J Am Soc Nephrol 13: 400–405, 2002

Mutations in NPHS2 Encoding Are a Prevalent Cause of Steroid-Resistant among Israeli-Arab Children

YAACOV FRISHBERG,* CHONI RINAT,* ORLI MEGGED,* ELI SHAPIRA,* SOFIA FEINSTEIN,* and ANNICK RAAS-ROTHSCHILD† *Division of Pediatric Nephrology, Shaare Zedek Medical Center, Jerusalem, Israel; and †Department of Human Genetics, Hadassah University Medical Center and Hadassah-Hebrew University School of Medicine, Jerusalem, Israel.

Abstract. Steroid-resistant nephrotic syndrome (SRNS) repre- tested (familial and nonfamilial), 15 patients (55%) were ho- sents a heterogeneous group of disorders that are often mozygous for the mutation (R138X). They all shared the same resistant to other immunosuppressive agents and tend to haplotype and were homozygous for the A1023G polymor- progress to end-stage renal failure. Mutations in the phism, thus pointing to a possible founder effect. Thirteen NPHS2 that encode a named podocin have recently children of Israeli-Jewish origin with SRNS and biopsy-proven been found in a recessive form of SRNS. Ten children from FSGS and 15 children of both ethnic groups with steroid- two inbred families of Israeli-Arab descent presented with responsive FSGS were tested, and none was found to have SRNS. Renal histologic findings were of diffuse mesangial mutations in NPHS2. The results of this study demonstrate that proliferation. Six patients reached end-stage renal failure, but mutations in NPHS2 are a common cause of SRNS in Israeli- nephrotic syndrome did not recur after renal transplantation. Arab children. Mutations in NPHS2 may cause SRNS in non- Mutation analysis of NPHS2 revealed that they were homozy- familial cases. The interethnic differences in the occurrence of gous for the C412T mutation (R138X). Eighteen children were NPHS2 mutations may explain, in part, the previous observa- subsequently analyzed with SRNS due to biopsy-proven focal tion that Arab patients with FSGS in Israel have a worse segmental glomerulosclerosis (FSGS) from unrelated families prognosis as compared with Jewish patients, despite similar of Israeli-Arab descent. Analysis disclosed six additional pa- presenting symptoms and medical management. Identifying tients (33%) bearing the same mutation in a homozygous the causing mutation will enable clinicians to avoid unneces- pattern. Three of them had no affected relatives, although they sary immunosuppressive therapeutic trials in newly diagnosed came from large families. Taken together, of the 27 patients patients and to provide prenatal diagnosis to families at risk.

The term “nephrotic syndrome” is applicable to any condition use of steroids (and subsequently other immunosuppressive with massive proteinuria, hypoalbuminemia, and edema. The medications) to treat nephrotic syndrome in children. The role glomerular capillary wall, which is composed of a basement of immune mechanisms was substantiated with the detection of membrane covered by fenestrated endothelium on the inner a glomerular permeability factor in a group of patients with part and visceral epithelial cells () on the outer part, focal segmental glomerulosclerosis (FSGS) who experienced is responsible for plasma ultrafiltration during urine formation. recurrence of nephrotic syndrome after renal transplantation Dysfunction of the glomerular barrier results in leakage of (10). The most common variety of nephrotic syndrome in plasma and the development of nephrotic syndrome children is characterized by minimal histologic changes in the (1). glomeruli on light microscopy (minimal change nephrotic syn- The exact pathogenesis of nephrotic syndrome in childhood drome), and indeed, the majority of patients respond to steroids remains elusive. Over the last three decades, much research has and have favorable long-term outcome. been directed toward the definition of linkage between the Steroid-resistant nephrotic syndrome (SRNS) represents a immune system and nephrotic syndrome (2–9). The accumu- heterogeneous group of kidney disorders that often are resistant lated knowledge, often circumstantial, has led to the successful to additional immunosuppressive agents and tend to progress to end-stage renal failure. The most prevalent histologic findings associated with SRNS in children are diffuse mesangial pro- Received August 7, 2001. Accepted October 6, 2001 liferation and FSGS. It has been suggested that a lack of Correspondence to Dr. Yaacov Frishberg, Division of Pediatric Nephrology, therapeutic response to steroids is a better prognostic indicator Shaare Zedek Medical Center, PO Box 3235, Jerusalem 91031, Israel. Phone: 972-2-6666144; Fax: 972-2-6555689; E-mail: [email protected] of outcome than the underlying histology (11). The exact factors that determine steroid responsiveness have not been 1046-6673/1302-0400 Journal of the American Society of Nephrology fully elucidated. Inter-ethnic differences in prevalence and Copyright © 2002 by the American Society of Nephrology outcome have been shown in the United States: children with J Am Soc Nephrol 13: 400–405, 2002 NPHS2 and Steroid-Resistant Nephrotic Syndrome 401

FSGS of African American or Hispanic descent have a worse disappearance of proteinuria (Ͻ4 mg/m2/h); steroid resistance was prognosis compared with white children (12). We have previ- defined as the lack of response to daily orally administered prednisone 2 ously shown in a study comparing Arab and Jewish children treatment at 60 mg/m for 1 mo. with FSGS in Israel that there is a much greater tendency We subsequently analyzed three additional groups of patients toward progressive renal disease in Arab children despite sim- with SRNS who had no family history of kidney disorders: (1) 18 children of Israeli-Arab origin with SRNS and biopsy-proven ilar presenting symptoms, medical management, and follow-up FSGS; (2) 13 children of Israeli-Jewish descent with SRNS and (13). The pathogenetic mechanisms underlying inter-ethnic biopsy-proven FSGS; and (3) 15 children of both ethnic groups differences in the course of the disease remains obscure. with biopsy-proven FSGS and a favorable course (steroid-respon- In recent years, the molecular bases of several conditions sive nephrotic syndrome). The biopsy findings included mild mes- leading to SRNS have been identified. The common denom- angial proliferation, interstitial alterations, and 5 to 10% segmen- inator shared by the following entities is that they all stem tally sclerosed glomeruli. from a structural defect in the glomerular barrier, thus After informed consent for the genetic studies was obtained from explaining their unresponsiveness to immunosuppressive the parents of all patients, blood samples were obtained from all medications (14). The congenital nephrotic syndrome of the patients for DNA analysis. The study was approved by the Shaare Finnish type is caused by mutations in the NPHS1 gene Zedek Medical Center Ethics Committee. encoding a protein called that is exclusively ex- pressed at the slit diaphragm joining the foot Homozygosity Mapping processes (15,16). Mutations in ACTN4, mapped to 19q13 Homozygosity mapping to the SRN1 locus on 1q25- and encoding ␣-actinin-4, an actin filament cross-linking q31 (20) was performed on the two kindreds with SRNS. The disease was assumed to be transmitted in an autosomal recessive mode with protein, have been found in dominantly inherited FSGS complete penetrance. Allele frequencies were assumed to be equal for (17). This gene is highly expressed in the podocyte and is each marker. upregulated during the evolution of nephrotic syndrome in animal models (18). More recently, the NPHS2 gene has Mutation Analysis in the NPHS2 Gene been cloned and found to encode a protein, podocin, that is Genomic DNA was extracted from whole blood collected in eth- specifically expressed in the podocytes of fetal and mature ylenediaminetetraacetate-containing tubes. It was performed by using kidney (19). It mapped to the 1q25-q31 locus, previously conventional molecular biology techniques with the EZ-DNA kit designated SRN1 (20), and was found to be mutated in (Genomic DNA Isolation Reagent; Biological Industries, Beit families with recessive inheritance of SRNS. Ha’emek, Israel). PCR was used to amplify individual exons of the The aims of this study were twofold: to confirm the genetic NPHS2 gene by using primers on the basis of published information basis of familial SRNS in two kindreds, and to determine the regarding intron-exon boundaries (19). PCR primers are listed in existence of mutations in the NPHS2 gene in a large group of Table 1. ␮ children with nonfamilial FSGS. Identification of the causing PCR reaction was performed in a total volume of 25 l containing ␮ ␮ mutation will enable us to provide prenatal diagnosis and to 1.5 l genomic DNA, 10 pg of each primer, 2.5 l of deoxynucleotide triphosphates (final concentration of 200 ␮M), 2.5 ␮lof10ϫ reaction avoid unnecessary immunosuppressive therapeutic trials in buffer, and 0.2 to 0.5 unit of thermostable DNA polymerase. DMSO newly diagnosed patients. was occasionally added to the reaction mixture. DNA was denaturated at 95°C for 1 min, followed by 39 cycles of Materials and Methods denaturation of 10 s at 95°C, annealing for 10 s at 55°C, extension of Patients 30sat72°C, and final extension of 5 min at 72°C. This protocol was Ten children from two heavily inbred families of Israeli-Arab implemented for the amplification of exons 1 to 4, 7, and 8. For exons descent were diagnosed with nephrotic syndrome and followed in the 5 and 6, DNA was denaturated at 94°C for 4 min, followed by 40 Division of Pediatric Nephrology at the Shaare Zedek Medical Center, cycles of denaturation at 94°C for 10 s, annealing at 55°C for 30 s, Jerusalem, Israel. Nephrotic syndrome consisted of massive protein- with a final extension time of 5 min. uria (Ͼ40 mg/m2 per hour), hypoalbuminemia (Ͻ2.5 g/dl), and PCR products were electrophoresed on a mutation detection edema. Remission of the nephrotic syndrome was defined as the enhancement gel. Gels were visualized by silver staining (21).

Table 1. Primers for DNA sequence analysis

Exon Forward Primer (5Ј-3Ј) Reverse Primer (5Ј-3Ј)

1 GCAGCGSCTCCACAGGGACT TCAGTGGGTCTCGTGGGGAT 2 AGGCAGTGAATACAGTGAAG GGCCTCAGGAAATTACCTA 3 TTCTGGGAGTGATTTGAAAG TGAAGAAATTGGCAAGTCAG 4 AAGGTGAAACCCAAACAGC CGGTAGGTAGACCATGGAAA 5 CATAGGAAAGGAGCCCAAGA TTCAGGCATATTGGCCATTA 6 CTCCCACTGACATCTGA AATTTAAAATGAAACCAGAA 7 CTAAATCAATGGCTGCACCACC TTCCTAAAGGGCAGTCTGG 8 GGTGAAGCCTTCAGGGAATG TTCTATGGCAGGCCCCTTTA 402 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 400–405, 2002

Variants seen on the single-strand conformation polymorphism Results (SSCP) gel were directly sequenced by the fluorescence dideoxy Study of Familial SRNS terminator method (22). Fluorometric sequences were analyzed Ten children from two heavily inbred families of Israeli- with the Applied Biosystem 3700 DNA Analyzer (Applied Arab descent presented at the mean age of 1.6 yr with Biosystem, Foster City, CA) according to the manufacturer’s nephrotic syndrome. Four children originated from two re- protocol. lated nuclear families, and the remaining six are the off- Comparison of the tested nucleotide sequence with the published spring of four consanguineous couples (Figure 1). Eight of sequence of NPHS2 gene was performed (GenBank database). the 10 children were biopsied within the first 3 mo after the Screening of the R138X mutation in the NPHS2 gene was performed by amplifying genomic DNA by using primers that selectively am- diagnosis of SRNS. Biopsy was deferred in two young plify normal or altered DNA sequences, a technique known as am- siblings of the proband in family 1B. The histologic findings plification refractory mutation system. The primers used for the on kidney biopsies were of diffuse mesangial proliferation detection of the mutation in exon 3 were as follows: 5'-GGTTGTA- without segmental sclerosis. In a few occasions, there were CAAGAGTAATGGAAAGAGTAATTATATTAT-3' for the mutated mesangial IgM deposits, C3 deposits, or both. The nephrotic DNA, and 5'-GGTTGTACAAGAGTAATGGAAAGAGTAATTA- syndrome was resistant to steroids in all children. In several TATTAC-3' for the wild-type DNA. The reverse primer is detailed in individuals, other immunosuppressive agents had been at- Table 1. DNA was denaturated at 95°C for 2 min, followed by 38 tempted, including therapy with cyclophosphamide and cy- cycles of denaturation at 94°C for 10 s, annealing at 54°C for 10 s, and closporine, without response. Renal function has gradually extension at 72°C for 10 s. The mutated DNA fragment was amplified deteriorated; six of the nine children reached end-stage renal only when the first primer was used. failure at the mean age of 6.6 yr. Five patients underwent

Figure 1. Pedigrees of kindreds of Israeli-Arab origin with steroid-resistant nephrotic syndrome. J Am Soc Nephrol 13: 400–405, 2002 NPHS2 and Steroid-Resistant Nephrotic Syndrome 403 renal transplantation without recurrence of the nephrotic gene with PCR-SSCP and direct sequencing, when indicated, syndrome. did not reveal mutations in any of the affected children of this group. Homozygosity Mapping Third, as a negative control, we tested 15 children (four Arab The high rate of consanguinity enabled us to perform ho- children and 11 Jewish children) with biopsy-proven FSGS mozygosity mapping in these two families. Fifteen members, who achieved long-lasting remission of their nephrotic syn- including four affected individuals from family B, and 23 drome with immunosuppressive protocols. As expected, none members, including five affected children from family A, were of them carried a mutation in the NPHS2 gene. enrolled in our study. The tenth affected patient died as a result of the disease before the initiation of this study, and a DNA Polymorphism in the NPHS2 Gene sample was not available for analysis. All of the affected The SSCP analysis, followed by direct sequencing, disclosed members studied were homozygotes for the following markers: a novel genetic polymorphism in exon 8 in both Arab and D1S2790, D1S242, D1S218, and D1S416; this confirms link- Jewish individuals. The A1023G transition did not result in a age to the SRN1 locus harboring the recently cloned NPHS2 change in the amino acid sequence. All 15 affected individuals gene with a maximum log of odds score of 2.99 (␪ ϭ 0.0). (both familial and nonfamilial cases) who were homozygous for the R138X mutation shared the same haplotype and were Mutation Analysis homozygous for the A1023G polymorphism. We detected ho- After linkage of SRNS was determined in both kindreds, the mozygosity for this polymorphism in 20% of healthy unrelated candidate gene NPHS2 was screened for mutations. The entire individuals of Arab or Jewish descent (80 in 8-exon coding region was screened by use of PCR-SSCP. each group were tested). This polymorphism was found in a SSCP analysis detected bandshift in exon 3 of one affected homozygous pattern in only 9% of children with FSGS who child from each family. Direct sequencing of the corresponding did not carry a mutation in the NPHS2 gene. Because all PCR product demonstrated that they all were homozygotes for patients are homozygotes for the same mutation, share a com- the C412T nonsense mutation leading to R138X. This transi- mon haplotype, and belong to the same ethnic group, our tion was found to be a heterozygous change in obligate findings point to a possible founder effect. carriers. Discussion Children with Biopsy-Proven Primary FSGS We have detected the same homozygous mutation in the To ascertain whether mutations in NPHS2 may cause SRNS NPHS2 gene in 15 children with SRNS of Israeli-Arab origin. in nonfamilial cases as well, we analyzed three distinct groups Twelve patients have affected siblings, and they originate from of patients with biopsy-proven FSGS. five unrelated kindreds. The remaining three are the first en- The first group included 18 children from unrelated families counter in their respective families. The identification of a of Israeli-Arab descent who presented with SRNS at the mean nonsense transition (C412T leading to R138X) segregating age of 5.9 yr. Eight children were hypertensive at diagnosis of with the disease in families, and resulting in premature termi- the nephrotic syndrome. All renal biopsies showed histologic nation of the protein in affected individuals, confirms its causal findings consistent with FSGS. None of the patients responded role in SRNS. Podocin, encoded by the NPHS2 gene, is an to other immunosuppressive agents, and 12 (67%) of 18 chil- integral structural protein of the podocyte, and a truncated dren showed declining renal function; they reached end-stage protein will presumably result in a glomerular barrier dysfunc- renal failure at the mean age of 10 yr. Our analysis disclosed tion, leading to nephrotic syndrome. Although its exact role has that six of 18 children in this group bore the same mutation in not been fully delineated, it clearly explains why children a homozygous pattern (C412T leading to R138X). Three of bearing homozygous mutations fail to respond to immunosup- them had no affected relatives despite stemming from very pressive agents, but also why the nephrotic syndrome does not large inbred families. In retrospect, each one of the remaining recur after renal transplantation. three had a sibling who had died as a result of a kidney Our affected population with the R138X mutation bear the disorder. SSCP analysis of NPHS2 in the remaining 12 chil- same haplotype and belong to a distinct ethnic group residing dren did not reveal any bandshift. After this study was com- in a confined geographic region—a phenomenon that points to pleted, we identified massive proteinuria on the first day of life a possible founder effect. This mutation has been previously in a sibling of an affected child. Renal biopsy was consistent identified in one consanguineous family from Egypt (19). This with minimal change nephrotic syndrome and homozygosity may be in favor of a Middle Eastern origin of this mutation. for the R138X mutation was confirmed. Nevertheless, we were unable to trace back the studied fami- The second group consisted of 13 children of Israeli-Jewish lies’ origin to a common ancestor. Data concerning the origin descent with SRNS secondary to biopsy-proven FSGS. They of the Israeli-Arab population are lacking, and political events originate from 13 nonconsanguineous unrelated families, and of many centuries may have resulted in population shifts. none had an affected relative. Their mean age at presentation Further attempts are underway to define a founder and the age was 8.4 yr, and two were hypertensive at the time of diagnosis. of this mutation. Five children (38%) reached end-stage renal failure at the mean We have noted a wide range of phenotypic variability be- age of 13 yr. Screening the entire coding region of the NPHS2 tween individuals with the same genotype in two respects: the 404 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 400–405, 2002 clinical characteristics and the histologic findings. Diagnosis of Mirsky Foundation. We appreciate the assistance of Dr. S. SRNS varied from the first day of life but more often was made Mitrani-Rosenbaum. between the ages of 1 to 4 yr. Podocin has been shown to be highly expressed in the fetal glomeruli (19), which may ac- count for our novel observation of a pattern consistent with References congenital nephrotic syndrome. It remains to be determined 1. Clark AG, Barratt TM: Steroid-responsive nephritic syndrome. why most cases are manifested beyond the first 3 mo of life. In: Pediatric Nephrology, 4th Ed., edited by Barratt TM, Avner ED, Harmon WE, Baltimore, Lippincott Williams & Wilkins, We assume that the ultimate phenotype is governed by inter- 1999, pp 731–747 actions between the NPHS2 gene and other , regarded as 2. Shalhoub RJ: Pathogenesis of lipoid nephrosis: A disorder of T modifier genes (23), genetic polymorphisms, or both. cell function. Lancet 2 (7880): 556–560, 1974 Our data confirm data of previous reports that demonstrated 3. 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