A novel Wilms' tumor 1 gene mutation in a child with ... - Springer Link

0 downloads 0 Views 990KB Size Report
May 31, 2008 - Mutations in this gene have been associated with Wilms' tumor, Frasier syndrome, and Denys–Drash syndrome, as well as isolated glomerular.
Pediatr Nephrol (2008) 23:1445–1453 DOI 10.1007/s00467-008-0845-7

ORIGINAL ARTICLE

A novel Wilms’ tumor 1 gene mutation in a child with severe renal dysfunction and persistent renal blastema Nicole Wagner & Kay-Dietrich Wagner & Mickael Afanetti & Fabien Nevo & Corinne Antignac & Jean-Francois Michiels & Andreas Schedl & Etienne Berard

Received: 4 January 2008 / Revised: 20 March 2008 / Accepted: 24 March 2008 / Published online: 31 May 2008 # IPNA 2008

Abstract The Wilms’ tumor suppressor gene WT1 is an important regulator of development. Mutations in this gene have been associated with Wilms’ tumor, Frasier syndrome, and Denys–Drash syndrome, as well as isolated glomerular disease. Here we report the case of a 4-month-old girl, who presented with end-stage renal disease, thrombopenia, anemia, and cardiac hypertrophy accompanied by severe hypertension. Histological analysis of kidney biopsies revealed a massive and diffuse nephroblastomatosis with a dramatic reduction in the number of glomeruli. Although no normal cortical nephrons could be detected, medullary organization was nearly normal. Sequence analysis demonstrated a heterozygous nonsense mutation in exon 9 of WT1, which leads to a truncation of the WT1 protein at the beginning of zinc finger 3. Given the requirement of WT1 for normal development of the kidney and heart, these data raise the hypothesis that the mutation identified was

responsible for the severe phenotype observed in our patient. Keywords Nephroblastomatosis . Wilms’ tumor suppressor gene . End-stage renal disease . Hypertension . Cardiac hypertrophy

Introduction The Wilms’ tumor 1 gene (WT1) encodes a zinc finger protein that has been identified on the basis of its mutational inactivation in 10–15% of all Wilms’ tumors or nephroblastomas. Alternative splicing in the zinc finger region of WT1 leads to the inclusion or omission of the three amino acids KTS, which influences the biochemical properties of the resulting protein. Isoforms that lack the

N. Wagner : K.-D. Wagner : A. Schedl INSERM, U636, Nice, France

C. Antignac Faculté de Médecine René Descartes, Université Paris Descartes, Paris, France

N. Wagner : K.-D. Wagner : A. Schedl Laboratoire de génétique du développement Normal et Pathologique, Université de Nice-Sophia Antipolis, Nice, France

J.-F. Michiels Service d’anatomie pathologique, Hôpital Pasteur, Nice, France

M. Afanetti : E. Berard (*) Service de Pédiatrie, Hôpital Archet-2, 151, route de Saint-Antoine-de-Ginestière, BP 3079, 06202 Nice, France e-mail: [email protected]

A. Schedl (*) Centre de Biochimie, INSERM U636, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 02, France e-mail: [email protected]

F. Nevo : C. Antignac Inserm, U574, Hôpital Necker-Enfants Malades, Paris, France

Present address: N. Wagner : K.-D. Wagner Faculté de Médecine, INSERM U907, Nice, France

1446

KTS sequence (Wt1−KTS) are potent transcriptional activators and bind preferentially to DNA, whereas Wt1+KTS proteins may interact with RNA. Analysis using mostly the mouse as a model system have demonstrated that WT1 is required for the development of the kidney, gonad [1], adrenal gland [2], heart [3], spleen [4], eye [5] and the olfactory epithelium [6]. In the kidney, expression of the Wt1 gene is developmentally regulated. It is found at low levels in un-induced mesenchymal cells, increases in the cap structure during nephron induction, and later becomes restricted to the presumptive podocyte layer. Once nephrogenesis has been completed, WT1 expression persists in the podocyte lineage throughout life. WT1 appears to act at multiple steps in kidney development: Firstly, it is required for the induction of the ureteric bud, and a complete loss of function in the early embryo leads to renal agenesis [1]. Secondly, experiments in vivo and in vitro have suggested a requirement for WT1 in nephron formation [2, 7]. Finally, WT1 remains to be expressed in the adult kidney within the podocyte, where it appears to fulfill an important function in maintaining this cell type in a highly differentiated state. As a consequence, reduced levels of WT1 expression or heterozygous mutations that interfere with the function of the protein lead to glomerular disease [8, 9]. Wt1 also seems to play an important role in heart development, at least in the mouse. Mice with targeted inactivation of Wt1 exhibit a partial defect of the epicardium, defective cardiac vascularization [3], and reduced thickness of the myocardium [1, 2]. This inactivation is lethal in the mutant embryos after mid-gestation, presumably due to a contractile failure of their hypoplastic hearts. Wt1 expression in early cardiac development is first seen in the epicardium; afterwards, Wt1-positive cells delaminate from the epicardium and migrate into the sub-epicardium, where they form the coronary vessels. In the adult, Wt1 expression is restricted to the epicardium, but, under certain pathophysiological conditions such as hypoxia, Wt1 expression in the cardiac vasculature is reactivated, facilitating the generation of new blood vessels [10]. Analysis of patients’ data has demonstrated an involvement of WT1 in a variety of human diseases. It was identified by positional cloning in children with WAGR syndrome, characterized by a predisposition to Wilms’ tumor (W), aniridia (A), genitourinary malformations (G), and mental retardation (R) [11, 12]. Afterwards, germline WT1 mutations in exons 8 or 9, coding for zinc fingers 2 or 3, were demonstrated in children suffering from Denys– Drash syndrome (DDS), which is characterized by a strong predisposition to Wilms’ tumors, early onset diffuse mesangial sclerosis with nephrotic syndrome and male pseudohermaphroditism [13]. Finally, heterozygous mutations that affect alternative splicing of WT1 have been

Pediatr Nephrol (2008) 23:1445–1453

found in patients with Frasier syndrome, which is characterized by the association of nephrotic syndrome with focal segmental glomerulosclerosis, male pseudohermaphroditism, and the development of gonadoblastoma [14, 15]. In contrast to DDS, Frasier mutations typically do not predispose the individual to Wilms’ tumor. For further detailed review see [9]. Here, we report on the case of a girl who suffered from severe kidney defects, which required bilateral nephrectomy. In addition, she had persistent cardiac hypertrophy, although hypertension is now well controlled. The case is associated with a novel mutation in the zinc finger region of WT1.

Methods Tissue samples and immunohistology The study adhered to the principles of the Declaration of Helsinki and to title 45, US code of Federal Regulations, part 46, protection of human subjects. Tissues were fixed in 10% buffered formalin and embedded in paraffin. The paraffin sections were de-waxed in xylene, hydrated in ethanol series, and washed in phosphatebuffered saline (PBS). Conventional histological examination was performed with hematoxylin–eosin staining, as described [3]. Immunohistological analysis was performed as described previously [16]. Antigens were detected with the EnVision+Dual Link System-HRP from Dako Cytomation, according to the manufacturer’s instructions, using Vector 3,3′-di-amino-benzidine (DAB) substrate (Vector Laboratories). The following primary antibodies were used: a monoclonal mouse anti-WT1 antibody [1:100 dilution in PBS, 0.1% Triton X-100, 3% bovine serum albumin (BSA); MAB4234, Chemicon], a monoclonal mouse antinestin antibody (1:100 dilution, MAB5326, Chemicon), a monoclonal mouse anti-synaptopodin antibody (ready-touse, G1D4, Progen), a polyclonal rabbit anti-Wt1 antibody (1:100 dilution, C-19, Santa Cruz Biotechnology), a polyclonal rabbit anti-podocin antibody (1:200 dilution, P0372, Sigma), a polyclonal rabbit anti-PAX2 antibody (1:50 dilution, 71-6000, Zymed), or a polyclonal guinea pig anti-nephrin antibody (1:50 dilution, GP-N2, Progen). In the case of nephrin, antigen was detected with an antiguinea pig biotinylated secondary antibody (Vector Laboratories), followed by incubation with peroxidase-coupled streptavidin (Sigma). Visualization was achieved with DAB substrate (Vector Laboratories). Slides were viewed under an epifluorescence microscope (DMLB, Leica) connected to a digital camera (Spot RT Slider, Diagnostic Instruments) with Spot Software (Universal Imaging Corp.).

Pediatr Nephrol (2008) 23:1445–1453

1447

Fig. 1 a Histological analysis of kidney material reveals massive and diffuse persistent renal blastema (nephroblastomatosis). Hematoxylin– eosin-staining was performed on paraffin-embedded sections of an age-matched control kidney (a, d), a Wilms’ tumor sample (b, e), and our patient (c, f). Note the almost complete absence of glomeruli

(arrowheads) and the papillary blastema formations (arrows) in the patient’s kidney, which resemble nephroblastoma (Wilms’ tumor, b, e). b, a–f The few glomeruli have an immature glomerular structure or present fibrotic or sclerotic lesions. Scale bars represent 50 μm

Detection of apoptosis

Mutational analysis

Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using the In situ Cell Death Detection Kit (Roche Molecular Biochemicals).

Blood samples for DNA studies were obtained after informed consent had been given by the parents. Mutational screening of WT1 was performed by direct sequencing of the ten coding exons, and the adjacent intronic junctions

1448

Pediatr Nephrol (2008) 23:1445–1453

(primers available upon request). Products of polymerase chain reaction (PCR) were treated with Exo-SAP IT (AP biotech), and both strands were sequenced using a BigDye terminator cycle sequencing kit (Applied Biosystems) with an ABI3130 automated sequencer (Applied Biosystems).

controlled, but the hypertrophic cardiomyopathy and some pericarditic extravasation (6 mm to 8 mm behind the heart) still persist, with no evidence of inflammatory or infectious diseases and without any other extravasations. In the parents, findings from clinical and ultrasound examination, and renal function measurements, were normal.

Results

Histological and genetic analysis

Case report

Macroscopically, the size and form of the kidneys were normal, with nodular, whitish, poorly defined lesions deforming the surface of the renal cortex. Histological examination of both kidneys revealed a very low number of glomeruli (maximal ten per kidney section compared to approximately 200 in the control section) embedded into nephroblastomatotic (persistent renal blastema) tissue, which was nearly confluent in the cortical region (Fig. 1a, slides c, f). We chose the term of nephroblastomatosis in its definition as the persistence of metanephric blastema into infancy. In our patient we saw massive diffuse pan-cortical lesions, which, occurring in early infancy, are rarely associated with the occurrence of Wilms’ tumor [17, 18]. The renal blastema lesions in the girl often showed a papillary organization, with focal calcifications. The rare glomeruli presented massive mesangiolytic or fibrotic lesions (Fig. 1b, slides a–f). In contrast, tubules were rather well developed, with only some de-differentiated tubules in a fibrotic interstitium. Moreover, thrombotic microangiopathy was present, with proliferating endarteritis and arteriolar lumen obstruction or complete obliteration of the vascular lumen. To test for the presence of glomeruli on a molecular level, we performed immuno-staining with antibodies against a variety of podocyte markers including WT1, nephrin, and nestin. WT1 was strongly expressed in the glomeruli of control kidneys and also in a sample from a Wilms’ tumour patient (Fig. 2a,b,d,e,g,h). In contrast, staining was almost absent from sections of the kidneys from our patient (Fig. 2c,f,i). Staining for nestin [19], nephrin [20, 21], podocin [22], and synaptopodin [23] (Fig. 2l,o, and data not shown) further confirmed an almost complete absence of glomeruli in the diseased kidney. Histological analysis suggested persistence of renal blastema in the diseased kidney (Fig. 1). PAX2 is a transcriptional regulator that is widely expressed during early kidney development. In normal mature kidneys, PAX2 is switched off, except within the collecting ducts. Glomeruli always exhibit negative findings for PAX2 (Fig. 3a,d). PAX2 and WT1 are thought to regulate each other’s expression during renal development. Immunohistochemical analysis in the diseased kidney demonstrated strong PAX2 expression in a proportion of cells that

Our female patient was the first child of non-consanguineous parents without any familial history of kidney disease. The pregnancy was uneventful. Results of prenatal ultrasound scans and neonatal examination were normal (birth weight 3,600 g, Apgar score 10/10/10). At 4 months of age, she presented with failure to thrive [weight 5,150 g, height 56 cm, −2 standard deviations (SD)], asthenia, high blood pressure (220/140 mmHg), and oliguria. According to her parents, since birth she had been tired, a poor feeder, had not grown well and exhibited pallor. Blood tests showed end-stage renal failure [plasma creatinine 499 μmol/l, blood urea nitrogen (BUN) 50.6 mmol/l, sodium (Na) 132 mmol/l, potassium (K) 4.0 mmol/l, bicarbonate (HCO3−) 14 mmol/l, calcium (Ca) 1.84 mmol/l, phosphorus (P) 3.86 mmol/l), anemia (hemoglobin 4.9 mmol/l] with schizocytes (4%), and thrombopenia (41 × 10 9 platelets/l). Kidney ultrasound scans demonstrated a slight increase in volume (+1 SD), diffuse hyperechogenicity, and irregular margins. Peritoneal dialysis was then started, and the diagnosis of atypical uremic and hemolytic syndrome was proposed. Testing of the alternative complement pathway showed no abnormalities. Evolution over the next months showed severe and resistant high blood pressure, despite multi-drug therapy (nicardipine, enalapril, labetalol). Bilateral nephrectomy was performed, which did not result in the normalization of the blood pressure values. Acute episodes of high blood pressure were associated with cardiogenic shock and acute dramatic increases in thrombopenia and anemia with schizocytosis (despite erythropoietin treatment). Ultrasound scans revealed a severe concentric myocardial hypertrophy, with moderate signs of heart failure and intermittent pericarditis. Minoxidil administration was necessary to regain blood pressure control. Karyotype analysis was 46, XX, and demonstrated no obvious chromosomal defects. Celioscopy demonstrated normal female internal genitalia. The patient is now 3.5 years old. Peritoneal dialysis is ongoing without notable events. She has just reached a weight of 12 kg and is waiting for kidney transplantation. For over 2.5 years, the blood pressure has been well

Pediatr Nephrol (2008) 23:1445–1453

Fig. 2 Immunohistochemical analysis demonstrates an almost complete absence of glomeruli. WT1 (a–i), nestin (j–l), and nephrin (m–o) were used as podocyte markers. Note that the almost complete absence of WT1 was confirmed with two antibodies recognizing different

1449

epitopes of the protein. Antigens were visualized using DAB (brown) as substrate. Nuclei were counterstained with hematoxylin (blue). Scale bars represent 50 μm

1450

Pediatr Nephrol (2008) 23:1445–1453

Fig. 3 High levels of expression of developmental regulator PAX2. PAX2 is restricted to collecting ducts in the normal mature kidney (a). Immunohistochemical analysis in Wilms’ tumor (b, e) and our

patient’s kidney (c, f) demonstrated strong PAX2 expression in the persistent renal blastema. Scale bars represent 50 μm

resembled blastema, thus corroborating the hypothesis of a developmental arrest in this tissue (Fig. 3c,f). TUNEL staining identified massive apoptosis in the kidneys (Fig. 4), however, as apoptotic signals were seen throughout the kidney (vessel wall in Fig. 4f) and were not restricted to cells normally expressing WT1. Thus, it is

likely that apoptosis, superimposed upon the primary defect, contributed to the severity of the phenotype. Given the presence of an arrest of nephron formation within the diseased kidney, we speculated that a mutation in WT1 might be responsible for the phenotype. Indeed, sequence analysis revealed a heterozygous nonsense muta-

Fig. 4 Apoptosis was dramatically increased in the patient’s kidney. TUNEL staining (a–f, brown) revealed a high number of apoptotic cells throughout the kidney of our patient. Note the massive apoptosis

in glomerular structures (arrows) and in the walls of kidney vessels (arrowheads, and example in f). Scale bars represent 50 μm

Pediatr Nephrol (2008) 23:1445–1453

1451

Fig. 5 a Sequence analysis of parents and the affected child. While WT1 sequences of both father and mother were normal, a heterozygous nonsense mutation (C→T) was detected at position 1165 in both the kidney and blood of the affected child. The mutation leads to a truncation of the WT1 protein after amino acid 389. b Schematic representation of the wild-type and mutant WT1 protein. Alternatively spliced exons are shown as orange boxes

tion (p.Q389X) in exon 9 (C→T at bp 29 of exon 9) in both blood and renal tissue of the patient (Fig. 5). The mutation was predicted to lead to a truncation of the WT1 protein at the beginning of zinc finger 3. Analysis of blood from both parents did not reveal any molecular changes, suggesting that the mutation in the child was de novo.

Discussion The clinical features of a failure to thrive, oliguria, and the constant weakness since birth in our patient led us to think that the renal insufficiency had already been present at birth. The presentation with severe anemia with schistocytosis prompted us, in the beginning, to consider a congenital hemolytic and uremic syndrome as the cause of her illness, which could not be confirmed. We now believe that these acute episodes of anemia were due to massive hypertension, because they ceased after we had regained control of her blood pressure. The hypertropic cardiomyopathy with recurrent pericarditis is much more difficult to explain. Also, if it could only be the consequence of a high and uncontrolled blood pressure, we do not understand its persistence after more than 2.5 years of controlled blood pressure and balanced hydration, always avoiding overhydration. To our knowledge, until now, WT1

mutations have never been associated with heart disease in humans. However, an important role for Wt1 in heart and coronary vessel development in mouse models has been established [1, 3]. The recurrent pericarditis is in agreement with the epicardial defects observed in the mouse mutant model. In contrast, cardiac hypertrophy is the opposite from the knockout mouse model, suggesting the involvement of yet unidentified WT1-dependent factors in human cardiac growth. Although we cannot prove a direct relationship between the WT1 mutation detected and the heart abnormality in our patient, we think it might be worth taking a possible link into consideration. However, at present, we cannot exclude the possibility of an additional genetic cause underlying cardiomyopathy. In contrary to the heart phenotype, WT1 mutations have been identified in several syndromes affecting urogenital development. The kidney phenotype in our case was much more severe than any of those so far described in the literature. The mutation found was very similar to truncations identified in some Wilms’ tumors or DDS patients [24]. The characteristic pathological lesion in DDS patients is a diffuse mesangial sclerosis of the glomerulus, shrunken glomeruli with hypertrophied podocytes, sometimes accompanied by a loss of Wt1 expression, and a de novo expression of PAX2 in the podocytes. In contrast, the leading histological feature in both of the kidneys of our

1452

patient was a massive and diffuse persistence of renal blastema (nephroblastomatosis), with a nearly complete absence of glomeruli, immense apoptosis concerning the whole kidney, and severe lesions of the arteries and arterioles, sometimes leading to complete obliteration of the vessels. WT1 expression, and also expression of other podocyte markers, were nearly lost in the few glomeruli of the patient. The high expression of PAX2 observed in the blastema rests, is consistent with a developmental arrest, but it may also be caused by a lack of suppression by WT1. Hypertension is a common finding in DDS, but it normalizes after nephrectomy, which was not the case in our patient. While it is conceivable that additional genetic components contribute to the disease, we believe that the mutation observed in WT1 played a major role in the almost complete absence of glomeruli. Indeed, in vitro experiments using small interfering RNA (siRNA) in organ cultures has demonstrated that a knockdown of Wt1 during kidney development can block nephron formation [7]. Similarly, mice expressing lower levels of Wt1 developed kidneys, but failed to form functional nephrons, clearly demonstrating the requirement of Wt1 for nephrogenesis [2]. The fact that the parents did not carry the mutation further supports the notion of the direct involvement of WT1 mutations in the disease. Why the phenotype observed here was more severe than in the WT1-caused renal diseases so far described is unclear. The mutated protein in DDS patients is proposed to act in a dominant negative manner, possibly by forming heterodimers with the wild-type protein, thus titrating away functional WT1. The mutation in our patient has not been described before, and it is possible that it renders the mutant protein more stable and, therefore, more potent in the suppression of wild-type protein function. Finally, it is conceivable that the genetic background in our patient caused a more dramatic phenotype than those found in classical DDS patients. Acknowledgments The authors thank Marie Claire Gubler for the kidney control tissue of a 2-month-old child. This work was supported by grants from Fondation pour la Recherche médicale (FRM), EuReGene (EU), FP6 and L’Agence Nationale de la Recherche (ANR) (ANR-05MRAR-019-01). N.W. is a fellow of Fondation - de France.

References 1. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R (1993) WT-1 is required for early kidney development. Cell 74:679–691 2. Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A (1999) YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development 126:1845–1857

Pediatr Nephrol (2008) 23:1445–1453 3. Wagner N, Wagner KD, Theres H, Englert C, Schedl A, Scholz H (2005) Coronary vessel development requires activation of the TrkB neurotrophin receptor by the Wilms’ tumor transcription factor Wt1. Genes Dev 19:2631–2642 4. Herzer U, Crocoll A, Barton D, Howells N, Englert C (1999) The Wilms tumor suppressor gene wt1 is required for development of the spleen. Curr Biol 9:837–840 5. Wagner KD, Wagner N, Vidal VP, Schley G, Wilhelm D, Schedl A, Englert C, Scholz H (2002) The Wilms’ tumor gene Wt1 is required for normal development of the retina. EMBO J 21:1398–1405 6. Wagner N, Wagner KD, Hammes A, Kirschner KM, Vidal VP, Schedl A, Scholz H (2005) A splice variant of the Wilms’ tumor suppressor Wt1 is required for normal development of the olfactory system. Development 132:1327–1336 7. Davies JA, Ladomery M, Hohenstein P, Michael L, Shafe A, Spraggon L, Hastie N (2004) Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumor suppressor is required for nephron differentiation. Hum Mol Genet 13:235–246 8. Guo JK, Menke AL, Gubler MC, Clarke AR, Harrison D, Hammes A, Hastie ND, Schedl A (2002) WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis. Hum Mol Genet 11:651–659 9. Niaudet P, Gubler MC (2006) WT1 and glomerular diseases. Pediatr Nephrol 21:1653–1660 10. Wagner KD, Wagner N, Bondke A, Nafz B, Flemming B, Theres H, Scholz H (2002) The Wilms’ tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction. FASEB J 16:1117–1119 11. Haber DA, Buckler AJ, Glaser T, Call KM, Pelletier J, Sohn RL, Douglass EC, Housman DE (1990) An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms’ tumor. Cell 61:1257–1269 12. Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GA (1990) Homozygous deletion in Wilms tumors of a zinc-finger gene identified by chromosome jumping. Nature 343:774–778 13. Pelletier J, Bruening W, Li FP, Haber DA, Glaser T, Housman DE (1991) WT1 mutations contribute to abnormal genital system development and hereditary Wilms’ tumor. Nature 353:431–434 14. Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P, Gessler M (1998) Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1+/ −KTS splice isoforms. Hum Mol Genet 7:709–714 15. Barbaux S, Niaudet P, Gubler MC, Grunfeld JP, Jaubert F, Kuttenn F, Fekete CN, Souleyreau Therville N, Thibaud E, Fellous M, McElreavey K (1997) Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat Genet 17:467–470 16. Wagner N, Panelos J, Massi D, Wagner KD (2008) The Wilms’ tumor suppressor WT1 is associated with melanoma proliferation. Pflugers Arch 455:839–847 17. White KS, Kirks DR, Bove KE (1992) Imaging of nephroblastomatosis. An overview. Radiology 82:1–5 18. Finegold M, Bennington JL (1986) Pathology of neoplasms of children and adolescents, Saunders, Philadelphia 19. Wagner N, Wagner KD, Scholz H, Kirschner KM, Schedl A (2006) The intermediate filament protein nestin is expressed in the developing kidney and heart and might be regulated by the Wilms’ tumor suppressor Wt1. Am J Physiol Regul Integr Comp Physiol 291:R779–R787 20. Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K (1998) Positionally cloned gene for a novel glomerular protein— nephrin—is mutated in congenital nephrotic syndrome. Mol Cell 1:575–582

Pediatr Nephrol (2008) 23:1445–1453 21. Wagner N, Wagner KD, Xing Y, Scholz H, Schedl A (2004) The major podocyte protein nephrin is transcriptionally activated by the Wilms’ tumor suppressor WT1. J Am Soc Nephrol 15:3044–3051 22. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C (2000) NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet 24:349–354

1453 23. Mundel P, Heid HW, Mundel TM, Kruger M, Reiser J, Kriz W (1997) Synaptopodin: an actin-associated protein in telencephalic dendrites and renal podocytes. J Cell Biol 139:193–204 24. Royer-Pokora B, Beier M, Henzler M, Alam R, Schumacher V, Weirich A, Huff V (2004) Twenty-four new cases of WT1 germline mutations and review of the literature: genotype/ phenotype correlations for Wilms tumor development. Am J Med Genet 127:249–257