==== Front Ann Saudi MedAnn Saudi MedAnnals of Saudi Medicine0256-49470975-4466King Faisal Specialist Hospital and Research Centre 2441385510.5144/0256-4947.2013.533asm-6-533Original ArticleSteroid-resistant nephrotic syndrome: impact of genetic testing Kari Jameela A. aEl-Desoky Sherif M. aGari Mamdooh bMalik Khalid aVega-Warner Virginia cLovric Svjetlana cBockenhauer Detlef d a Department of Pediatrics, King Abdulaziz University, Jeddah, Saudi Arabia b Center of Excellence in Denomic Medicine Research, Jedah, Saudi Arabia c Pediatrics, University of Michigan, Ann Arbor, Michigan, United States d Renal Unit, Great Ormond Street Hospital, London, United KingdomCorrespondence: Jameela Abdulaziz Kari, Pediatrics, King Abdulaziz University, PO Box 13042 Jeddah 21943 Saudi Arabia, T: 966-505-677-904, F: 966-22-408-8353, jkari@doctors.org.ukNov-Dec 2013 33 6 533 538 Copyright © 2013, Annals of Saudi Medicine2013This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.BACKGROUND AND OBJECTIVES Mutations in several genes are known to cause steroid-resistant nephrotic syndome (SRNS), most commonly in NPHS1, NPHS2, and WT1. Our aims were to determine the frequency of mutations in these genes in children with SRNS, the response of patients with SRNS to various immunosuppressants, and the disease outcome, and to review the predictive value of genetic testing and renal biopsy result. DESIGN AND SETTINGS A retrospective review was performed of the medical records for all children with SRNS who were treated and followed-up in the Pediatric Nephrology Unit of King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia from 2002–2012. PATIENTS AND METHODS We retrospectively reviewed the medical records of children above 1 year of age, who presented with SRNS to KAUH, Jeddah, Saudi Arabia, in the 10-year interval from 2002–2012 and for whom the results of genetic testing for NPHS1, NPHS2, and WT1 were available. We compared the clinical phenotype, including response to treatment and renal outcome to genotype data. RESULTS We identified 44 children with a clinical diagnosis of SRNS in whom results of genetic testing were available. Presumably disease-causing mutations were detected in 5 children (11.4%) of which 3 (6.8%) had NPHS2 mutation and 2 (4.5%) had NPHS1 mutation. Renal biopsy revealed minimal change disease (MCD) or variants in 17 children, focal segmental glomerulosclerosis (FSGS) in 23 children, membranoproliferative changes (MPGN) in 2 children, and IgA nephropathy in another 2 children. Children with MCD on biopsy were more likely to respond to treatment than those with FSGS. None of those with an identified genetic cause showed any response to treatment. CONCLUSION The frequency of identified disease-causing mutations in children older than 1 year with SRNS presented to KAUH was 11.4%, and these patients showed no response to treatment. Initial testing for gene mutation in children with SRNS may obviate the need for biopsy, and the use of immunosuppressive treatment in children with disease due to NPHS1 or NPHS2 mutations. Renal biopsy was useful in predicting response in those without genetic mutations. ==== Body The diagnosis and management of steroid-resistant nephrotic syndrome (SRNS) remains a challenge to pediatric nephrologists. Several genes have been found to cause SRNS. NPHS2 and NPHS1 mutations are the most common genetic causes in children with SRNS and congenital nephrotic syndrome, respectively.1 Many different mutations of the NPHS2 gene have been reported in several Western, European, Middle Eastern, and Asian countries.1–9 The prevalence of NPHS2 mutations varies in these different populations, and underlies 26% of cases with SRNS in American2 and 24.7% of Turkish patients,3 compared to 9% in Greek,4 4 % in Indian5 3.4% in Pakistani,6 4.3 % in Chinese7 and 0% in Japanese8 and Korean9 children with SRNS. Mutations in WT1, NPHS3, TRPC6, CD2AP, PLCE1, INF2, ACTN4, and ITGA3 have also been reported as causes of SRNS.5,10–13 Identification of an underlying genetic basis allows clinical observations in molecularly defined patient cohorts, which impacts prognosis and treatment.14 Indeed, several reports so far indicate that children with SRNS caused by mutations in NPHS1 and NPHS2 do not respond to immunosuppressive treatment,2,15–17 whereas some with an underlying mutation in WT1 appear to respond to treatment with cyclosporin.18 In this study, we reviewed clinical features of children older than 1 year with SRNS presenting to a King Abdulaziz University Hospital (KAUH) in Jeddah, Saudi Arabia, including response to treatment, biopsy findings, disease outcome, and the frequency of NPHS1, NPHS2, and WT1 mutations in this cohort. PATIENTS AND METHODS A retrospective review was performed of the medical records for all children with SRNS who were treated and followed-up in the Pediatric Nephrology Unit of AUH, Jeddah, Saudi Arabia, from 2002–2012. The diagnosis of NS was based on clinical and laboratory findings of nephrotic range proteinuria, hypoalbuminemia, and hyperlipidemia. Only children with primary nephrotic syndrome (NS) who had undergone renal biopsy and had genetic testing results available for NPHS2, NPHS1, and WT1 were included in the study. We excluded children with (a) an underlying, secondary cause for NS (such as lupus nephritis, infections, or neoplasm), (b) congenital and infantile NS, or (c) steroid-sensitive nephrotic syndrome (SSNS). Permission to conduct the study was granted by the Ethics Research Committee of King Abdulaziz University. For all children included in the study, blood urea and serum creatinine measurements were performed regularly to monitor kidney function. Other investigations included hepatitis B and C serology, human immunodeficiency virus 1 and 2 serology, and complement C3 and C4 levels; antinuclear antibody assays were performed for selected cases with older age at presentation. The diagnosis of SRNS was made if the child did not respond to the standard steroid therapy with oral prednisone 60 mg/m2/d for 4 weeks. Secondary steroid resistance was defined as no response after 4 weeks of prednisone 60 mg/m2/d in a child previously known to have a steroid-sensitive course. All children received intravenous pulse methyl prednisolone 600 mg/m2 daily for 3 consecutive days after failure of the 4-week treatment with oral prednisolone. Ultrasound-guided kidney biopsy was performed in all patients. The biopsy specimens were examined by light, electron, and immunofluorescence microscopy. An adequate biopsy was defined as the presence of at least 5 glomeruli in the specimen on light microscopy. Based on the histopathologic diagnosis, children were treated with 1 or more of the following regimens: For children with focal segmental glomerulosclerosis (FSGS), the following treatments were given consecutively: (1) calcineurin inhibitor (CNI), mainly cyclosporin, 4 to 6 mg/kg/d in 2 divided doses, (2) mycophenolate mofetil (MMF) 500 mg/m2/dose twice daily, and (3) intravenous rituximab (375 mg/m2/dose) 2 doses given 2 weeks apart, in addition to alternate-day oral prednisolone. For children with minimal change disease (MCD), the following treatments were given consecutively: (1) oral cyclophosphamide 2.5 mg/kg/d was given for 8 weeks, (2) children who did not respond to cyclophosphamide were treated with cyclosporin for 3 to 6 months, and (3) if no response to cyclosporine was observed, then the children were treated with MMF for 3 to 6 months. Children with membranoproliferative glomerulonephritis (MPGN) were treated with low-dose aspirin, 8-week course of cyclophosphamide (2.5 mg/kg/d), and prolonged alternate day course of prednisolone at 40 mg/m2 for 6 months, followed by 30 mg/m2 for another 6 months, then 20 mg/m2 for 6 months, and 10 mg/m2 for 6 months. Genetic testing for mutations in the NPHS1, NPHS2, and WT1 genes was performed as detailed previously.19 The treatment response was evaluated by measuring the degree of proteinuria and serum albumin levels. The reports can be listed as follows: (1) complete remission was defined as negative dipstick test result (normal protein excretion) in a spot first-morning urine sample or quantitative urine protein test result of ≤300 mg/L and serum albumin level ≥35 g/L. (2) Partial remission was defined as a reduction in urine protein excretion in a spot morning urine sample below the nephrotic range in the pediatric age group (i.e., <1000 mg/m2/d) and serum albumin between 25 and 34 g/L. (3) Failure to respond was defined as persistent proteinuria (≥1000 mg/m2/d) and failure of serum albumin to increase to 25 g/L or more. The follow-up was done twice monthly initially and subsequently every 1 to 3 months. On each visit, the child was evaluated clinically for the evidence of disease activity and complications (infections and drug adverse effects). When the child did not respond to a given drug for 3 to 6 months it was discontinued. Statistical analysis Statistical analysis was performed using the SPSS, version 16.0 (SPSS Inc., Chicago, IL USA). For categorical variables, descriptive statistics were performed and the results were expressed as frequencies and percentages. Descriptive statistics were performed using median and inter-quartile range. RESULTS Patient characteristics A total of 242 children with NS were followed up at the Pediatric Nephrology Unit of KAUH between 2002 and 2012. Idiopathic NS was diagnosed in 214 patients older than 1 year of age (88.4%), SSNS was diagnosed in150 patients (62.0%), and SRNS in 64 patients (26.4%). Patients with SRNS were classified to have either primary or secondary SRNS; 53 patients (82.8%) had primary SRNS while 11(17.2%) had secondary SRNS. Genetic testing results were available for 44 children of which 36 had primary SRNS and 8 had secondary SRNS. Five children (11.4%) had presumably disease-causing mutations identified (mut+). The 2 patients with NPHS1 mutations were sisters homozygous for the same missense variant c.2215G>A (p.A739T). The pathogenicity of this variant is unclear, but is not a recognized single nucleotide polymorphism (SNP), and the amino acid is reasonably conserved. They also carried the known SNP in NPHS2 G688A (p.R229Q). Another 3 children had homozygous mutations in NPHS2 (Table 1). There was no underlying WT1 mutation identified in any patient. Table 1 gives a summary of the mutations detected. All mut+ children presented with primary SRNS and did not respond to treatment with immunsuppressives (Table 1). There were 2 children with a history of affected siblings who tested negative for mutations in NPHS1, NPHS2, and WT1 and thus their disease was likely caused by another gene not tested for. Biopsy findings were as follows: MCD25 or MCD variants (immunoglobulin M [IgM] nephropathy and C1q nephropathy) in 17 children, FSGS in 23 children, MPGN in 2 children, and IgA nephropathy in another 2 children. All mut+ children presented a histological picture of FSGS on biopsy. Treatment Initially all patients received oral prednisone 60 mg/m2/d for 4 to 8 weeks. All patients received intravenous methyl prednisolone pulse therapy (600 mg/m2 body surface area/d) for 3 consecutive days, followed by 3 doses on alternate days if the child did not respond after the 3 initial doses. Seventeen children received oral cyclophosphamide 2.5 mg/kg/d for 8 weeks. Thirty-four patients received cyclosporin for a mean duration of 2.91 years. Twelve patients received MMF for a mean duration of 2.57 years, and 5 patients received rituximab 375 mg/m2 body surface area, of which only 1 had a partial remission while the other 4 patients had no clinical or laboratory response. Table 2 summarizes the treatment modalities and the response according to the histopathologic examination.” Children who relapsed after remission were managed by pulse methyprednisolone, ranging from 3 to18 pulses (median, 6 pulses) during followup, in addition to changing the steroid-sparing immunosuppressive therapy. A total of 26 children received 2 immunosuppressants, in addition to methyl prednisolone, 5 children received 3, and 1 child received 4 different immunosuppressive drugs (apart from steroids) during the course of their illness. Complications The following complications, due to SRNS or as a result of treatment, were observed: cushingoid appearance in 20 patients (45%), growth retardation in 17 patients (38.6%), hypertension in 9 patients (27.3%), recurrent infections in 9 patients (20.5%), acute renal failure in 8 patients (18.2%), 2 resistant edema in 2 patients, and simultaneous onset of type 1 diabetes mellitus in 2 patients (4.5%); however, 4 patients (9.1%) did not develop overt complications. Disease outcome A total of 29 children without identified genetic cause responded to immunosuppressive treatment and achieved remission (19 complete, 10 partial). Sixteen children who achieved remission had MCD or MCD variants (94%), 10 had FSGS (55%), 2 with MPGN, and 1 with IgA nephropathy. None of the 5 mut+ children responded to a 3- to 6-month course of cyclosporin and one of them had an extra course of MMF for another 3 months without apparent response. Two patients (4.5%) progressed to chronic kidney disease (stage 3 and 4), 3 (6.8%)progressed to end-stage kidney disease and 2 (4.5%) died. None of the mut+ children achieved remission. DISCUSSION The primary goals of this study were to determine the frequency of NPHS2 and WT1 mutations in children with SRNS, the response to various immunosuppressants, and the disease outcome. Our results showed that 4.5% and 6.8% of children with primary SRNS had mutations in the NPHS1 and NPHS2 genes, respectively; none of the patients with secondary SRNS had a mutation in the NPHS1 or NPHS2 genes. This is lower than the podocin mutation frequency of 18.1% reported by Hinkes et al in 430 children with SRNS.16 This could be explained by the small number of our cohort or more likely reflects the lower frequency of these mutations in Saudi Arabia. Indeed, our results are comparable to those of Abid et al from Pakistan who found a frequency of 5.5% NPHS1 and 3.4% NPHS2 mutations in children with SRNS.6 This low mutation frequency indicates that the availability of rapid genetic testing for NPHS1, NPHS2, and WT1 would influence the management of only a minority of patients. However, we observed non-response in these children and therefore recognizing these children would spare them a renal biopsy and prolonged exposure to immunosuppressive drugs. We did not test for other potential recessive genes such as PLCE1 (NPHS3) that could be the underlying genetic cause in some of our patients. Other unidentified autosomal recessive genes could also be the underlying cause in other nonresponding children. We had 2 children (40%) with biallelic mutations explaining their disease. Biallelic and triallelic mutations were reported to be implicated in genotype/phenotype correlations.20,21 Schultheiss et al20 reported 5 patients with mutations in both the NPHS1 and the NPHS2 genes out of 62 children with SRNS and CNS. Koziell et al22 showed that an overlap in the NPHS1/NPHS2 mutation spectrum with the characterization of a unique digenic inheritance of NPHS1 and NPHS2 mutations, which results in a multiple allelic hits that modify an autosomal recessive disease phenotype in humans from CNS to FSGS. This may result from an epistatic gene interaction and functional inter-relationship between NPHS1 and NPHS2 in human nephrotic disease.) In those children without identified mutations, a biopsy can still direct treatment. Our results here are biased, as not all patients received the same treatment from the outset. Nevertheless, the high subsequent response rate to cyclosporin and cyclophosphamide in patients with MCD supports the initiation of treatment with these agents. More than half of the patients in our cohort (23 of 44; 52.3%) had a histologic diagnosis of FSGS. Similar to our findings, other data show that FSGS is the most common histopatholgical subtype in children with SRNS.23,24 Approximately 75% of patients with SRNS exhibit renal histologic features of FSGS while 20% show MCD.25 Although the role of kidney biopsy has increasingly become restricted,26 the underlying histologic features in NS are of significance in determining the outcome27 as children with SRNS and the histological picture of MCD are more likely to achieve remission. In one report on 136 patients with SRNS, it was shown that following extended immunosuppressive therapy, children with MCD had significantly greater remission rates compared to those with other histopathologic subtypes.23 The treatment options in our study were also based on the histopathologic findings, and the response was better in children with MCD. About 80% of children with idiopathic NS responded to corticosteroids;28 however, some authors suggest that testing for NPHS2 mutations should be performed in children with a first episode of NS to avoid an unnecessary steroid course in those testing positive.4,16 In our current practice, it is not possible to base initial treatment decisions on genetic testing, as results become available only after several months. In our case, we had to weigh the benefit of initiating treatment with corticosteroids against the potential complications that could arise as a result of non-treatment. At present, no optimal treatment has been reported to meet the goals of therapy for SRNS, which are to achieve complete resolution of proteinuria and to preserve kidney function. Immunosupressive therapy has been used to induce complete remission in patients with SRNS; however, there is only partial remission in some cases, especially in genetic forms of SRNS, which are typically refractory to immunosuppressive therapy. In one report on patients with SRNS, none of the 29 cases with NPHS2 mutations who were treated with cyclosporin or cyclophosphamide demonstrated complete remission.4 Similar observations were made by other authors who found that none of the 43 patients with an identified genetic cause of SRNS demonstrated complete response to cyclosporin therapy, yet 2 achieved a partial response.29 A considerable number of our cohort achieved either complete or partial remission on cyclosporin therapy. This is in line with the Cochrane review of 14 randomized controlled trials of 449 children with SRNS, which found that cyclosporin when compared with placebo or no treatment significantly increased the number of children who achieved complete remission. 29 Other investigators also reported a high response rate to cyclosporin in SRNS without identified disease-causing mutations. Recently, cyclosporin was reported as having an antiproteinuric effect independent of its immunosuppression action.31 A total of 10 of the 12 patients (83%) who received MMF in this study demonstrated complete remission of NS, and one additional patient (8.5%) demonstrated partial remission. Moreover, all of those patients received MMF after relapsing or not achieving remission with either cyclophosphamide and/or cyclosporin. In the published reports, there are inconsistent results regarding the efficacy of MMF in patients with SRNS. In one clinical trial of 138 patients with SRNS, only 33% of the patients achieved a partial or complete response. However, the patients in their study were randomly assigned to a combination therapy comprising oral pulse dexamethasone and MMF.32 Other observational studies demonstrate that MMF has variable benefits in children with SRNS.33,34 The role of rituximab in SRNS is still uncertain: It has been demonstrated in several case series that rituximab, in combination with a corticosteroid and/or CNI, improves remission rates in patients with SRNS.35,36 We cannot, however, compare our findings with those of other authors because the proportion of patients who received rituximab in our study was very small. In conclusion, the frequency of identified disease-causing mutations (NPHS1 and NPHS2) in children with SRNS presented to KAUH is 11.4%, and they show no response to treatment. Initial testing for gene mutation in children with SRNS would save them unnecessary treatment. Renal biopsy was useful in deciding immunosuppression regimen and predicting prognosis for those with negative mutations. MMF was a highly effective treatment in those children with no response to cyclophosphamide and or cyclosporin. Acknowledgments This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under the grant number (432/003/d) for the pediatric nephrology research. The authors, therefore, acknowledge with thanks the DSR technical and financial support. The authors also thank the clinical research unit at KAUH for their assistance with the statistics and writing up this paper. Table 1 Summary of the mutations detected in children with SRNS. Age at presentation Follow-up duration Histopathologic examination Primary or secondary Mutation found Consequence for protein Homozygous (H) or heterozygous (h) Response to treatment 1 1.5 10 FSGS Primary NPHS1: c.2215G>A NPHS2: G688A A739T R229Q H h Did not respond to cyclosporine or MMF 2 2 5 FSGS Primary NPHS1: c.2215G>A NPHS2: G688A A739T R229Q H h Did not respond to cyclosporine 3 3.5 1.5 FSGS Primary NPHS2: Ex6:779 T>A V260E (Weber et al37) H Did not respond to cyclosporin 4 1 3 FSGS Primary NPHS2:Exon 6 779T>A=V260E (Weber et al37) H Did not respond to cyclosporin 5 4 11 FSGS Primary NPHS2: Ex5:538 G>A V180 M (Boute et al22) H Did not respond to cyclosporin SRNS: Steroid-resistant nephrotic syndrome, FSGS: focal segmental sclerosis. Table 2 Summary of the therapeutic Interventions performed and the response according to the histopathologic examination. Histopathologic examination IV MP Cyclophosphamide Cyclosporine MMF Rituximab MCD (n=17) Number received the drug 17 15 9 2 1 Response (complete + partial) 2 (2+0) 6 (5+1) 7 (5+2) 1 (1+0) 0 FSGS (n=23-with negative genetic testing= 18) Number received the drug 23 0 23 9 4 Response (complete + partial) 1(0+1) 8 (5+3) 8 (7+1) 1(0+1) MPGN n=(2) Number received the drug 2 2 0 0 0 Response (complete + partial) 0 2 (2+0)a IgA nephropathy (n=2) Number received the drug 2 0 2 1 0 Response (complete + partial) 0 1 (0+1) 1 (1+0) IV: Intravenous, MMF: mycophenolate mofetil, MCD: minimal change disease, FSGS: focal segmental sclerosis, MPGN: membranoproliferative glomerulonephritis, IgA: immunoglobulin A. a Patients also received alternate days prednisolone at 40 mg/m2 for 6 months, followed by 30 mg/m2 for another 6 months, then 20 mg/m2 for 6 months and 10 mg/m2 for 6 months. ==== Refs REFERENCES 1 Santin S Bullich G Tazon-Vega B Garcia-Maset R Gimenez I Silva I Ruiz P Ballarin J Torra R Ars E Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome Clin J Am Soc Nephrol 2011 5 6 5 1139 48 21415313 2 Ruf RG Lichtenberger A Karle SM Patients with mutations in NPHS2 (podocin) do not respond to standard steroid treatment of nephrotic syndrome J Am Soc Nephrol 2004 15 722 732 14978175 3 Berdeli A Mir S Yavascan O NPHS2 (Podicin) mutations in Turkish children with idiopathic nephrotic syndrome Pediatr Nephrol 2007 22 2031 2040 17899208 4 Megremis S Mitsioni A Mitsioni AG Nucleotide variations in the NPHS2 gene in Greek children with steroid-resistant nephrotic syndrome Genet Test Mol Biomarkers 2009 13 249 256 19371226 5 Vasudevan A Siji A Raghavendra A Sridhar TS Phadke KD NPHS2 mutations in Indian children with sporadic early steroid resistant nephrotic syndrome Indian Pediatr 2012 49 231 233 22080622 6 Abid A Khaliq S Shahid S A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic syndrome patients from Pakistan Gene 2012 502 133 137 22565185 7 Yu Z Ding J Huang J Mutations in NPHS2 in sporadic steroid-resistant nephrotic syndrome in Chinese children Nephrol Dial Transplant 2005 20 902 908 15769810 8 Maruyama K Iijima K Ikeda M NPHS2 mutations in sporadic steroid-resistant nephrotic syndrome in Japanese children Pediatr Nephrol 2003 18 412 416 12687458 9 Cho HY Lee JH Choi HJ WT1 and NPHS2 mutations in Korean children with steroid-resistant nephrotic syndrome Pediatr Nephrol 2008 23 63 70 17934764 10 Gigante M Caridi G Montemurno E TRPC6 mutations in children with steroid-resistant nephrotic syndrome and atypical phenotype Clin J Am Soc Nephrol 2011 6 1626 1634 21734084 11 Mir S Yavascan O Berdeli A Sozeri B TRPC6 gene variants in Turkish children with steroid-resistant nephrotic syndrome Nephrol Dial Transplant 2012 27 205 209 21511817 12 Li J Ding J Zhao D WT1 gene mutations in Chinese children with early onset nephrotic syndrome Pediatr Res 2010 68 155 158 20442690 13 Benoit G Machuca E Nevo F Gribouval O Lepage D Antignac C Analysis of recessive CD2AP and ACTN4 mutations in steroid-resistant nephrotic syndrome Pediatr Nephrol 2010 25 445 451 19956976 14 Bockenhauer D Medlar AJ Ashton E Kleta R Lench N Genetic testing in renal disease Pediatr Nephrol 2012 27 873 883 21617915 15 Santin S Tazon-Vega B Silva I Clinical value of NPHS2 analysis in early- and adult-onset steroid-resistant nephrotic syndrome Clin J Am Soc Nephrol 2011 6 344 354 20947785 16 Hinkes B Vlangos C Heeringa S Specific podocin mutations correlate with age of onset in steroid-resistant nephrotic syndrome J Am Soc Nephrol 2008 19 365 371 18216321 17 Buscher AK Kranz B Buscher R Immunosuppression and renal outcome in congenital and pediatric steroid-resistant nephrotic syndrome Clin J Am Soc Nephrol 2010 5 2075 2084 20798252 18 Gellermann J Stefanidis CJ Mitsioni A Querfeld U Successful treatment of steroid-resistant nephrotic syndrome associated with WT1 mutations Pediatr Nephrol 2010 25 1285 1289 20191369 19 Hinkes BG Mucha B Vlangos CN Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2) Pediatrics 2007 119 e907 e919 17371932 20 Schultheiss M Ruf RG Mucha BE No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations Pediatr Nephrol 2004 19 1340 1348 15338398 21 Koziell A Grech V Hussain S Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration Hum Mol Genet 2002 11 379 388 11854170 22 Boute N Gribouval O Roselli S NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome Nat Genet 2000 24 349 354 10742096 23 Gulati S Sengupta D Sharma RK Steroid resistant nephrotic syndrome: role of histopathology Indian Pediatr 2006 43 55 60 16465008 24 Nammalwar BR Vijayakumar M Prahlad N Experience of renal biopsy in children with nephrotic syndrome Pediatr Nephrol 2006 21 286 288 16328540 25 Fuchshuber A Gribouval O Ronner V Clinical and genetic evaluation of familial steroid-responsive nephrotic syndrome in childhood J Am Soc Nephrol 2001 12 374 378 11158229 26 Gulati S Sharma AP Sharma RK Gupta A Gupta RK Do current recommendations for kidney biopsy in nephrotic syndrome need modifications? Pediatr Nephrol 2002 17 404 408 12107803 27 Niaudet P Gagnadoux MF Broyer M Treatment of childhood steroid-resistant idiopathic nephrotic syndrome Adv Nephrol Necker Hosp 1998 28 43 61 9889985 28 Niaudet P Long-term outcome of children with steroid-sensitive idiopathic nephrotic syndrome Clin J Am Soc Nephrol 2009 4 1547 1548 19808239 29 Tarshish P Tobin JN Bernstein J Edelmann CM Jr Prognostic significance of the early course of minimal change nephrotic syndrome: report of the International Study of Kidney Disease in Children J Am Soc Nephrol 1997 8 769 776 9176846 30 Hamasaki Y Yoshikawa N Hattori S Cyclosporin and steroid therapy in children with steroid-resistant nephrotic syndrome Pediatr Nephrol 2009 24 2177 2185 19714370 31 Faul C Donnelly M Merscher-Gomez S The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporin A Nat Med 2008 14 931 938 18724379 32 Gipson DS Trachtman H Kaskel FJ Clinical trial of focal segmental glomerulosclerosis in children and young adults Kidney Int 2011 80 868 878 21734640 33 Li Z Duan C He J Mycophenolate mofetil therapy for children with steroid-resistant nephrotic syndrome Pediatr Nephrol 2010 25 883 888 19953275 34 Cattran DC Wang MM Appel G Matalon A Briggs W Mycophenolate mofetil in the treatment of focal segmental glomerulosclerosis Clin Nephrol 2004 62 405 411 15630898 35 Gulati A Sinha A Jordan SC Efficacy and safety of treatment with rituximab for difficult steroid-resistant and -dependent nephrotic syndrome: multicentric report Clin J Am Soc Nephrol 2010 5 2207 2212 20798255 36 Bagga A Sinha A Moudgil A Rituximab in patients with the steroid-resistant nephrotic syndrome N Engl J Med 2007 356 2751 2752 17596616