Summary
Background and objectives
To date, very few cases with adult-onset focal segmental glomerulosclerosis (FSGS) carrying NPHS2 variants have been described, all of them being compound heterozygous for the p.R229Q variant and one pathogenic mutation.
Design, setting, participants, & measurements
Mutation analysis was performed in 148 unrelated Spanish patients, of whom 50 presented with FSGS after 18 years of age. Pathogenicity of amino acid substitutions was evaluated through an in silico scoring system. Haplotype analysis was carried out using NPHS2 single nucleotide polymorphism and microsatellite markers.
Results
Compound heterozygous or homozygous NPHS2 pathogenic mutations were identified in seven childhood-onset steroid-resistant nephrotic syndrome (SRNS) cases. Six additional cases with late childhood- and adult-onset SRNS were compound heterozygotes for p.R229Q and one pathogenic mutation, mostly p.A284V. p.R229Q was more frequent among SRNS cases relative to controls (odds ratio = 2.65; P = 0.02). Significantly higher age at onset of the disease and slower progression to ESRD were found in patients with one pathogenic mutation plus the p.R229Q variant in respect to patients with two NPHS2 pathogenic mutations.
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 alleles are present in conserved haplotypes, suggesting a common origin for these substitutions. Patients carrying this specific NPHS2 allele combination did not respond to corticoids or immunosuppressors and showed FSGS, average 8-year progression to ESRD, and low risk for recurrence of FSGS after kidney transplant.
Introduction
Nephrotic syndrome (NS) is characterized by edema, massive proteinuria, hypoalbuminemia, and hyperlipidemia. Clinically, NS has been divided into two categories based on the response to steroid therapy: steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS) (1). In children and adults with SRNS, renal histology typically shows focal segmental glomerulosclerosis (FSGS), and 50 to 70% of patients progress to ESRD (2,3). In the last few years, mutations in genes encoding podocyte proteins have been identified in several forms of hereditary SRNS (4–10).
To date, the main player in the genetic forms of SRNS has been podocin, encoded by the NPHS2 gene (11). Podocin is a 383-amino acid lipid-raft–associated protein localized at the slit diaphragm, where it is required for the structural organization and regulation of the glomerular filtration barrier. Its interaction with nephrin, NEPH1, CD2AP, and TRPC6 manage mechanosensation signaling, podocyte survival, cell polarity, and cytoskeletal organization (12). The NPHS2 gene was identified 10 years ago in early-onset familial cases of autosomal-recessive SRNS (5). Nearly all patients with two NPHS2 pathogenic mutations develop NS before the age of 6 years, present mostly with FSGS, do not respond to immunosuppressant treatment, reach ESRD before the end of the first decade of life, and have a reduced risk for recurrence of FSGS after kidney transplant (8 versus 33%) (13–19). In addition, Tsukaguchi et al. (20) reported NPHS2 variants in 23% of late-onset familial cases and in 2% of sporadic ones. In contrast, NPHS2 mutations were not found in four large cohorts of adult-onset cases published subsequently (21–24). Recently, Machuca et al. (25) identified NPHS2 substitutions in 14% of cases presenting with SRNS after 18 years of age. Fifteen sporadic and 11 families with adult-onset FSGS carrying NPHS2 variants have been reported thus far, and affected individuals were compound heterozygous for a particular variant, p.R229Q, and one pathogenic mutation, which was frequently the p.A284V substitution among South American patients. Although p.R229Q is one of the most common nonsynonymous NPHS2 variants in Caucasians (26), its pathogenic role in SRNS is not clear because it is observed with similar allele frequencies in SRNS and normal control subjects (5.13 and 3.75%, respectively) (17,18). Support for a functional role of this variant comes from in vitro studies showing decreased nephrin binding to mutant p.R229Q-podocin (20).
The goals of this study were (1) to assess the utility of NPHS2 testing in Spanish children and adults with SRNS or FSGS, (2) to determine whether the p.A284V pathogenic mutation and the p.R229Q variant occur on conserved haplotypes, (3) to evaluate genotype–phenotype correlation among patients with NPHS2 variants, focusing on adult patients with FSGS, and (4) to study the association with SRNS of the relatively common p.P20L, p.R229Q, and p.E264Q NPHS2 variants in a case-control study.
Materials and Methods
Patients
From a group of 239 Spanish patients with NS referred for NPHS2 mutation analysis, we selected patients affected by SRNS (1,2) to evaluate NPHS2 genotype–phenotype correlations. We excluded patients with a potential underlying immune disorder defined by remission after steroid (n = 37) or immunosuppressive (n = 18) therapy or late steroid resistance (n = 7). Moreover, individuals with evidence of autosomal-dominant disease (n = 6), as well as those in whom we identified mutations in NPHS1, WT1, or TRPC6 (n = 23), were excluded. Renal biopsy was available in all patients with adult-onset NS and all showed FSGS. Secondary forms of FSGS were not included. The cohort analyzed in this study thus represented 148 patients belonging to 139 families with SRNS. Patients originating from a consanguineous marriage (n = 4) or those with an additional affected sibling (n = 8) were considered as familial cases. The remaining 127 were sporadic SRNS cases. Age at onset of NS, response to treatment, histopathologic findings, progression to ESRD, and recurrence after kidney transplantation were obtained (Table 1). We classified our population according to the age at onset of the disease in early childhood onset (0 to 5 years; 34.4 ± 17.4 months, n = 65), late childhood onset (6 to 17 years; 11.1 ± 3.5 years, n = 33), and adult onset (>18 years; 32.9 ± 10.8 years, n = 50). To calculate mutation frequency, we used the number of families; when evaluating phenotype, we considered number of patients.
Table 1.
Genotype–phenotype correlation according to NPHS2 mutation status for 148 patients from 139 families with SRNS
| Two Pathogenic Mutations | One Pathogenic Mutation + One WT | One Pathogenic Mutation + p.R229Q | Pathogenic Mutations (Total) | No Pathogenic Mutation | Total Cases/Patients | |
|---|---|---|---|---|---|---|
| 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 (>18 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 ± SD; years) | 2.8 ± 2.6, n = 10 | 8.5 ± 2.1, n = 2 | 21.8 ± 9.1, n = 9 | 11.5 ± 11.1, n = 21 | 15.3 ± 15.3, n = 127 | 14.7 ± 14.8, n = 148 |
| Response to immunosuppressant/ACEI (no. of patients with NR/PR/NA) | 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) |
| 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 = 10 | 1 (50), n = 2 | 7 (78), n = 9 | 14 (67), n = 21 | 64 (57), n = 111 | 78 (59), n = 132 |
| Age at ESRD (mean ± SD; years) | 7.6 ± 2.5, n = 6 | 8, n = 1 | 30.4 ± 11.8, n = 7 | 19.1 ± 14.3, n = 14 | 27.8 ± 18.6, n = 64 | 26.2 ± 18.1, n = 78 |
| Progression time to ESRD (mean ± SD; years) | 4.2 ± 2.7, n = 6 | 1, n = 1 | 8.4 ± 3.7, n = 7 | 6.1 ± 3.9, n = 14 | 5.2 ± 5.8, n = 64 | 5.3 ± 5.5, n = 78 |
| Recurrence after kidney transplant (no. of patients) | 0 (0), n = 4 | — | 0 (0), n = 6 | 0 (0), n = 10 | 16 (33), n = 49 | 16 (27), n = 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.
Mutation Analysis
Genomic DNA was isolated from peripheral blood cells using a standard method (27) after obtaining signed informed consent from participants. The study was approved by the institutional review boards of each participating hospital. Mutation analysis was performed by direct sequencing of all eight exons of NPHS2 using exon-flanking primers, as described elsewhere (5). Segregation of the detected substitutions in families was assessed in all available family members. Unpublished missense mutations were screened in 300 control chromosomes either by direct sequencing or by specific restriction enzyme digestion.
Classification of Sequence Variants
We developed an in silico scoring system to evaluate the pathogenicity of amino acid substitutions (missense mutations) identified in the NPHS2 gene. This scoring system takes into consideration a number of in silico predictors (28–30) and population data. We scored each of these factors, the sum of which resulted in an overall variant score (VS). These were classified into four groups (30): VS ≥ 11 (highly likely pathogenic, mutation group [MG] = B); 10 ≥ VS ≤ 5 (likely pathogenic, MG = C), 4 ≥ VS ≤ 0 (indeterminate, MG = I), and VS ≤ −1 (highly likely neutral, MG = NV). Nonsense and frameshift mutations were classed as definitely pathogenic mutations (MG = A) because they are predicted to result in truncated proteins.
We considered “pathogenic mutations” to be those sequence variants predicted to result in a truncated protein (MG = A) and those amino acid substitutions not found in healthy controls, segregated with the disease in families, and expected to severely alter the protein sequence using in silico predictors (MG = B). Missense substitutions classified as MG = C or I were designated as “variants of unknown clinical significance.”
Haplotype Analysis
We genotyped family members of patients carrying the p.R229Q variant and p.A284V mutation using NPHS2 microsatellite markers (D1S3758, D1S3760, D1S215, D1S3759, and D1S2883). Haplotype construction was also carried out using eight single nucleotide polymorphisms (SNPs): 5′UTR-52C>G, 5′UTR-51G>T (rs12406197), c.102G>A (rs1079292), c.288C>T (rs3738423), IVS3–21C>T (rs12401708), IVS7+7A>G, c.954T>C (rs1410592), and c.1038A>G (rs3818587). Moreover, three informative SNPs (5′UTR-51G>T, IVS3–21C>T, and c.954T>C) were chosen for further analysis in patients and controls carrying the p.R229Q variant.
Statistical Analyses
Data are expressed as mean ± SD. Comparisons between two continuous variables were made using t tests. Genetic associations between NPHS2 variants and SRNS were assessed by comparing genotypic frequencies between patients and control subjects (matched by ethnicity and geography with the study cohort) using χ2 or Fisher's exact test. The odds ratio was calculated with 95% confidence interval. All tests were two-sided. P < 0.05 was considered significant. Statistical analyses were performed using SNPstats software (31).
Results
Pathogenic NPHS2 Mutations and Their Frequency in Patients with SRNS
NPHS2 mutation analysis was performed in 148 patients from 139 families with SRNS, representing 91% sporadic and 9% familial cases. We identified seven cases (five familial and two sporadic) carrying NPHS2 pathogenic mutations in the homozygous or compound heterozygous state. In addition, only one pathogenic NPHS2 mutation was identified in two patients with sporadic SRNS; one of these (patient 103) also carried a predicted highly likely neutral NPHS2 variant in heterozygosity (p.M187I). Finally, six unrelated cases presented with one pathogenic NPHS2 mutation in exon 7 in compound heterozygosity with the p.R229Q variant, of which three were familial and three were sporadic cases (Table 1).
Two pathogenic NPHS2 mutations were identified in 42% (5/12) of familial cases, but only 1.6% (2/127) of the sporadic ones. If patients with one heterozygous pathogenic NPHS2 mutation with the p.R229Q in compound heterozygosity were also included, the mutation detection rate rose to 67% for familial SRNS (8 of 12) and to 4% for sporadic cases (5 of 127).
In the subset of cases with adult-onset NS, six patients from four families carried mutations (9%, 4 of 47 cases), and all of them were compound heterozygous for one disease-causing mutation and the p.R229Q variant. In the late childhood-onset SRNS group, two cases bore one pathogenic mutation plus the p.R229Q variant and only one had two pathogenic NPHS2 mutations (10%, 3 of 31 cases). In contrast, in patients with early childhood-onset SRNS, pathogenic NPHS2 mutations were present in 10% (6 of 61) of cases and always in homozygous or compound heterozygous state.
Eleven different NPHS2 pathogenic mutations were detected in the present study; nine were missense and two were frameshift mutations leading to a premature stop codon (Table 2). Seven of these pathogenic mutations have been described elsewhere. The newly identified mutations include three missense and one indel substitution in exon 8, which represents the first indel mutation described in this gene thus far (c.971_987delinsACAG [p.L324fsX343]). Interestingly, this mutation consisted in an insertion of 4 nucleotides and a deletion of 17 nucleotides, which also is the largest deletion in the NPHS2 gene described to date. The three novel missense mutations were c.346A>C (p.T116P), c.662C>T (p.T221I), and c.842A>C (p.E281A). p.T116P and p.T221I were moderately conservative amino acid substitutions but at highly conserved sites in the multi-sequence alignment (MSA) of ortholog podocin proteins (except for zebrafish). p.E281A was a nonconservative change in a highly conserved position among podocin orthologs. All of them were predicted to be deleterious (Polyphen, Sorting Intolerant From Tolerant [SIFT]) and were not found in 300 control chromosomes. The large majority of pathogenic mutations hereby detected were located in the C-terminal part of podocin (9/11), mostly in the alanine- and glutamate-rich region that is highly conserved within the stomatin protein family (p.E381A, p.A284V, p.A288T, p.Q285fsX302). Only one mutation (p.T116P) was detected in the membrane domain and one (p.G92C) in the N-terminal region of podocin.
Table 2.
Classification of NPHS2 substitutions
| NPHS2 Substitutions | Exon | Previous Descriptiona | Control Chrsb | Segregation Analysisc | GDd | GVe | GD/GV Matrix Scoref | GDevg | Polyphen Predictionh | SIFT Predicted Toleratedi | Splicing Predictionj | Described in Single Nucleotide Polymorphism Databasek | VSl | MGm |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Previously reported NPHS2 pathogenic mutations | ||||||||||||||
| G92C | 1 | refs. 5 and 17 (+1) | 0/320 (ref. 17) (+2) | FP (+4) | 159 | 0 | +7 | 159 (+2) | 1.7 (+1) | No (+2) | LD (+1) | No (+1) | 21 | B |
| R138Q | 3 | refs. 5, 11, 13–18, 20, 25, 35, 48, and 49 (+1) | 0/320 (ref. 17) (+2) | F (+4) | 43 | 0 | +5 | 43 (+2) | 2.1 (+2) | No (+2) | NP (0) | No (+1) | 19 | B |
| L169P | 4 | refs. 13, 16, 18, and 35 (+1) | 0/200 (ref. 16) (+2) | FP (+4) | 98 | 0 | +6 | 98 (+2) | 1.5 (+1) | Yes (−2) | NP (0) | No (+1) | 15 | B |
| V260E | 6 | refs. 17 (+1) | 0/320 (ref. 17) (+2) | FP (+4) | 121 | 0/22 [pl] | +5 | 121 (+2) | 2.4 (+2) | No (+2) | NP (0) | No (+1) | 19 | B |
| A284V | 7 | refs. 17, 18, 20, 25, 32, 49, and 50 (+1) | 0/320 (ref. 17) (+2) | F (+4) | 64 | 0 | +5 | 64 (+2) | 1.9 (+1) | No (+2) | NP (0) | No (+1) | 18 | B |
| A288T | 7 | refs. 20, 17, and 25 (+1) | 0/320 (ref. 17) (+2) | FP (+4) | 58 | 0 | +5 | 58 (+2) | 1.9 (+1) | No (+2) | NP (0) | No (+1) | 18 | B |
| Previously reported NPHS2 variants of unknown clinical significance | ||||||||||||||
| P20L | 3 | refs. 5, 16–18, 35, and 51 | 2/360n (−1) | — | 98 | 155 | −2 | 14 (−2) | 2.3 (+2) | No (+2) | NP (0) | No (+1) | 0 | I |
| R229Q | 5 | refs. 14–18, 20, 23–25, 32, 35, 49, 52, and 53 | 9/454n (−2) | — | 43 | 0/29 [fi] | +4 | 20 (+1) | 0.4 (−2) | Yes (−2) | NP (0) | No (+1) | 0 | I |
| Novel missense amino acid NPHS2 substitutions identified in our cohort | ||||||||||||||
| T116P | 2 | Novel | 0/300n (+2) | F (+4) | 38 | 0/103 [fi] | –2 | 29 (+2) | 2.3 (+2) | No (+2) | NP (0) | No (+1) | 11 | B |
| M187I | 5 | Novel | 0/300n (+2) | — | 10 | 0/92 [ch,fi] | –4 | 0 (−2) | 0.6 (−2) | Yes (−2) | NP (0) | No (+1) | –7 | NV |
| T221I | 5 | Novel | 0/300n (+2) | F (+4) | 89 | 0/58 [fi] | +3 | 89 (+2) | 2.3 (+2) | No (+2) | NP (0) | No (+1) | 16 | B |
| E264Q | 6 | Novel | 2/454n (−1) | — | 29 | 0 | +2 | 29 (+2) | 1.8 (+1) | No (+2) | NP (0) | No (+1) | 7 | C |
| E281A | 7 | Novel | 0/300n (+2) | — | 107 | 0 | +6 | 107 (+2) | 1.5 (+1) | No (+2) | NP (0) | No (+1) | 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.
When a change was described previously in the literature as a pathogenic mutation or in the HGM database (+1).
When a sequence variant was not present in the control chromosomes (+2), if present <1% (−1) or >1% (−2).
Segregation shown in family (+4, F, affected and not affected siblings and their parents; FP, segregation in family in previous description).
GD (Grantham distance), score of chemical difference between the normal and mutated residue (high score, greater difference).
GV (Grantham variation), score of chemical difference between 11 orthologs (ranging from chimpanzee to zebrafish, 0 = completed conserved among podocin orthologs, [pl] = conserved among orthologs except in Platypus, [fi] = conserved among orthologs except in Zebrafish, [ch,fi] = conserved among orthologs except in Chiken and Zebrafish).
GD/GV matrix score, ranging from −2 to +8 (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 MSA]).
GDev (Grantham deviation), score of chemical difference between the mutated residue and the range of variation between orthologs (GD similar to GDev, higher difference, +2).
Polyphen assessment, ratio Polyphen >2 (probably damaging, +2), ratio Polyphen 1 to 2 (possibly damaging, +1), ratio Polyphen <1 (benign, −2).
SIFT prediction: not tolerated (+2), tolerated (−2).
Not predicted (NP) by splice site prediction neural network (0), if predicted loss of donor site (LD, +1).
When a change was not described in the single nucleotide polymorphism database (+1), if described (−1).
Variant score (VS); VS > 11 →.
Mutation group (MG) = B; VS = 5 to 10 → MG = C; VS = 0 to 4 → MG = I; VS < −1 → MG = NV; A, definitely pathogenic; B, highly likely pathogenic; C, likely pathogenic; I, unknown pathogenicity; NV, highly likely neutral.
This report.
Sequence NPHS2 Variants of Unknown Clinical Significance and Their Association with SRNS
Several amino acid substitutions (p.P20L, p.M187I, p.R229Q, p.E264Q) were not predicted to be clearly pathogenic using our in silico analysis (Table 2). Subsequently, these variants were screened in a minimum of 360 control chromosomes. None of the healthy controls showed the p.M187I sequence variant, indicating that it is probably not a polymorphism, although it was classified as a highly likely neutral variant (MG = NV) by our in silico NPHS2 scoring system. Moreover, it was found in an SRNS patient with another pathogenic NPHS2 mutation in compound heterozygosity.
On the contrary, p.P20L, p.E264Q, and p.R229Q were found in controls. To assess the putative association of these variants with SRNS, we excluded those cases carrying two pathogenic NPHS2 mutations (n = 7) because they had a clear disease cause. p.P20L and p.E264Q were present in single heterozygous state in both controls and patients with SRNS. The p.P20L variant (MG = I) is a nonconservative amino acid exchange and was predicted to be deleterious (Polyphen, SIFT) but at a nonconserved site in the MSA. The allele frequency observed in SRNS cases was 0.75% (2/264), which was not significantly different from the 0.55% (2/360) in controls (Table 3). The p.E264Q novel variant (MG = C) is conservative but at a highly conserved site in the MSA. Despite being more frequent among patients (0.75%; 2/264) than among controls (0.44%; 2/454), the difference was not statistically significant.
Table 3.
Genotype frequencies for NPHS2 variants of unknown clinical significance in patients with SRNS and control subjects
| NPHS2 Variants | Genotype, Number (Frequency) | Total | MAF | P (HWE)a | P [OR (95% CI)]b | ||
|---|---|---|---|---|---|---|---|
| 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.
Test for deviation from Hardy-Weinberg equilibrium (HWE) law.
Genotype frequency difference test (χ2) under dominant model.
Finally, the p.R229Q variant was identified in heterozygous state in 12 unrelated cases with SRNS, of which 6 carried the p.R229Q variant associated with one pathogenic NPHS2 mutation on the other allele. We also detected one patient with sporadic FSGS that was homozygous for p.R229Q. This position was conserved during evolution until chicken, but Polyphen and SIFT predicted that the p.R229Q variant was “benign.” In this study, p.R229Q was more frequent among cases compared with controls, showing association with SRNS: 5.30% (14/264) versus 1.98% (9/454) (odds ratio = 2.65; P = 0.02).
Haplotype Analysis of p.A284V and p.R229Q Alleles
We found six cases from different areas of Spain carrying the p.R229Q variant in compound heterozygous state with one pathogenic NPHS2 mutation in exon 7 (p.A284V, n = 5; p.A288T, n = 1). Segregation analysis performed in all available family members confirmed that all affected patients were compound heterozygotes for p.R229Q and the pathogenic mutation, and no unaffected individual carried both NPHS2 variants. We tried to investigate whether p.R229Q/p.A284V substitutions occurred on conserved haplotypes within our cohort of Spanish families to discern between a hot spot and a founder effect. The analysis of five microsatellites showed conserved haplotypes for the p.A284V mutation (171;172;208;192;186) and for the p.R229Q variant (171;186;208;188;197). Moreover, for three informative SNPs (5′UTR-51G>T, IVS3–21C>T, and c.954T>C), a common haplotype was also identified: (G;C;T) and (T;T;T), respectively. Phases for p.A284V/p.R229Q and each of the four microsatellites and three SNPs were confirmed in the whole group of families. We extended the study to include 15 p.R229Q-heterozygous carriers from SRNS and control subjects and 1 p.R229Q-homozygous patient, and we found that all these individuals also shared the same common haplotype.
Clinical Data for Patients Carrying One NPHS2 Pathogenic Mutation and the p.R229Q Variant
Patients carrying p.R229Q and one NPHS2 mutation (n = 9) developed NS significantly later than those carrying two pathogenic mutations (mean, 21.8 ± 9.1 versus 2.8 ± 2.6 years; P < 0.01; Table 1). This group included two patients (60-1, 164) with subnephrotic proteinuria at the time of diagnosis (Table 4). Renal biopsy showed mesangioproliferative lesions with FSGS in two patients and FSGS in seven patients. All of them were resistant to corticosteroids and immunosuppressant drugs. Angiotensin-converting enzyme inhibitors showed inconsistent effects in the majority of this group; however, in one patient (61-1), these agents decreased proteinuria from 4.5 to 1 g/day. Seven patients had developed ESRD at a mean age of 30.4 years (range: 15 to 50 years) and in a mean time of 8.4 years (range: 4 to 13 years) after the onset of the disease, which was also significantly later than the group of two pathogenic NPHS2 mutations (P < 0.01 and P = 0.03, respectively). No disease recurrence was observed in six patients who received renal allograft.
Table 4.
Clinical data of patients with SRNS and NPHS2 mutations
| Patient | Gender | Age at Onset of NS (years) | Renal Biopsy | Therapy | Evolution | Tx/Recurrence | NPHS2 Mutations |
|---|---|---|---|---|---|---|---|
| Patients with two pathogenic mutations | |||||||
| 10–1a | M | 4 | FSGS | Cs, CsA− | ESRD at 12 years | Yes/noc | [c.274G>T (p.G92C)]+[c.506T>C (p.L169P)] |
| 10–2a | M | 2 | MCNS | Cs, CsA, ACEI± | Normal Cr at 18 years | No | |
| 26–1a | F | 4.5 | FSGS | Cs, CsA, MMF− | ESRD at 7 years | Yes/no | [c.274G>T (p.G92C)]+[c.413G>A (p.R138Q)] |
| 26–2a | M | 2.5 | FSGS | Cs, CsA, MMF− | ESRD at 5 years | Yes/noc | |
| 71 | M | 1 | FSGS* | Cs, CP, CsA− | ESRD at 6 years | Yes/no | [c.413G>A (p.R138Q)]+[c.971_987delinsACAG (p.L324fsX343)] |
| 77 | F | 5.1 | FSGS* | Cs, CP, CsA, MMF− | CKD stage III at 9 years | No | [c.855_856delAA (p.Q285fsX302)]+[c.855_856delAA (p.Q285fsX302)] |
| 102–1a | M | 0.6 | FSGS* | Cs, CsA− | ESRD at 7 years | No | [c.346A>C (p.T116P)]+[c.346A>C (p.T116P)] |
| 102–2a | F | 0.02 | Not performed | Cs− | Normal Cr at 5 years | No | |
| 225b | M | 0.3 | Not performed | Cs, ACEI± | Normal Cr at 2 years | No | [c.842A>C (p.E281A)]+[c.842A>C (p.E281A)] |
| 228b | F | 8 | FSGS | Cs, CsA, MMF− | ESRD at 9 years | No | [c.779T>A (p.V260E)]+[c.779T>A (p.V260E)] |
| Patients with one mutation +WT | |||||||
| 103 | M | 10 | FSGS | Cs, MMF− | Normal Cr at 16 years | No | [c.561G>A (p.M187I)]+[c.862G>T (p.A288T)] |
| 227 | F | 7 | FSGS | Cs, CsA, ACEI− | ESRD at 8 years | No | [c.662 C>T (p.T221I)]+[?] |
| Patients with one mutation +R229Q | |||||||
| 44 | F | 39 | FSGS* | Cs, CsA, MMF− | ESRD at 50 years | No | [c.686G>A (p.R229Q)]+[c.851C>T (p.A284V)] |
| 59 | M | 10 | FSGS | Cs, CsA, MMF− | ESRD at 15 years | Yes/noc | [c.686G>A (p.R229Q)]+[c.851C>T (p.A284V)] |
| 60–1a | F | 16 | FSGS | Cs− | ESRD at 25 years | Yes/noc | [c.686G>A (p.R229Q)]+[c.851C>T (p.A284V)] |
| 60–2a | M | 13 | FSGS | Cs, CsA− | ESRD at 26 years | Yes/no | |
| 61–1a | M | 24 | FSGS* | Cs, CP, ACEI± | CKD stage II at 34 years | No | [c.686G>A (p.R229Q)]+[c.851C>T (p.A284V)] |
| 61–2a | M | 18 | FSGS | Cs, CP, CsA, MMF, ACEI− | CKD stage II at 29 years | No | |
| 121–1a | M | 28 | FSGS | Cs, CsA− | ESRD at 34 years | Yes/no | [c.686G>A (p.R229Q)]+[c.851C>T (p.A284V)] |
| 121–2a | M | 19 | FSGS | Cs, CsA− | ESRD at 23 years | Yes/no | |
| 164 | F | 28 | FSGS | Cs, CP− | ESRD at 40 years | Yes/no | [c.686G>A (p.R229Q)]+[c.862G>T (p.A288T)] |
Therapy effect categories: (−) no response, (±) 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.
Siblings with the same parents.
Only child of consanguineous parents.
These patients presented with chronic rejection nephropathy.
The subset of adult patients with FSGS carrying one pathogenic mutation plus the p.R229Q variant (n = 6) had an earlier onset of NS compared with those without NPHS2 mutations (26.2 ± 7.7 versus 33.9 ± 11.0 years; P = 0.05); however, no difference was observed in the age at onset of ESRD (36.8 ± 11.3 versus 39.6 ± 13.8 years; P = 0.65). In the group of adult FSGS patients without mutations, the recurrence in a renal transplant was 22% (6 of 27 patients); conversely, in the group with mutations, no recurrence was observed.
Discussion
NPHS2 mutations were initially described in patients developing NS from birth to 6 years of age (17,19). However, the utility of NPHS2 testing in adults with FSGS has not been fully studied. Our study represents the first cohort of Spanish SRNS patients evaluated for the NPHS2 gene, including early childhood-, late childhood-, and adult-onset cases. In contrast to the literature, we found that Spanish patients with late childhood- and adult-onset SRNS had a similar NPHS2 mutation detection rate than those with early childhood-onset (12%, 9 of 78 cases versus 10%, 6 of 61 cases, respectively). In the subset of cases with NPHS2 variants, our data suggest that the age at onset of the disease could be correlated with the genotype. Patients with early childhood onset (<6 years) carried two pathogenic mutations, patients with late childhood onset (6 to 18 years) carried two pathogenic or one pathogenic mutation in heterozygous state with the p.R229Q variant, and patients with adult onset (>18 years) carried one pathogenic mutation plus the p.R229Q variant. Quite interestingly, we confirmed that p.R229Q in compound heterozygous state with p.A284V mutation is the most common allelic combination causing late-onset SRNS in Spanish patients, in agreement with data previously reported by Machuca et al. (25) in patients from South America.
In addition, we showed that the p.A284V pathogenic mutation occurs in a conserved haplotype in Spanish patients, which supports the idea of a single origin for this variant. Because this mutation has mostly been detected in South American patients (Table 5), we could hypothesize that a Spanish founder might have introduced it into the Hispanic population studied by other groups (20,25,32), as suggested by Hildebrandt et al. (33). On the other hand, the high frequency of p.R229Q could mean that this variant arose by a recurrent event or that it is an ancient mutation present worldwide caused by population expansion. The shared haplotype among Spanish p.R229Q carriers gives further evidence of an ancient origin for this variant. In accordance, Tsukaguchi et al. (20) also found a common haplotype among p.R229Q carriers of African, Brazilian, and European descent. There is an uneven p.R229Q allele distribution throughout different populations: it is more frequent among Spaniards, South Americans, Europeans, and European Americans (∼4 to 7%) (16–18,23–25,34,35) than among Africans, African Americans, and Asians (∼0.0 to 1.5%) (21,36–40), suggesting that this variant emerged in Europe, although it is not possible to discern a specific geographic origin.
Table 5.
Allele frequencies for p.A284V and p.R229Q among patients and controls from different geographic areas
| Area/Ethnicity | p.A284V |
p.R229Q |
References | |||
|---|---|---|---|---|---|---|
| Cases | Allele Frequency | Cases | Controls | Allele 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 |
| Canada | 0/87 | 0/174 (0.000) | 8/87j | 3/108 | 11/390 (0.028) | 53 |
| South America | ||||||
| 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 |
One case in homozygous state and two cases in compound heterozygous state.
Four cases in compound heterozygous state, 2 cases in homozygous state, and 10 cases in heterozygous state. This study includes 120 steroid-sensitive cases.
Four cases in compound heterozygous state, two cases in homozygous state, and six cases in heterozygous state. This study includes 59 steroid-sensitive cases.
Four cases in compound heterozygous state, 5 cases in homozygous state, and 13 cases in heterozygous state.
Eight cases in compound heterozygous state, five cases in homozygous state, and two cases in heterozygous state.
Three cases in homozygous state and 32 cases in heterozygous state or in compound heterozygous state.
One case from Dominican Republic in homozygous state and two sporadic cases from unknown origin in compound heterozygous state.
Six families in compound heterozygous state and 11 sporadic cases in heterozygous state, of whom 2 had the p.A284V in compound heterozygous state.
Thirty-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).
This study includes 15 steroid-sensitive cases.
All South American cases and controls were of Spanish descent.
Fifteen different NPHS2 sequence variants have been identified in this study, 13 of which were missense. The high percentage of NPHS2 missense variants represents a diagnostic challenge because in some cases it is difficult to differentiate between a disease-causing mutation and a neutral variant. We describe here an in silico sequence variant classification strategy for the NPHS2 amino acid substitutions based on the combination of different approaches previously reported for other genes (30,41,42), which takes into account (1) the analysis of control chromosomes, (2) the cosegregation with the disease in a family, (3) the biophysical and biochemical difference between wild-type and mutant amino acids (28), (4) the evolutionary conservation of the amino acid residue in orthologs (29,43,44), and (5) software that uses in silico predictors of pathogenic effect [Polyphen (45), SIFT (46)] and splice site [Neural Network (47)]. The accuracy of this in silico analysis was tested using previously described and classified podocin-amino acid substitutions. Afterward, we evaluated the novel missense substitutions identified in our study cohort, and we found that three of five were clearly pathogenic mutations.
Finally, we identified three variants with unknown clinical significance. The p.M187I is a novel sequence variant that is predicted to be a highly likely neutral variant by our in silico scoring system, but we could speculate that this variant is implicated in the pathogenesis of late-onset SRNS because it was not found in 360 control chromosomes and was present in compound heterozygous state with a pathogenic mutation (p.A288T) in a patient with late childhood-onset SRNS. Heterozygosity for p.P20L and p.E264Q did not increase the risk for SRNS because the frequency of these alleles was similar in both patient and control subjects. On the other hand, p.R229Q was present in 4.54% (12 of 132) of cases in heterozygosity and in 0.75% (1 of 132) of cases in homozygosity. p.R229Q has been extensively reported in a higher frequency among patients than in controls, but without statistical significance (17,18,20,21,26). In our Spanish study population, the frequency of the p.R229Q allele was significantly higher in SRNS patients than in controls (5.3 versus 1.9%), which supports the results obtained by Machuca et al. (25) among Europeans and South American patients.
Conclusions
NPHS2 mutations were not uncommon in our cohort of Spanish patients with late- and adult-onset SRNS. For genetic diagnostic purposes in European adults with FSGS, the first step should be to screen for p.R229Q, and only in those carrying this variant would further analysis be indicated to identify the second mutation, usually p.A284V for patients of Spanish descent. Age at onset of the disease and progression to ESRD in patients with one pathogenic mutation plus the p.R229Q variant was significantly higher and slower than in patients with two NPHS2 pathogenic mutations. Furthermore, because compound heterozygous patients with the p.R229Q variant do not respond to either corticoids or immunosuppressants and do not relapse after kidney transplantation, genetic counseling in these families should be as follows: (1) to avoid unnecessary steroid and immunosuppressive treatment, (2) to promote living donor kidney transplantation, and (3) to provide the possibility to screen couples carrying NPHS2 mutation for p.R229Q.
Disclosures
None.
Acknowledgments
We thank the patients and their families for taking part in this study. This work was funded by grants from the Spanish Health Ministry (FIS-05/0761 and FIS-09/01506) and the REDinREN (Red Renal de Investigación Española 16/06, RETICS, Instituto de Investigación Carlos III). Other investigators in the FSGS Spanish Study Group: Hospital de la Santa Creu i Sant Pau- Gloria Fraga; Hospital Universitario La Fe- Santiago Mendizábal, Isabel Zamora; Hospital Infantil La Paz- Antonia Peña, Laura Espinosa, Carmen García, Marta Melgosa, Mercedes Navarro; Hospital Vall d'Hebron- Joan López-Hellin, Sara Chocrón, Álvaro Madrid, Ramón Vilalta, José Luciano Nieto, Clara Ventura; Hospital Sant Joan de Déu- Antonio Giménez, Jorge Vila Cots, Juan Antonio Camacho; Hospital Infantil Universitario Virgen del Rocío- Ana Sánchez-Moreno, Francisco de la Cerda; Hospital Universitario de Canarias- Eduardo Salido; Fundación Jiménez Díaz- Alberto Ortiz, Simona Alexandra, Carlos Caramelo†, Jesús Egido; Hospital Universitario de La Princesa- Carmen Bernis; Hospital General Universitario Gregorio Marañón- Augusto Luque de Pablos, Ma Dolores Morales San José; Hospital Germans Trias i Pujol- Guillem Pintos; Hospital de Barcelona- Pere Sala, Frederic Raspall, Ángel Vila; Hospital Torrecárdenas- Antonio María Daza; Hospital Niño Jesús- Mercedes Vázquez, José Luis Écija; Hospital Universitario Reina Sofía- Mario Espinosa; Hospital Princeps d'España- Rafael Poveda; Hospital Clínic de Barcelona- Eduard Mirapeix; Hospital de niños Ricardo Gutiérrez- Graciela Vallejo; Hospital Universitario de Getafe- Cristina Aparicio; Hospital Materno-Infantil Son Dureta- Jordi Rosell, Diego de Sotto; Hospital Infantil doce de Octubre- Rafael Muley; Hospital de Galdakao- Jesús Montenegro; Hospital Universitario Marqués de Valdecilla- Domingo González; Hospital Universitario Virgen de las Nieves- David Barajas de Frutos; Hospital Son Llàtzer- Esther Trillo; Hospital Universitario Virgen de la Arrixaca- Salvador Gracia; Hospital de Cruces- Francisco Javier Gainza de los Ríos; Hospital Miguel Servet- Ma Luisa Justa; Hospital Materno-Infantil de Badajoz- Emilia Hidalgo-Barquero.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
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