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Novel Mutations in NPHS2 Detected in Both Familial and Sporadic Steroid-Resistant Nephrotic Syndrome

University Children’s Hospital, Freiburg, Germany.

Correspondence to Dr. Arno Fuchshuber, University Children’s Hospital, Mathildenstrasse 1, 79106 Freiburg, Germany. Phone: 497612704301; Fax: 497612704533; E-mail: fuchshub{at}kk1200.ukl.uni-freiburg.de

	Abstract

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ABSTRACT. Autosomal recessive steroid-resistant nephrotic syndrome (SRINS) belongs to the heterogeneous group of familial nephrotic syndrome and represents a frequent cause of end-stage renal disease in childhood. This kidney disorder is characterized by early onset of proteinuria, progression to end-stage renal disease, and histologic findings of focal segmental glomerulosclerosis, minimal change nephrotic syndrome, or both. A causative gene, NPHS2, has been mapped to chromosome 1q25-q31 and was recently identified by positional cloning. This study reports five novel NPHS2 mutations: A284V, R196P, V290M, IVS4-1GT, and 460-467insT in 12 (46%) of 26 multiplex families and in 7 (28%) of 25 single patients with the clinical diagnosis of a SRINS. Because NPHS2 mutations were found in nearly 30% of these patients with "sporadic" SRINS, mutational analysis should also be performed in these patients. Besides better classification of the disease entity, identification of NPHS2 mutations may save some of these patients from unnecessary steroid treatment and also permit the prediction of absence of disease recurrence after kidney transplantation.

	Introduction

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Autosomal recessive steroid-resistant nephrotic syndrome (SRINS) belongs to the heterogeneous group of familial nephrotic syndrome. It is characterized by childhood onset of proteinuria, progression to end-stage renal disease, and histologic findings of focal segmental glomerulosclerosis (FSGS), or minimal change nephrotic syndrome (1). Several causative genes for familial SRINS are known. For a subgroup with an autosomal recessive trait, the responsible gene (NPHS2, formerly SRN1) has been mapped to chromosome 1q25-q31 (1) and recently identified by positional cloning (2). It encodes a 383–amino acid protein, podocin, that is almost exclusively expressed in glomerular podocytes. From database comparisons, podocin is predicted to be an integral membrane protein linking the plasma membrane and the cytoskeleton (2). Podocin is supposed to interact with nephrin, -actinin-4, and CD2AP (3). Nephrin (NPHS1) causes the most severe form of nephrotic syndrome, the congenital nephrotic syndrome of the Finnish type. A less severe form of SRINS with autosomal-dominant inheritance and FSGS1, adult onset, and slow progression to end-stage renal failure has been described (4). FSGS1 is caused by defects in the -actinin-4 gene (ACTN4) located on chromosome 19q13 (5). A second autosomal-dominant FSGS gene locus (FSGS2) maps on chromosome 11q21-q22. It has not yet been identified (6).

In this study, we performed mutational analysis in families and single patients with SRINS to determine whether NPHS2 mutations are also a cause of sporadic SRINS.

	Materials and Methods

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Patients
We analyzed 27 multiplex families (53 affected individuals were included) and 25 patients with "sporadic" SRINS originating mainly from Germany but also from other European countries. Parental consanguinity was not reported. For clinical evaluation, we used a standard questionnaire as previously described (7). The characteristic features defining the clinical diagnosis of SRINS included familial occurrence, age at onset in early childhood, resistance to steroid therapy, progression to end-stage renal disease within a few years, and absence of recurrence after renal transplantation (1).

Haplotype Analysis
Genomic DNA was extracted from leukocytes according to standard laboratory protocols. PCR was performed with five polymorphic markers spanning the critical region of NPHS2 on chromosome 1q25-q31 (1,2,7). The respective order from the centromeric to the telomeric border was: cen, D1S416, D1S1640, D1S2791, NPHS2, D1S215, D1S2883, tel (flanking markers are underlined). In the multiplex families, we first performed haplotype analysis. In case of consistency with linkage to NPHS2, mutational analysis was performed.

Mutational Analysis
We performed mutational analysis by single-strand conformation polymorphism (SSCP) and direct sequencing of both strands as previously described (8) in 17 SRINS families compatible with linkage to the NPHS2 gene locus on chromosome 1q21 and 25 patients with sporadic SRINS. The PCR of exons 3, 4, and 8 was carried out as previously described (2). For the remaining exons, the following primers and conditions were applied: exon 1: 5'-GCAGCGACTCCACAGGGACT-3' and 5'-TCCACCTTATCTGACGCCCC-3'; exon 2: 5'-AGAATTGGACCAACAGATGC-3' and 5'-AAGTGAGAATGGGCATGGTG-3'; exon 5: 5'-AAAGGAGCCCAAGAATCAAG-3' and 5'-AAATATTTCAGCATATTGGCC-3'; exon 6: 5'-GTTTAGGCATGCTC TCCTC-3' and 5'-GATATGGCTATAGTACTCAGTG-3'; exon 7: 5'-GTCTGTGTGAAAGCTTTGGC-3' and 5'-GCAAAGGGGAAATGTTCTCC-3'; at annealing temperatures of 52°C (exon 5) and 60°C (exons 1, 2, 6, and 7). Because of the high CG content of exon 1, PCR was performed with Taq polymerase and Q solution according to manufacturer’s instructions (Qiagen, Hilden, Germany). To rule out polymorphism in 100 chromosomes of healthy individuals, we used SSCP for R138Q, SSCP and direct sequencing for V290M, digestion with HhaI (Amersham Pharmacia Biotech, Freiburg, Germany) for A284V and with ClaI (MBI Fermentas GmbH, St. Leon-Rot, Germany) for R229Q, polyacrylamide gel electrophoresis for 460-467insT (8), and allele-specific PCR for IVS-1 GT (5'-TCCAAACTTTTTTCTGCCTAT-3' and 5'-AAATATTTCAGCATATTGGCC-3' [59°C]) and A196P (5'-GGAGATAGATGCCATTTGCTACTACCC-3' and 5'-TAAGTACCTTTGCATCTTGGGCGATGC-3' [62°C]). SSCP electrophoresis was performed for 6 h at 150 V with Power Pack P25 (Biometra, Göttingen, Germany). Direct sequencing of both strands was carried out as described previously (8).

	Results

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We report on 12 (46%) of 27 multiplex SRINS families and 7 (28%) of 25 single patients with SRINS with defects in the NPHS2 gene (Table 1). All 31 patients showed initially proteinuria exceeding 40 mg/m² per hour and two patients (67 II:2 and 83 II:1) low-grade gross proteinuria exceeding 1000 mg/m² per hour. Edema was present in all of them, except affected individuals of INS 28, 29, 43, 92, and 72, who had normal renal function. The age at onset varied from 0.1 to 16.6 yr (median, 3.0 yr; Table 1). Sixty-eight percent (21 of 31) of the patients progressed into end-stage renal failure within a median of 7.4 yr after diagnosis (range, 1.6 to 19.5 yr). No recurrence was reported in the 16 patients who underwent renal transplantation. The histologic findings in the 28 patients on whom renal biopsy was performed showed FSGS in 75% (21 of 28) and minimal change nephrotic syndrome in 21% (6 of 28). One biopsy result was unspecific (INS 46 II:1) (Table 1).

View larger version (26K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Pedigrees of seven patients with steroid-resistant nephrotic syndrome. Haplotypes were generated by using five consecutive microsatellite markers spanning the critical genetic NPHS2 region on chromosome 1q25-q31. Affected individuals are indicated as black symbols. Male family members are shown as squares and female family members as circles. The differently shaded bars indicate heterozygous (white) and homozygous (black) haplotypes. The respective order from top to bottom was as follows: cen, D1S416, D1S1640, D1S2791, NPHS2, D1S215, D1S2883, tel (flanking markers are underlined). Note that in all families, haplotype data were compatible with homozygosity by descent, with the exception of INS 86. The haplotypes of the respective parents, analyzed in four patients (INS 72, 74, 86, and 90), were congruent with these findings (data not shown).

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Detection of five novel NPHS2 mutations by direct sequencing. The corresponding nucleotide sequence with the respective amino acids are shown at the bottom of each chromatogram. The left column exemplifies the respective chromatograms of healthy control individuals (1A to 5A). The right column shows the chromatograms of patients with steroid-resistant nephrotic syndrome with missense mutations (1B to 3B), a splice site mutation (4B), and a frameshift mutation (5B). In patient INS 76, we found a homozygous CT substitution at position 851 leading to A284V (1B). Patient INS 86 showed two missense mutations in the heterozygous state. The first GC substitution at position 587 leads to R196P (2B), whereas the GA substitution at position 868 leads to V290M (3B). A homozygous splice site mutation IVS4-1GT was detected in patient INS 74 (4B). A heterozygous frameshift mutation 460-467insT leading to a premature stop codon T181X was found in patient INS 92 (5B). The mutations cosegregated with the disease within the families, and none of them were found in at least 100 control chromosomes.

Splice Site and Frameshift Mutations
We identified the first splice site mutation in NPHS2. IVS4-1GT involves the 3' acceptor splice site of intron 4 and was detected in a homozygous state in patient INS 74 (Figure 2). Another novel mutation results in a 1-bp insertion at position 460 to 467 with a consecutive frameshift and premature stop codon T181X. This is the second potential loss-of-function mutation in family INS 92. In addition, we detected two further frameshift mutations due to deletions that have previously been described (2). In family INS 50, we found the paternal 419delG in both affected siblings in a heterozygous state. 855–856delAA was identified in the patient INS 83 in a homozygous state.

Polymorphisms
Aside from the potential loss-of-function mutations described, we detected a number of nucleotide exchanges that are probably without any influence on NPHS2 function (Table 2). Some were silent without an amino acid exchange: 954TC (A318A), 102GA (G34G), 288CT (S96S), and 1038AG (L346L). In fact, one led to an amino acid exchange but was also detected in 3% of all chromosomes in 100 healthy controls: 686GA (R229Q). Interestingly, the R229Q exchange was found in a heterozygous state in three of four families with only one mutation (INS 18, 29, and 46) whereby the respective missense mutations were on the opposite chromosome. Furthermore, none of the healthy control individuals carrying R229Q was homozygous for this exchange. A functional role of R229Q is unlikely; however, it cannot completely been ruled out. The polymorphisms 288CT, 954TC, and 1038AG have recently been published by Wu et al. (10,11).

	Discussion

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This study reports the identification of 5 novel NPHS2 mutations A284V, R196P, V290M, IVS4-1GT, and 460-467insT in families and patients with sporadic SRINS with the clinical diagnosis of SRINS. None of the novels were found in at least 100 control chromosomes. In total, both potential loss of function NPHS2 mutations were detected in eight SRINS families and seven patients with sporadic SRINS. In four families, only one heterozygous mutation was found, suggesting mutations in the promoter region in these patients.

The novel missense mutations are predicted to cause either conservative (A284V and V290M) or nonconservative amino acid substitutions (R196P) affecting the carboxy-terminal cytoplasmic tail of podocin. Furthermore, the latter R196P substitution is highly conserved among the stomatin-like protein family members (2). The absence of the mutations in at least 100 control chromosomes and the consistency with cosegregating in the families suggest a pathogenic role even for the conservative amino acid substitutions. In addition to the six R138Q mutations reported by Boute et al. (2), we observed 11 R138Q mutations within our cohort. A common haplotype did not emerge significantly from haplotype analysis in these families (data not shown) or in the patients (see INS 73 and 90; Figure 1). But because these patients originate mainly from northern Europe, we support a founder effect hypothesis in Europe (2). The novel splice site mutation IVS4-1GT affects a guanine residue 100% conserved at splice acceptor sites of vertebrates. Because no renal specimen from the patient was available, we could not clarify its effect in further RNA analysis. But it seems likely that this mutation results in either skipping of exon 5 or in activating a cryptic splice site. The novel frameshift mutation 460-467insT results in a premature termination at position T181X and will most likely lead to a premature truncation of the protein.

In four families (INS 18, 28, 29, and 46), only one heterozygous mutation was found. Assuming a causative role for NPHS2 in these families and regarding the fact, that autosomal dominant inheritance is not likely (no more affected in the extended family pedigree), these patients may have mutations elsewhere in the promoter or in intron areas of the NPHS2 gene.

All five novel NPHS2 mutations reported here comprise missense, splice site, and frameshift mutation and confirm the crucial role of podocin in the function of the glomerular filtration barrier. We found NPHS2 mutations not only in familial cases, but also in seven patients with sporadic SRINS. In six of the seven tested patients, homozygous mutations were found that suggested a common founder in these families. Haplotype analysis demonstrating homozygosity of additional flanking genetic markers corroborates this conclusion (Figure 1).

SRINS represents an important cause of end-stage renal disease in childhood. Because we found NPHS2 mutations not only in familial cases but also in nearly 30% of patients with sporadic SRINS, we propose to perform mutational analysis in these patients as well. Besides better classification of the disease entity, identification of NPHS2 mutations may prevent some of these patients from unnecessary steroid treatment and also permit the prediction of absence of disease recurrence after kidney transplantation. We are in the process of establishing systems to evaluate podocin function (e.g., by testing protein-protein interactions of podocin with potential binding proteins such as nephrin, CD2AP, and ZO-1).

	Acknowledgments

 
Members of the Study Group of the Arbeitsgemeinschaft für Pädiatrische Nephrologie include: C. Aufricht, T. Arbeiter, K. Müller (Vienna, Austria); M. Bulla, E. Kuwertz-Bröking, S. Fründ (Münster, Germany); D. Drozdz, A. Pogan (Krakau, Poland); J.H.H. Ehrich, C. Strehlau (Hannover, Germany); P. Hoyer, K.E. Bonzel, M. Bald, (Essen, Germany); J. Janda, T. Seeman (Prag, Czech Republic); M. Kamm (Erlangen, Germany); B. Klare (München, Germany); O. Mehls, B. Tönshoff (Heidelberg, Germany); J. Misselwitz, U. John, L. Patzer, G. Rönnefarth (Jena, Germany); L. Monnens (Nijmegen, The Netherlands); D.E. Müller-Wiefel, H. Altrogge, K. Timmermann (Hamburg, Germany); T. Neuhaus, M. Kemper (Zürich, Switzerland); U. Querfeld, T. Lennert, M. Zimmering (Berlin, Germany); W. Rascher, P. Haas (Erlangen, Germany); H.-J. Stolpe, Wigger, G. Adomssent, (Rostock, Germany).

We thank R. Waldherr for reference pathology results and B. Schönfeld for technical assistance. FH is a Heisenberg-Scholar of the Deutsche Forschungsgemeinschaft (Hi 381/7-2)

	References

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