variant in respect to patients with two NPHS2 Instituto de pathogenic mutations. Investigacio´n Carlos III, Barcelona, Spain Conclusions NPHS2 analysis has a clinical value in both childhood- and adult-onset SRNS patients. For adult-onset patients, the first step should be screening for p.R229Q and, if positive, for p.A284V. These al- Correspondence: Dr. Roser Torra or Dr. leles are present in conserved haplotypes, suggesting a common origin for these substitutions. Patients car- Elisabet Ars, Fundacio´ rying this specific NPHS2 allele combination did not respond to corticoids or immunosuppressors and Puigvert, Cartagena 340- showed FSGS, average 8-year progression to ESRD, and low risk for recurrence of FSGS after kidney 350, 08025 Barcelona, ϩ transplant. Spain. Phone: 34 93 416 97 00; Fax: ϩ34 93 Clin J Am Soc Nephrol 6: 344–354, 2011. doi: 10.2215/CJN.03770410 416 97 30; E-mail: rtorra@fundacio- puigvert.es or ears@ Introduction onset familial cases of autosomal-recessive SRNS (5). fundacio-puigvert.es Nephrotic syndrome (NS) is characterized by edema, Nearly all patients with two NPHS2 pathogenic mu- massive proteinuria, hypoalbuminemia, and hyper- tations develop NS before the age of 6 years, present lipidemia. Clinically, NS has been divided into two mostly with FSGS, do not respond to immunosup- categories based on the response to steroid therapy: pressant treatment, reach ESRD before the end of the steroid-sensitive NS (SSNS) and steroid-resistant NS first decade of life, and have a reduced risk for recur- (SRNS) (1). In children and adults with SRNS, renal rence of FSGS after kidney transplant (8 versus 33%) histology typically shows focal segmental glomerulo- (13–19). In addition, Tsukaguchi et al. (20) reported sclerosis (FSGS), and 50 to 70% of patients progress to NPHS2 variants in 23% of late-onset familial cases and ESRD (2,3). In the last few years, mutations in genes in 2% of sporadic ones. In contrast, NPHS2 mutations encoding podocyte proteins have been identified in were not found in four large cohorts of adult-onset several forms of hereditary SRNS (4–10). cases published subsequently (21–24). Recently, Ma- To date, the main player in the genetic forms of chuca et al. (25) identified NPHS2 substitutions in 14% SRNS has been podocin, encoded by the NPHS2 gene of cases presenting with SRNS after 18 years of age. (11). Podocin is a 383-amino acid lipid-raft–associated Fifteen sporadic and 11 families with adult-onset protein localized at the slit diaphragm, where it is FSGS carrying NPHS2 variants have been reported required for the structural organization and regula- thus far, and affected individuals were compound tion of the glomerular filtration barrier. Its interaction heterozygous for a particular variant, p.R229Q, and with nephrin, NEPH1, CD2AP, and TRPC6 manage one pathogenic mutation, which was frequently the mechanosensation signaling, podocyte survival, cell p.A284V substitution among South American pa- polarity, and cytoskeletal organization (12). The tients. Although p.R229Q is one of the most common NPHS2 gene was identified 10 years ago in early- nonsynonymous NPHS2 variants in Caucasians (26),

344 Copyright © 2011 by the American Society of Nephrology www.cjasn.org Vol 6 February, 2011 Clin J Am Soc Nephrol 6: 344–354, February, 2011 NPHS2 in Childhood- and Adult-Onset SRNS, Santín et al. 345

its pathogenic role in SRNS is not clear because it is members. Unpublished missense mutations were observed with similar allele frequencies in SRNS and screened in 300 control chromosomes either by direct normal control subjects (5.13 and 3.75%, respectively) sequencing or by specific restriction enzyme diges- (17,18). Support for a functional role of this variant tion. comes from in vitro studies showing decreased neph- rin binding to mutant p.R229Q-podocin (20). Classification of Sequence Variants The goals of this study were (1) to assess the We developed an in silico scoring system to evalu- utility of NPHS2 testing in Spanish children and ate the pathogenicity of amino acid substitutions adults with SRNS or FSGS, (2) to determine (missense mutations) identified in the NPHS2 gene. whether the p.A284V pathogenic mutation and the This scoring system takes into consideration a num- p.R229Q variant occur on conserved haplotypes, (3) ber of in silico predictors (28–30) and population data. to evaluate genotype–phenotype correlation among We scored each of these factors, the sum of which patients with NPHS2 variants, focusing on adult resulted in an overall variant score (VS). These were Ն patients with FSGS, and (4) to study the association classified into four groups (30): VS 11 (highly likely ϭ Ն Յ with SRNS of the relatively common p.P20L, pathogenic, mutation group [MG] B); 10 VS 5 ϭ Ն Յ p.R229Q, and p.E264Q NPHS2 variants in a case- (likely pathogenic, MG C), 4 VS 0 (indetermi- ϭ Յ Ϫ control study. nate, MG I), and VS 1 (highly likely neutral, MG ϭ NV). Nonsense and frameshift mutations were classed as definitely pathogenic mutations (MG ϭ A) Materials and Methods because they are predicted to result in truncated pro- Patients teins. From a group of 239 Spanish patients with NS We considered “pathogenic mutations” to be those referred for NPHS2 mutation analysis, we selected sequence variants predicted to result in a truncated patients affected by SRNS (1,2) to evaluate NPHS2 protein (MG ϭ A) and those amino acid substitutions genotype–phenotype correlations. We excluded pa- not found in healthy controls, segregated with the tients with a potential underlying immune disorder disease in families, and expected to severely alter the defined by remission after steroid (n ϭ 37) or immu- ϭ ϭ protein sequence using in silico predictors (MG B). nosuppressive (n 18) therapy or late steroid resis- Missense substitutions classified as MG ϭ C or I were tance (n ϭ 7). Moreover, individuals with evidence of ϭ designated as “variants of unknown clinical signifi- autosomal-dominant disease (n 6), as well as those cance.” in whom we identified mutations in NPHS1, WT1, or ϭ TRPC6 (n 23), were excluded. Renal biopsy was Haplotype Analysis available in all patients with adult-onset NS and all We genotyped family members of patients carrying the showed FSGS. Secondary forms of FSGS were not p.R229Q variant and p.A284V mutation using NPHS2 mi- included. The cohort analyzed in this study thus rep- crosatellite markers (D1S3758, D1S3760, D1S215, D1S3759, resented 148 patients belonging to 139 families with and D1S2883). Haplotype construction was also carried SRNS. Patients originating from a consanguineous out using eight single nucleotide polymorphisms (SNPs): marriage (n ϭ 4) or those with an additional affected 5ЈUTR-52CϾG, 5ЈUTR-51GϾT(rs12406197), c.102GϾA sibling (n ϭ 8) were considered as familial cases. The (rs1079292), c.288CϾT(rs3738423), IVS3–21CϾT remaining 127 were sporadic SRNS cases. Age at on- (rs12401708), IVS7ϩ7AϾG, c.954TϾC(rs1410592), and set of NS, response to treatment, histopathologic find- c.1038AϾG(rs3818587). Moreover, three informative SNPs ings, progression to ESRD, and recurrence after kid- (5ЈUTR-51GϾT, IVS3–21CϾT, and c.954TϾC) were cho- ney transplantation were obtained (Table 1). We sen for further analysis in patients and controls carrying classified our population according to the age at onset the p.R229Q variant. of the disease in early childhood onset (0 to 5 years; 34.4 Ϯ 17.4 months, n ϭ 65), late childhood onset (6 to Statistical Analyses 17 years; 11.1 Ϯ 3.5 years, n ϭ 33), and adult onset Data are expressed as mean Ϯ SD. Comparisons (Ͼ18 years; 32.9 Ϯ 10.8 years, n ϭ 50). To calculate between two continuous variables were made using t mutation frequency, we used the number of families; tests. Genetic associations between NPHS2 variants when evaluating phenotype, we considered number and SRNS were assessed by comparing genotypic of patients. frequencies between patients and control subjects (matched by ethnicity and geography with the study cohort) using ␹2 or Fisher’s exact test. The odds ratio Mutation Analysis was calculated with 95% confidence interval. All tests Genomic DNA was isolated from peripheral blood were two-sided. P Ͻ 0.05 was considered significant. cells using a standard method (27) after obtaining Statistical analyses were performed using SNPstats signed informed consent from participants. The study software (31). was approved by the institutional review boards of each participating hospital. Mutation analysis was Results performed by direct sequencing of all eight exons of Pathogenic NPHS2 Mutations and Their Frequency in NPHS2 using exon-flanking primers, as described Patients with SRNS elsewhere (5). Segregation of the detected substitu- NPHS2 mutation analysis was performed in 148 tions in families was assessed in all available family patients from 139 families with SRNS, representing 4 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 346

Table 1. Genotype–phenotype correlation according to NPHS2 mutation status for 148 patients from 139 families with SRNS

Two Pathogenic One Pathogenic Mutation One Pathogenic Mutation Pathogenic Mutations No Pathogenic Mutation Total Cases/Patients Mutations ϩ One WT ϩ p.R229Q (Total)

SRNS no. of cases 7/139 (5) 2/139 (1.5) 6/139 (4.5) 15/139 (11) 124/139 (89) 139 no. of familial cases 5/12 (42) 0/12 (0) 3/12 (25) 8/12 (67) 4/12 (33) 12 no. of sporadic cases 2/127 (1.5) 2/127 (1.5) 3/127 (3) 7/127 (6) 120/127 (94) 127 Early childhood-onset SRNS (0 to 5 years) no. of cases 6/61 (10) 0/61 (0) 0/61 (0) 6/61 (10) 55/61 (90) 61 no. of familial cases 4/8 (50) 0/8 (0) 0/8 (0) 4/8 (50) 4/8 (50) 8 no. of sporadic cases 2/53 (4) 0/53 (0) 0/53 (0) 2/53 (4) 51/53 (96) 53 Late childhood-onset SRNS (6 to 17 years) no. of cases 1/31 (3) 2/31 (6.5) 2/31 (6.5) 5/31 (16) 26/31 (84) 31 no. of familial cases 1/2 (50) 0/2 (0) 1/2 (50) 2/2 (100) 0/2 (0) 2 no. of sporadic cases 0/29 (0) 2/29 (7) 1/29 (3) 3/29 (10) 26/29 (90) 29 Adult-onset FSGS (Ͼ18 years) no. of cases 0/47 (0) 0/47 (0) 4/47 (9) 4/47 (9) 43/47 (91) 47 no. of familial cases 0/2 (0) 0/2 (0) 2/2 (100) 2/2 (100) 0/2 (0) 2 no. of sporadic cases 0/45 (0) 0/45 (0) 2/45 (5) 2/45 (5) 43/45 (95) 45 Age at onset of NS (mean Ϯ SD; years) 2.8 Ϯ 2.6, n ϭ 10 8.5 Ϯ 2.1, n ϭ 2 21.8 Ϯ 9.1, n ϭ 9 11.5 Ϯ 11.1, n ϭ 21 15.3 Ϯ 15.3, n ϭ 127 14.7 Ϯ 14.8, n ϭ 148 Response to immunosuppressant/ACEI (no. of patients 8/2/0 (80/20/0) 2/0/0 (100/0/0) 8/1/0 (89/11/0) 18/3/0 (86/14/0) 43/7/77 (34/6/60) 61/10/77 (41/7/52) with NR/PR/NA) Histology (no. of patients with FSGS/MCNS/NA) 7/1/2 (70/10/20) 2/0/0 (100/0/0) 9/0/0 (100/0/0) 18/1/2 (86/5/9) 105/13/9 (83/10/7) 123/14/11 (83/10/7) ESRD (no. of patients) 6 (60), n ϭ 10 1 (50), n ϭ 2 7 (78), n ϭ 9 14 (67), n ϭ 21 64 (57), n ϭ 111 78 (59), n ϭ 132 Age at ESRD (mean Ϯ SD; years) 7.6 Ϯ 2.5, n ϭ 68,n ϭ 1 30.4 Ϯ 11.8, n ϭ 7 19.1 Ϯ 14.3, n ϭ 14 27.8 Ϯ 18.6, n ϭ 64 26.2 Ϯ 18.1, n ϭ 78 Progression time to ESRD (mean Ϯ SD; years) 4.2 Ϯ 2.7, n ϭ 61,n ϭ 1 8.4 Ϯ 3.7, n ϭ 7 6.1 Ϯ 3.9, n ϭ 14 5.2 Ϯ 5.8, n ϭ 64 5.3 Ϯ 5.5, n ϭ 78 Recurrence after kidney transplant (no. of patients) 0 (0), n ϭ 4 — 0 (0), n ϭ 6 0 (0), n ϭ 10 16 (33), n ϭ 49 16 (27), n ϭ 59

Percentage is noted in parentheses. ACEI, angiotensin-converting enzyme inhibitor; MCNS, minimal change nephrotic syndrome; NA, not available; NR, no response; PR, partial response; WT, wild type. Clin J Am Soc Nephrol 6: 344–354, February, 2011 NPHS2 in Childhood- and Adult-Onset SRNS, Santín et al. 347

91% sporadic and 9% familial cases. We identified brane domain and one (p.G92C) in the N-terminal re- seven cases (five familial and two sporadic) carrying gion of podocin. NPHS2 pathogenic mutations in the homozygous or compound heterozygous state. In addition, only one Sequence NPHS2 Variants of Unknown Clinical pathogenic NPHS2 mutation was identified in two Significance and Their Association with SRNS patients with sporadic SRNS; one of these (patient Several amino acid substitutions (p.P20L, p.M187I, 103) also carried a predicted highly likely neutral p.R229Q, p.E264Q) were not predicted to be clearly NPHS2 variant in heterozygosity (p.M187I). Finally, pathogenic using our in silico analysis (Table 2). Sub- six unrelated cases presented with one pathogenic sequently, these variants were screened in a mini- NPHS2 mutation in exon 7 in compound heterozygos- mum of 360 control chromosomes. None of the ity with the p.R229Q variant, of which three were healthy controls showed the p.M187I sequence vari- familial and three were sporadic cases (Table 1). ant, indicating that it is probably not a polymorphism, Two pathogenic NPHS2 mutations were identified although it was classified as a highly likely neutral in 42% (5/12) of familial cases, but only 1.6% (2/127) variant (MG ϭ NV) by our in silico NPHS2 scoring of the sporadic ones. If patients with one heterozy- system. Moreover, it was found in an SRNS patient gous pathogenic NPHS2 mutation with the p.R229Q with another pathogenic NPHS2 mutation in com- in compound heterozygosity were also included, the pound heterozygosity. mutation detection rate rose to 67% for familial SRNS On the contrary, p.P20L, p.E264Q, and p.R229Q (8 of 12) and to 4% for sporadic cases (5 of 127). were found in controls. To assess the putative associ- In the subset of cases with adult-onset NS, six pa- ation of these variants with SRNS, we excluded those ϭ tients from four families carried mutations (9%, 4 of cases carrying two pathogenic NPHS2 mutations (n 47 cases), and all of them were compound heterozy- 7) because they had a clear disease cause. p.P20L and gous for one disease-causing mutation and the p.E264Q were present in single heterozygous state in both controls and patients with SRNS. The p.P20L p.R229Q variant. In the late childhood-onset SRNS ϭ group, two cases bore one pathogenic mutation plus variant (MG I) is a nonconservative amino acid exchange and was predicted to be deleterious (Poly- the p.R229Q variant and only one had two pathogenic phen, SIFT) but at a nonconserved site in the MSA. NPHS2 mutations (10%, 3 of 31 cases). In contrast, in The allele frequency observed in SRNS cases was patients with early childhood-onset SRNS, pathogenic 0.75% (2/264), which was not significantly different NPHS2 mutations were present in 10% (6 of 61) of from the 0.55% (2/360) in controls (Table 3). The cases and always in homozygous or compound p.E264Q novel variant (MG ϭ C) is conservative but heterozygous state. at a highly conserved site in the MSA. Despite being Eleven different NPHS2 pathogenic mutations more frequent among patients (0.75%; 2/264) than were detected in the present study; nine were mis- among controls (0.44%; 2/454), the difference was not sense and two were frameshift mutations leading to statistically significant. a premature stop codon (Table 2). Seven of these Finally, the p.R229Q variant was identified in pathogenic mutations have been described else- heterozygous state in 12 unrelated cases with SRNS, where. The newly identified mutations include of which 6 carried the p.R229Q variant associated three missense and one indel substitution in exon 8, with one pathogenic NPHS2 mutation on the other which represents the first indel mutation described allele. We also detected one patient with sporadic in this gene thus far (c.971_987delinsACAG FSGS that was homozygous for p.R229Q. This posi- [p.L324fsX343]). Interestingly, this mutation consisted tion was conserved during evolution until chicken, in an insertion of 4 nucleotides and a deletion of 17 but Polyphen and SIFT predicted that the p.R229Q nucleotides, which also is the largest deletion in the variant was “benign.” In this study, p.R229Q was NPHS2 gene described to date. The three novel mis- more frequent among cases compared with controls, sense mutations were c.346AϾC (p.T116P), c.662CϾT showing association with SRNS: 5.30% (14/264) ver- (p.T221I), and c.842AϾC (p.E281A). p.T116P and sus 1.98% (9/454) (odds ratio ϭ 2.65; P ϭ 0.02). p.T221I were moderately conservative amino acid substitutions but at highly conserved sites in the Haplotype Analysis of p.A284V and p.R229Q Alleles multi-sequence alignment (MSA) of ortholog podocin We found six cases from different areas of Spain proteins (except for zebrafish). p.E281A was a noncon- carrying the p.R229Q variant in compound heterozy- servative change in a highly conserved position among gous state with one pathogenic NPHS2 mutation in podocin orthologs. All of them were predicted to be exon 7 (p.A284V, n ϭ 5; p.A288T, n ϭ 1). Segregation deleterious (Polyphen, Sorting Intolerant From Tolerant analysis performed in all available family members [SIFT]) and were not found in 300 control chromosomes. confirmed that all affected patients were compound The large majority of pathogenic mutations hereby de- heterozygotes for p.R229Q and the pathogenic muta- tected were located in the C-terminal part of podocin tion, and no unaffected individual carried both (9/11), mostly in the alanine- and glutamate-rich region NPHS2 variants. We tried to investigate whether that is highly conserved within the stomatin protein p.R229Q/p.A284V substitutions occurred on con- family (p.E381A, p.A284V, p.A288T, p.Q285fsX302). served haplotypes within our cohort of Spanish fam- Only one mutation (p.T116P) was detected in the mem- ilies to discern between a hot spot and a founder 4 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 348

Table 2. Classification of NPHS2 substitutions

Described in Single GD/GV SIFT Segregation Polyphen Splicing Nucleotide NPHS2 Substitutions Exon Previous Descriptiona Control Chrsb GDd GVe Matrix GDevg Predicted VSl MGm Analysisc Predictionh Predictionj Polymorphism Scoref Toleratedi Databasek

Previously reported NPHS2 pathogenic mutations G92C 1 refs. 5 and 17 (ϩ1) 0/320 (ref. 17) (ϩ2) FP (ϩ4) 159 0 ϩ7 159 (ϩ2) 1.7 (ϩ1) No (ϩ2) LD (ϩ1) No (ϩ1) 21 B R138Q 3 refs. 5, 11, 13–18, 20, 25, 35, 48, 0/320 (ref. 17) (ϩ2) F (ϩ4) 43 0 ϩ543(ϩ2) 2.1 (ϩ2) No (ϩ2) NP (0) No (ϩ1) 19 B and 49 (ϩ1) L169P 4 refs. 13, 16, 18, and 35 (ϩ1) 0/200 (ref. 16) (ϩ2) FP (ϩ4) 98 0 ϩ698(ϩ2) 1.5 (ϩ1) Yes (Ϫ2) NP (0) No (ϩ1) 15 B V260E 6 refs. 17 (ϩ1) 0/320 (ref. 17) (ϩ2) FP (ϩ4) 121 0/22 ͓pl͔ϩ5 121 (ϩ2) 2.4 (ϩ2) No (ϩ2) NP (0) No (ϩ1) 19 B A284V 7 refs. 17, 18, 20, 25, 32, 49, 0/320 (ref. 17) (ϩ2) F (ϩ4) 64 0 ϩ564(ϩ2) 1.9 (ϩ1) No (ϩ2) NP (0) No (ϩ1) 18 B and 50 (ϩ1) A288T 7 refs. 20, 17, and 25 (ϩ1) 0/320 (ref. 17) (ϩ2) FP (ϩ4) 58 0 ϩ558(ϩ2) 1.9 (ϩ1) No (ϩ2) NP (0) No (ϩ1) 18 B Previously reported NPHS2 variants of unknown clinical significance P20L 3 refs. 5, 16–18, 35, and 51 2/360n (Ϫ1) — 98 155 Ϫ214(Ϫ2) 2.3 (ϩ2) No (ϩ2) NP (0) No (ϩ1) 0 I R229Q 5 refs. 14–18, 20, 23–25, 32, 35, 49, 52, 9/454n (Ϫ2) — 43 0/29 ͓fi͔ϩ420(ϩ1) 0.4 (Ϫ2) Yes (Ϫ2) NP (0) No (ϩ1) 0 I and 53 Novel missense amino acid NPHS2 substitutions identified in our cohort T116P 2 Novel 0/300n (ϩ2) F(ϩ4) 38 0/103 ͓fi͔ –2 29 (ϩ2) 2.3 (ϩ2) No (ϩ2) NP (0) No (ϩ1) 11 B M187I 5 Novel 0/300n (ϩ2) — 10 0/92 ͓ch,fi͔ –4 0 (Ϫ2) 0.6 (Ϫ2) Yes (Ϫ2) NP (0) No (ϩ1) –7 NV T221I 5 Novel 0/300n (ϩ2) F(ϩ4) 89 0/58 ͓fi͔ϩ389(ϩ2) 2.3 (ϩ2) No (ϩ2) NP (0) No (ϩ1) 16 B E264Q 6 Novel 2/454n (Ϫ1) —290 ϩ229(ϩ2) 1.8 (ϩ1) No (ϩ2) NP (0) No (ϩ1) 7 C E281A 7 Novel 0/300n (ϩ2) — 107 0 ϩ6 107 (ϩ2) 1.5 (ϩ1) No (ϩ2) NP (0) No (ϩ1) 14 B Frameshift NPHS2 substitutions Q285fsX302 7 refs. 5, 14, 18, 25, 53, and 49 NR NR NR NR NR NR NR NR NR NR NR A L324fsX343 8 Novel NR NR NR NR NR NR NR NR NR NR NR A

NR, not required. aWhen a change was described previously in the literature as a pathogenic mutation or in the HGM database (ϩ1). bWhen a sequence variant was not present in the control chromosomes (ϩ2), if present Ͻ1% (Ϫ1) or Ͼ1% (Ϫ2). cSegregation shown in family (ϩ4, F, affected and not affected siblings and their parents; FP, segregation in family in previous description). dGD (Grantham distance), score of chemical difference between the normal and mutated residue (high score, greater difference). eGV (Grantham variation), score of chemical difference between 11 orthologs (ranging from chimpanzee to zebrafish, 0 ϭ completed conserved among podocin orthologs, ͓pl͔ϭconserved among orthologs except in Platypus, ͓fi͔ϭconserved among orthologs except in Zebrafish, ͓ch,fi͔ϭconserved among orthologs except in Chiken and Zebrafish). fGD/GV matrix score, ranging from Ϫ2toϩ8 (lower matrix scores corresponded to low GD and high GV ͓conservative change and strong variation within the MSA), whereas higher matrix scores corresponded to high GD and low GV ͓nonconservative change and strong conservation within the MSA͔). gGDev (Grantham deviation), score of chemical difference between the mutated residue and the range of variation between orthologs (GD similar to GDev, higher difference, ϩ2). hPolyphen assessment, ratio Polyphen Ͼ2 (probably damaging, ϩ2), ratio Polyphen 1 to 2 (possibly damaging, ϩ1), ratio Polyphen Ͻ1 (benign, Ϫ2). iSIFT prediction: not tolerated (ϩ2), tolerated (Ϫ2). jNot predicted (NP) by splice site prediction neural network (0), if predicted loss of donor site (LD, ϩ1). kWhen a change was not described in the single nucleotide polymorphism database (ϩ1), if described (Ϫ1). lVariant score (VS); VS Ͼ 11 3. m Mutation group (MG) ϭ B; VS ϭ 5to103 MG ϭ C; VS ϭ 0to43 MG ϭ I; VS ϽϪ1 3 MG ϭ NV; A, definitely pathogenic; B, highly likely pathogenic; C, likely pathogenic; I, unknown pathogenicity; NV, highly likely neutral. nThis report. Clin J Am Soc Nephrol 6: 344–354, February, 2011 NPHS2 in Childhood- and Adult-Onset SRNS, Santín et al. 349

Table 3. Genotype frequencies for NPHS2 variants of unknown clinical significance in patients with SRNS and control subjects

P b NPHS2 Variants a P ͓ ͔ Genotype, Number (Frequency) Total MAF (HWE) OR (95% CI)

p.P20L C/C C/T T/T SRNS 130 (0.985) 2 (0.015) 0 (0.000) 132 0.007 1 controls 178 (0.989) 2 (0.011) 0 (0.000) 180 0.005 1 0.76 ͓1.37 (0.19 to 9.85)͔ p.E264Q G/G G/C C/C SRNS 130 (0.985) 2 (0.015) 0 (0.000) 132 0.007 1 controls 225 (0.991) 2 (0.009) 0 (0.000) 227 0.004 1 0.59 ͓1.73 (0.24 to 12.43)͔ p.R229Q G/G G/A A/A SRNS 119 (0.901) 12 (0.091) 1 (0.008) 132 0.053 0.3 controls 218 (0.961) 9 (0.039) 0 (0.000) 227 0.019 1 0.02 ͓2.65 (1.10 to 6.37)͔

Frequencies were calculated using one affected case per family. In the group of SRNS, we excluded those patients carrying two pathogenic mutations in the NPHS2 gene. MAF, minor allele frequency; OR, odds ratio; 95% CI, 95% confidence interval. aTest for deviation from Hardy-Weinberg equilibrium (HWE) law. bGenotype frequency difference test (␹2) under dominant model. effect. The analysis of five microsatellites showed con- The subset of adult patients with FSGS carrying one served haplotypes for the p.A284V mutation (171;172; pathogenic mutation plus the p.R229Q variant (n ϭ 6) 208;192;186) and for the p.R229Q variant (171;186;208; had an earlier onset of NS compared with those with- 188;197). Moreover, for three informative SNPs out NPHS2 mutations (26.2 Ϯ 7.7 versus 33.9 Ϯ 11.0 (5ЈUTR-51GϾT, IVS3–21CϾT, and c.954TϾC), a com- years; P ϭ 0.05); however, no difference was observed mon haplotype was also identified: (G;C;T) and (T;T; in the age at onset of ESRD (36.8 Ϯ 11.3 versus 39.6 Ϯ T), respectively. Phases for p.A284V/p.R229Q and 13.8 years; P ϭ 0.65). In the group of adult FSGS each of the four microsatellites and three SNPs were patients without mutations, the recurrence in a renal confirmed in the whole group of families. We ex- transplant was 22% (6 of 27 patients); conversely, in tended the study to include 15 p.R229Q-heterozygous the group with mutations, no recurrence was ob- carriers from SRNS and control subjects and served. 1 p.R229Q-homozygous patient, and we found that all these individuals also shared the same common hap- lotype. Discussion NPHS2 mutations were initially described in pa- Clinical Data for Patients Carrying One NPHS2 tients developing NS from birth to 6 years of age Pathogenic Mutation and the p.R229Q Variant (17,19). However, the utility of NPHS2 testing in Patients carrying p.R229Q and one NPHS2 muta- adults with FSGS has not been fully studied. Our tion (n ϭ 9) developed NS significantly later than study represents the first cohort of Spanish SRNS those carrying two pathogenic mutations (mean, patients evaluated for the NPHS2 gene, including 21.8 Ϯ 9.1 versus 2.8 Ϯ 2.6 years; P Ͻ 0.01; Table 1). early childhood-, late childhood-, and adult-onset This group included two patients (60-1, 164) with cases. In contrast to the literature, we found that subnephrotic proteinuria at the time of diagnosis Spanish patients with late childhood- and adult-onset (Table 4). Renal biopsy showed mesangioprolifera- SRNS had a similar NPHS2 mutation detection rate tive lesions with FSGS in two patients and FSGS in than those with early childhood-onset (12%, 9 of 78 seven patients. All of them were resistant to cortico- cases versus 10%, 6 of 61 cases, respectively). In the steroids and immunosuppressant drugs. Angioten- subset of cases with NPHS2 variants, our data suggest sin-converting enzyme inhibitors showed inconsis- that the age at onset of the disease could be correlated tent effects in the majority of this group; however, in with the genotype. Patients with early childhood on- one patient (61-1), these agents decreased proteinuria set (Ͻ6 years) carried two pathogenic mutations, pa- from 4.5 to 1 g/day. Seven patients had developed tients with late childhood onset (6 to 18 years) carried ESRD at a mean age of 30.4 years (range: 15 to 50 two pathogenic or one pathogenic mutation in years) and in a mean time of 8.4 years (range: 4 to 13 heterozygous state with the p.R229Q variant, and pa- years) after the onset of the disease, which was also tients with adult onset (Ͼ18 years) carried one patho- significantly later than the group of two pathogenic genic mutation plus the p.R229Q variant. Quite inter- NPHS2 mutations (P Ͻ 0.01 and P ϭ 0.03, respec- estingly, we confirmed that p.R229Q in compound tively). No disease recurrence was observed in six heterozygous state with p.A284V mutation is the patients who received renal allograft. most common allelic combination causing late-onset 5 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 350

Table 4. Clinical data of patients with SRNS and NPHS2 mutations

Age at Onset Patient Gender Renal Biopsy Therapy Evolution Tx/Recurrence NPHS2 Mutations of NS (years)

Patients with two pathogenic mutations 10–1a M 4 FSGS Cs, CsAϪ ESRD at 12 years Yes/noc ͓c.274GϾT (p.G92C)͔ϩ͓c.506TϾC (p.L169P)͔ 10–2a M 2 MCNS Cs, CsA, ACEIϮ Normal Cr at 18 years No 26–1a F 4.5 FSGS Cs, CsA, MMFϪ ESRD at 7 years Yes/no ͓c.274GϾT (p.G92C)͔ϩ͓c.413GϾA (p.R138Q)͔ 26–2a M 2.5 FSGS Cs, CsA, MMFϪ ESRD at 5 years Yes/noc 71 M 1 FSGS* Cs, CP, CsAϪ ESRD at 6 years Yes/no ͓c.413GϾA (p.R138Q)͔ϩ͓c.971_987delinsACAG (p.L324fsX343)͔ 77 F 5.1 FSGS* Cs, CP, CsA, MMFϪ CKD stage III at 9 years No ͓c.855_856delAA (p.Q285fsX302)͔ϩ͓c.855_856delAA (p.Q285fsX302)͔ 102–1a M 0.6 FSGS* Cs, CsAϪ ESRD at 7 years No ͓c.346AϾC (p.T116P)͔ϩ͓c.346AϾC (p.T116P)͔ 102–2a F 0.02 Not performed CsϪ Normal Cr at 5 years No 225b M 0.3 Not performed Cs, ACEIϮ Normal Cr at 2 years No ͓c.842AϾC (p.E281A)͔ϩ͓c.842AϾC (p.E281A)͔ 228b F 8 FSGS Cs, CsA, MMFϪ ESRD at 9 years No ͓c.779TϾA (p.V260E)͔ϩ͓c.779TϾA (p.V260E)͔ Patients with one mutation ϩWT 103 M 10 FSGS Cs, MMFϪ Normal Cr at 16 years No ͓c.561GϾA (p.M187I)͔ϩ͓c.862GϾT (p.A288T)͔ 227 F 7 FSGS Cs, CsA, ACEIϪ ESRD at 8 years No ͓c.662 CϾT (p.T221I)͔ϩ͓?͔ Patients with one mutation ϩR229Q 44 F 39 FSGS* Cs, CsA, MMFϪ ESRD at 50 years No ͓c.686GϾA (p.R229Q)͔ϩ͓c.851CϾT (p.A284V)͔ 59 M 10 FSGS Cs, CsA, MMFϪ ESRD at 15 years Yes/noc ͓c.686GϾA (p.R229Q)͔ϩ͓c.851CϾT (p.A284V)͔ 60–1a F 16 FSGS CsϪ ESRD at 25 years Yes/noc ͓c.686GϾA (p.R229Q)͔ϩ͓c.851CϾT (p.A284V)͔ 60–2a M 13 FSGS Cs, CsAϪ ESRD at 26 years Yes/no 61–1a M 24 FSGS* Cs, CP, ACEIϮ CKD stage II at 34 years No ͓c.686GϾA (p.R229Q)͔ϩ͓c.851CϾT (p.A284V)͔ 61–2a M 18 FSGS Cs, CP, CsA, MMF, ACEIϪ CKD stage II at 29 years No 121–1a M 28 FSGS Cs, CsAϪ ESRD at 34 years Yes/no ͓c.686GϾA (p.R229Q)͔ϩ͓c.851CϾT (p.A284V)͔ 121–2a M 19 FSGS Cs, CsAϪ ESRD at 23 years Yes/no 164 F 28 FSGS Cs, CPϪ ESRD at 40 years Yes/no ͓c.686GϾA (p.R229Q)͔ϩ͓c.862GϾT (p.A288T)͔

Therapy effect categories: (Ϫ) no response, (Ϯ) partial reduction of proteinuria; ACEI, angiotensin-converting enzyme inhibitor; Cs, corticosteroids; CP, cyclophosphamide; CsA, cyclosporin A; Cr, creatinine; CKD, chronic kidney disease; F, female; FSGS*, FSGS with mesangioproliferative lesions; MCNS, minimal change nephrotic syndrome; M, male; MMF, mycophenolate mophetil; Tx, kidney transplantation; WT, wild type. aSiblings with the same parents. bOnly child of consanguineous parents. cThese patients presented with chronic rejection nephropathy. lnJA o eho :3434 eray 2011 February, 344–354, 6: Nephrol Soc Am J Clin

Table 5. Allele frequencies for p.A284V and p.R229Q among patients and controls from different geographic areas p.A284V p.R229Q Area/Ethnicity Allele Allele References Cases Cases Controls Frequency Frequency Europe Germans/central Europeans and Turks 3/285a 4/570 (0.007) 16/285b 9/80 27/730 (0.036) 18 Italians 0/179 0/358 (0.000) 12/179c 5/100 19/558 (0.034) 16 Italians 0/33 0/66 (0.000) 3/33 7/124 10/314 (0.032) 23 French and North Africans 3/272a 4/544 (0.007) 22/272d 12/160 39/864 (0.045) 17 Turks 0/295 0/590 (0.000) 15/295e — 15/590 (0.025) 35 Europeans 2/214 2/428 (0.005) 35/214f 16/308 54/1044 (0.050) 25 United States Caucasians, Africans, Hispanics 3/121g 4/242 (0.016) 17/121h 1/32;3/49;9/124i 30/652 (0.046) 20 African descent — — — 1/96 0/192 (0.005) 36 European Americans 0/129 0/258 (0.000) 12/129 21/271 33/800 (0.041) 21 African Americans 0/247 0/494 (0.000) 5/247 16/634 21/1762 (0.012) 21 Asia Japanese 0/36 0/72 (0.000) 0/36 — 0/72 (0.000) 37 Chinese 0/45 0/90 (0.000) 0/45 — 0/90 (0.000) 38, 39

Koreans 0/70 0/140 (0.000) 0/70 — 0/140 (0.000) 40 NPHS2 Canada 0/87 0/174 (0.000) 8/87j 3/108 11/390 (0.028) 53

South America 351 al. et Santín SRNS, Adult-Onset and Childhood- in Brazilians — — — 85/1577 85/3154 (0.027) 34 Brazilians 0/39 0/78 (0.000) 2/39 — 2/78 (0.026) 24 Chileans and Argentineansk 13/47 13/94 (0.138) 16/47 1/70 17/234 (0.073) 25 Spain 5/139 5/278 (0.018) 13/139 9/227 23/732 (0.031) Present study aOne case in homozygous state and two cases in compound heterozygous state. bFour cases in compound heterozygous state, 2 cases in homozygous state, and 10 cases in heterozygous state. This study includes 120 steroid-sensitive cases. cFour cases in compound heterozygous state, two cases in homozygous state, and six cases in heterozygous state. This study includes 59 steroid-sensitive cases. dFour cases in compound heterozygous state, 5 cases in homozygous state, and 13 cases in heterozygous state. eEight cases in compound heterozygous state, five cases in homozygous state, and two cases in heterozygous state. fThree cases in homozygous state and 32 cases in heterozygous state or in compound heterozygous state. gOne case from Dominican Republic in homozygous state and two sporadic cases from unknown origin in compound heterozygous state. hSix families in compound heterozygous state and 11 sporadic cases in heterozygous state, of whom 2 had the p.A284V in compound heterozygous state. iThirty-two control individuals of African descent (0.01), 49 from Brazil (0.03), and 124 from the Western panel DNA samples (majority Europeans) (0.036). jThis study includes 15 steroid-sensitive cases. kAll South American cases and controls were of Spanish descent. 352 Clinical Journal of the American Society of Nephrology

SRNS in Spanish patients, in agreement with data SRNS. Heterozygosity for p.P20L and p.E264Q did previously reported by Machuca et al. (25) in patients not increase the risk for SRNS because the frequency from South America. of these alleles was similar in both patient and control In addition, we showed that the p.A284V patho- subjects. On the other hand, p.R229Q was present in genic mutation occurs in a conserved haplotype in 4.54% (12 of 132) of cases in heterozygosity and in Spanish patients, which supports the idea of a single 0.75% (1 of 132) of cases in homozygosity. p.R229Q origin for this variant. Because this mutation has has been extensively reported in a higher frequency mostly been detected in South American patients among patients than in controls, but without statisti- (Table 5), we could hypothesize that a Spanish cal significance (17,18,20,21,26). In our Spanish study founder might have introduced it into the Hispanic population, the frequency of the p.R229Q allele was population studied by other groups (20,25,32), as sug- significantly higher in SRNS patients than in controls gested by Hildebrandt et al. (33). On the other hand, (5.3 versus 1.9%), which supports the results obtained the high frequency of p.R229Q could mean that this by Machuca et al. (25) among Europeans and South variant arose by a recurrent event or that it is an American patients. ancient mutation present worldwide caused by pop- ulation expansion. The shared haplotype among Conclusions Spanish p.R229Q carriers gives further evidence of an NPHS2 mutations were not uncommon in our co- ancient origin for this variant. In accordance, Tsuka- hort of Spanish patients with late- and adult-onset guchi et al. (20) also found a common haplotype SRNS. For genetic diagnostic purposes in European among p.R229Q carriers of African, Brazilian, and adults with FSGS, the first step should be to screen for European descent. There is an uneven p.R229Q allele p.R229Q, and only in those carrying this variant distribution throughout different populations: it is would further analysis be indicated to identify the more frequent among Spaniards, South Americans, second mutation, usually p.A284V for patients of Europeans, and European Americans (ϳ4 to 7%) (16– Spanish descent. Age at onset of the disease and 18,23–25,34,35) than among Africans, African Ameri- progression to ESRD in patients with one pathogenic cans, and Asians (ϳ0.0 to 1.5%) (21,36–40), suggest- mutation plus the p.R229Q variant was significantly ing that this variant emerged in Europe, although it is higher and slower than in patients with two NPHS2 not possible to discern a specific geographic origin. pathogenic mutations. Furthermore, because com- Fifteen different NPHS2 sequence variants have pound heterozygous patients with the p.R229Q vari- been identified in this study, 13 of which were mis- ant do not respond to either corticoids or immuno- sense. The high percentage of NPHS2 missense vari- suppressants and do not relapse after kidney ants represents a diagnostic challenge because in transplantation, genetic counseling in these families some cases it is difficult to differentiate between a should be as follows: (1) to avoid unnecessary steroid disease-causing mutation and a neutral variant. We and immunosuppressive treatment, (2) to promote describe here an in silico sequence variant classifica- living donor kidney transplantation, and (3) to pro- tion strategy for the NPHS2 amino acid substitutions vide the possibility to screen couples carrying NPHS2 based on the combination of different approaches mutation for p.R229Q. previously reported for other genes (30,41,42), which takes into account (1) the analysis of control chromo- Acknowledgments somes, (2) the cosegregation with the disease in a We thank the patients and their families for taking part in family, (3) the biophysical and biochemical difference this study. This work was funded by grants from the Spanish between wild-type and mutant amino acids (28), (4) Health Ministry (FIS-05/0761 and FIS-09/01506) and the the evolutionary conservation of the amino acid resi- REDinREN (Red Renal de Investigacio´n Espan˜ola 16/06, due in orthologs (29,43,44), and (5) software that uses RETICS, Instituto de Investigacio´n Carlos III). Other inves- in silico predictors of pathogenic effect [Polyphen (45), tigators in the FSGS Spanish Study Group: Hospital de la SIFT (46)] and splice site [Neural Network (47)]. The Santa Creu i Sant Pau- Gloria Fraga; Hospital Universitario La accuracy of this in silico analysis was tested using Fe- Santiago Mendiza´bal, Isabel Zamora; Hospital Infantil La previously described and classified podocin-amino Paz- Antonia Pen˜a, Laura Espinosa, Carmen García, Marta acid substitutions. Afterward, we evaluated the novel Melgosa, Mercedes Navarro; Hospital Vall d’Hebron- Joan missense substitutions identified in our study cohort, Lo´pez-Hellin, Sara Chocro´n,A´ lvaro Madrid, Ramo´n Vilalta, and we found that three of five were clearly patho- Jose´Luciano Nieto, Clara Ventura; Hospital Sant Joan de De´u- genic mutations. Antonio Gime´nez, Jorge Vila Cots, Juan Antonio Camacho; Finally, we identified three variants with unknown Hospital Infantil Universitario Virgen del Rocío- Ana Sa´nchez- clinical significance. The p.M187I is a novel sequence Moreno, Francisco de la Cerda; Hospital Universitario de Ca- variant that is predicted to be a highly likely neutral narias- Eduardo Salido; Fundacio´n Jime´nez Díaz- Alberto Or- variant by our in silico scoring system, but we could tiz, Simona Alexandra, Carlos Caramelo†, Jesu´s Egido; speculate that this variant is implicated in the patho- Hospital Universitario de La Princesa- Carmen Bernis; Hospital genesis of late-onset SRNS because it was not found in General Universitario Gregorio Maran˜o´n- Augusto Luque de 360 control chromosomes and was present in com- Pablos, Ma Dolores Morales San Jose´; Hospital Germans Trias pound heterozygous state with a pathogenic muta- i Pujol- Guillem Pintos; Hospital de Barcelona- Pere Sala, Fred- tion (p.A288T) in a patient with late childhood-onset eric Raspall, A´ ngel Vila; Hospital Torreca´rdenas- Antonio Clin J Am Soc Nephrol 6: 344–354, February, 2011 NPHS2 in Childhood- and Adult-Onset SRNS, Santín et al. 353

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Yu Z, Ding J, Huang J, Yao Y, Xiao H, Zhang J, Liu J, Received: April 29, 2010 Accepted: September 13, 2010 Yang J: Mutations in NPHS2 in sporadic steroid-resis- tant nephrotic syndrome in Chinese children. Nephrol Published online ahead of print. Publication date available Dial Transplant 20: 902–908, 2005 at www.cjasn.org. 40. Cho HY, Lee JH, Choi HJ, Lee BH, Ha IS, Choi Y, Cheong HI: WT1 and NPHS2 mutations in Korean chil- R.T. and E.A. contributed equally to this work.