Pediatr Nephrol (2006) 21:1393–1398 DOI 10.1007/s00467-006-0225-0
ORIGINAL ARTICLE
WT1 mutations in nephrotic syndrome revisited. High prevalence in young girls, associations and renal phenotypes Filippo Aucella & Luigi Bisceglia & Patrizia De Bonis & Maddalena Gigante & Gianluca Caridi & Giancarlo Barbano & Gerolamo Mattioli & Francesco Perfumo & Loreto Gesualdo & Gian Marco Ghiggeri Received: 27 February 2006 / Revised: 9 May 2006 / Accepted: 9 May 2006 / Published online: 15 August 2006 # IPNA 2006
Abstract WT1 mutations have been considered a rare cause of nephrotic syndrome but recent reports challenge this assumption. Exclusion of inherited forms is a basic point in any therapeutic strategy to nephrotic syndrome since they do not respond to drugs. We screened for WT1 mutations in 200 patients with nephrotic syndrome: 114 with steroid resistance (SRNS) and 86 with steroid dependence (SDNS) for whom other inherited forms of nephrotic syndrome (NPHS2, CD2AP) had been previously excluded. Three girls out of 32 of the group with steroid resistance under 18 years presented classical WT1 splice mutations (IVS9+5G>A, Filippo Aucella and Luigi Bisceglia contributed equally to the work. F. Aucella Nephrology Unit, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy L. Bisceglia : P. De Bonis Medical Genetics Service, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy G. Caridi : G. M. Ghiggeri (*) Laboratory on Pathophysiology of Uremia, G. Gaslini Children’s Hospital, Largo G. Gaslini 5, 16148 Genoa, Italy e-mail:
[email protected] G. Barbano : F. Perfumo Department of Nephrology, G. Gaslini Children’s Hospital, Genoa, Italy
IVS9+4C>T) of Frasier syndrome. Another one presented a mutation coding for an amino acid change (D396N) at exon 9 that is typical of Denys-Drash syndrome. All presented resistance to drugs and developed end stage renal failure within 15 years. Two girls of the Frasier group presented a 46 XY karyotype with streak gonads while one was XX and had normal gonad morphology. In the two cases with IVS9+5G>A renal pathology was characterized by capillary wall thickening with deposition of IgG and C3 in one that was interpreted as a membrane pathology. Foam cells were diffuse in tubule-interstitial areas. In conclusion, WT1 splice mutations are not rare in females under 18 years with SRNS. This occurs in absence of a clear renal pathology picture and frequently in absence of phenotype change typical of Frasier syndrome. In adults and children with SDNS, screening analysis is of no clinical value. WT1 hot spot mutation analysis should be routinely done in children with SRNS; if the molecular screening anticipates any further therapeutic approach it may modify the long term therapeutic strategy. Keywords WT1 . Nephrotic syndrome . Focal segmental glomerulosclerosis . Frasier syndrome . Denys-Drash syndrome Abbreviations FSGS Focal segmental glomerulosclerosis FS Frasier syndrome DDS Denys-Drash syndrome
G. Mattioli Department of Pediatric Surgery, G. Gaslini Children’s Hospital, Genoa, Italy M. Gigante : L. Gesualdo Department of Biomedical Sciences-Nephrology Section, University of Foggia, Foggia, Italy
Introduction The WT1 gene encodes a transcriptional factor of the zincfinger protein family that is involved in kidney and gonadal
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development [1]. WT1 has been localized to chromosome 11q13; it consists of ten exons and generates four different isoforms resulting from alternative splicings [2, 3]. After birth, WT1 protein expression is restricted to renal podocytes where it probably contributes to maintain an adult differentiation [4]. Germline heterozygous WT1 mutations have been extensively reported in the literature as the cause of Denys-Drash (DDS) and Frasier (FS) syndromes that are characterized by nephrotic syndrome, genitalia anomalies and pseudo-hermaphroditism [5, 6]. WT1 is also known as the cause of sporadic and familial Wilms tumor [2, 7] that is the most common solid tumor in childhood and represents a potential evolution of DDS. Genetic and clinical phenotypes allow an easy differentiation between DDS and FS since more than 95% of DDS cases carry missense mutations in exons 8 and 9, while FS is more frequently associated with specific splice mutations at IVS9 resulting in the presence or absence of a lysinethreonine-serine (KTS) tri-peptide (KTS) in exon 9 [5, 6, 8, 9]. Renal findings in DDS are predominantly characterized by diffuse mesangial sclerosis of early onset and rapid evolution to end stage renal failure while FS usually presents slow progressive focal segmental sclerosis (FSGS) but rare variants without typical histology lesions have been recently reported [10]. The real impact of WT1 mutations in children with steroid resistant nephrotic syndrome (SRNS) has been recently evaluated by Ruf et al. [11] and in an extended cohort by Mucha et al. [12] where an overall incidence of 7% with a prevalence of FS (IVS9 and FSGS) over DDS (missense mutations at exons 8 and 9) was found. It was striking that only 1 female out of 8 with FS presented XY inversion and sexual anomalies, considered a key sign of the syndrome. This finding strongly supports the need of mutational analysis of WT1 in children with
nephrotic syndrome with steroid resistance in order to avoid potentially harmful therapeutic approaches. In view of the alarming high prevalence of WT1 mutation in children with nephrotic syndrome, we screened a large cohort of 115 SRNS and 86 patients with steroid dependent nephritic syndrome (SDNS) that included both children and adults and could confirm to some extent the Ruf report [11]. Our findings also extend to unusual renal findings of the basic pathology picture of FS describing two cases with thinning of capillary walls and with the presence of foam cells in the interstitium that appears to represent the prevalent early feature in FS patients.
Materials and methods Patients This study was approved by the local institutional review board and informed consent for genetic studies was obtained from each patient or legal tutor in the case of minors. Two hundred-thirteen patients with nephrotic syndrome were enrolled in the study. After the initial screening for NPHS2 and CD2AP mutations, 13 carriers of NPHS2 homozygous/compound heterozygous (n 10) or of heterozygous CD2AP mutations were excluded. Therefore, 200 patients were screened for WT1 mutations at the two hot spots at exons 8 and 9 including the classical splice site at intron 9. They were divided into four groups according to the clinical outcome and age of onset of proteinuria (Table 1). Overall, 114 patients presented steroid resistance, 64 were younger than 18 years; 86 presented steroid dependence with a predominance of people under 18 years.
Table 1 General features relative to 114 patients with steroid resistant and 86 steroid dependent nephrotic syndrome. Both cohorts were subdivided according to the age of presentation of proteinuria N
Gender
Age onset (years)
18 years
50
F 20 M 30
37.7
18 years
35
F 20 M 15
39.8
Biopsy (FSGS/IgM/MCN/Other)
ESRF(n)
SRNS FSGS 42 IgM 11 MCN 7 FSGS 46 IgM 2 MCN 1
28
27
SDNS FSGS 14 IgM 5 MCN 2 FSGS 28 IgM 1 MCN 4
SRNS, steroid resistant nephrotic syndrome; SDNS, steroid dependent nephrotic syndrome; ESRF, end stage renal failure
–
1
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Steroids were given according to consolidated schemes that utilize starting doses of prednisolone 2 mg/kg followed by tapering [13, 14]. Steroid resistance was considered when proteinuria persisted after 2 month therapy. Cyclosporine was given at 5 mg/kg starting dose, followed by tapering to reach the minimum dose required for maintaining serum drug levels between 50 and 100 ng/ml. Two patients were receiving a combined treatment with cyclosporine plus angiotensin converting enzyme inhibitors, three others had received combined treatments as above and in one case plasmapheresis had been performed at the onset of proteinuria. Based on immune-histological findings, 88 SRNS patients were classified as FSGS for the presence of at least one segmental area of glomerulosclerosis associated with diffuse mesangial IgM deposition; 13 patients presented mesangial IgM deposition at immunofluorescence with mesangial matrix proliferation and had a diagnosis of mesangial proliferative glomerulonephrites with IgM deposits (Mes IgM), eight presented minimal change lesions MCN. Four patients presented capillary wall thickening instead and granular membrane deposits that could not be interpreted since no electron microscopy analysis was available. Forty-two SDNS had FSGS, 6 and 6 had instead a diagnosis of IgM and MCN, respectively. NPHS2 and CD2AP molecular analysis Genomic DNA was extracted from peripheral blood samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Molecular analyses of NPHS2 coding regions was performed by direct sequencing as already described [15]. Molecular analyses of the 17 exons of the CD2AP gene were performed by polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. CD2AP flanking intronic primers were selected on the basis of published sequences or designed using the primer3 program (http:// frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). WT1 molecular analysis Samples for DHPLC analysis were amplified in a final reaction volume of 25 μl, using 70 ng of genomic DNA, PCR buffer with 15 mM MgCl2, 200 mM dNTPs, 0.30 mM primer, and 1.0 unit AmpliTaq Gold TM (Applied Biosystems Foster City, California, USA). PCR cycling conditions consisted of an initial denaturation at 95°C for 12 min followed by 35 cycles at 94°C for 30 s; the annealing temperature range was 58°C for 30 s, followed by 72°C for 30 s, and ending with a final elongation step at 72°C for 8 min. The following primers were used for PCR amplification: exon 8 forward: 5’-ccttaggcattttgggatct-3’;
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exon 8 reverse: 5’-cacatggctgactctctcatt-3’; exon 9 forward: 5’-gtgaggcagatgcagacatt-3; exon 9 reverse: 5’-tagccacgcac tattccttc-3’. DHPLC analysis was carried out on an automated DHPLC instrument (Transgenomic Inc, Santa Clara, California, USA). Heteroduplex formation was induced by mixing an aliquot of unpurified PCR amplicon from patients and wild type sample in a 1:1 ratio. The mixed samples were heated to 95°C for 5 min and cooled slowly over 25 min to 35°C. Sample 5–8 ul of re-annealed DNA duplexes were injected onto the column and then run at two different temperatures: 56.2°C and 59.2°C for exon 8; 55.7°C and 60.2°C for exon 9. Fragments showing variation in retention time or chromatogram shape were sequenced following the Big Dye Terminator protocol (PE ABI, Foster City, California, USA) and analyzed on an ABI 3100 DNA sequencer (Applied Biosystems). Genotype Genotype was evaluated by classical karyotype as well with amelogenin amplification.
Results We screened 200 patients with nephrotic syndrome (114 with SRNS, 86 with SDNS) for WT1 who did not show, at a previous analysis, mutations of one of the slit-diaphragm genes (NPHS2, CD2AP) causing nephrotic syndrome. Clinical details of the study cohort are given in Table 1. Three girls presented two classical mutations for FS at the splice site of exon 9 (IVS 9+5G>A; IVS 9+4C>T); in one case a mutation more typical for DDS at exon 9 (D396N) was found. All three cases with FS had an early onset of proteinuria resistant to treatments (Table 2) and progressing to end stage renal failure that required dialysis at 10, 14 and 3 years, respectively. Two also had an XY genotype inversion with female phenotype and streak gonads that were removed. The third FS girl is instead XX and presents a normal genital status. Immune-fluorescence and pathology findings are reported in Table 3; one carrier of IVS9+5G>A mutation (VR) had an early biopsy showing thickening of capillary walls with images of double contours and deposition of IgG and C3 (Fig. 1); no typical lesions of FSGS were present at this early stage. Numerous foam cells were found in tubule-interstitial areas. The second girl carrying the same mutation had two renal biopsies over a period of 2 years. In the first biopsy the same capillary wall thickening was confirmed in absence of any early IgM/C3 deposition. Lacking electron microscope images, no final pathology conclusions were reached but lacking segmental areas of sclerosis FSGS was not suspected. In the second biopsy some important changes
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Table 2 Clinical data and WT1 mutations in patients with SRNS Pts
Sex
Mutation
Age at onset (years)
Therapy sensitivity steroids/ CsA / Cycloph.
ESRF (years)
Genotype
Genital status
IE VR
F F
IVS9+ 5G>A IVS9+ 5G>A
6 5
Res. /Res./ Res. Res. /Res./ Res
10 14
XX XY
TV
F
IVS9+ 4C>T
3
Res. /Res./ Res
3
XY
DF
F
D396N
2
nd/nd/nd
2
XX
female, normal female, streak gonads female, streak gonads female, normal
F, female; Res, Resistant
were found characterized by mesangial deposition of IgM and C3 in association with diffuse glomerulosclerosis. The patient carrying IVS9+4C>T mutation presented FSGS with negative immune-fluorescence. The young girl with D396N mutation and DDS phenotype had an abrupt onset of renal failure and presented with generic end stage kidney lesions. Therefore, atypical pathology changes were present in 2 out 3 patients at an early biopsy thus supporting the concept that capillary wall changes and images of double contours should be included in the pathology picture of FS.
Discussion SRNS is a major cause of uremia in humans that is emerging as a real medical problem in western countries. It is not an homogeneous disease but rather includes genetic and immunological subsets that require adequate clinical diagnosis and differentiate therapeutic approaches [16]. Drug responsiveness to major immune modulators such as cyclosporine represents the more definite distinguishing feature between benign conditions and others that evolve to uremia [17]. Familial forms of SRNS are usually refractory to drugs and potentially coincide with most evolutionary subsets. The real impact of genetically mediated sporadic cases [16] can be calculated in 20–25% of SRNS under 18 years, an estimate that considers altogether genes of the slit-
diaphragm such as NPHS1 and NPHS2, genes encoding for protein of the cytoskeleton (α-actinin 4), CD2AP and transcriptional factors such as WT1. The last one was recognized as a rare cause of SRNS in early reports, but the real prevalence correctly determined in a representative sample of 115 SRNS patients was only recently extended to a significant mean of 7% by Ruf et al. [11]. These authors described prevalently WT1 splice mutations at intron-exon junction 9 that were associated with a pathologic picture of FSGS. In an extension of the screening, Mucha et al. [12], from the same group, described three new children with WT1 splice mutations and six others with mutations at exons 8 and 9 with an overall incidence of almost 10% equally distributed between FS (n=8) and DDS (n=7). Besides the data on unexpected prevalence, the two reports above clearly demonstrated that WT1 mutations associated with nephrotic syndrome are restricted to exons 8 and 9, that represent a sort of hot-spot that may be easily investigated. The alarming finding in the Ruf and Mucha reports was that almost all females with WT1 splice mutation at intron 9 exhibited only a partial phenotype lacking sexual anomalies that are the most apparent sign of FS and did not present any differential sign from classical FSGS. This means that most patients with SRNS and WT1 mutations remain unrecognized and are treated with potentially toxic drugs in spite of clear resistance. If the above data is confirmed, a basic molecular characterization
Table 3 Immune-fluorescence and pathology findings in patients with WT1 mutations Pts
Mutation
Age at biopsy
Immunofluorescence
Mesangial proliferation
Focal sclerosis
Capillary wall thickening
Double contours
Foam cells
IE a
IVS9+ 5G>A IVS9+ 5G>A IVS9+ 5G>A IVS9+ 4C>T D396N
8
Neg
Yes
No
Focal
Focal
No
10
IgM / C3
No
Diffuse sclerosis
–
–
Numerous
6
IgG / C3
Mild
No
Focal
Focal
Numerous
3
neg
–
Yes
No
No
No
2
End stage kidneys
IE b VR TV DF
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Fig. 1 Pathological features of the renal biopsy in one of the patients with IVS9+5G>A splice mutation of WT1 (patient 2 in Tables 2 and 3). Masson staining of two different glomeruli showing membrane thickening with magnification of 40X (a) and 100X (b). Masson staining of tubule-interstitial spaces showing foam cells with magnification of 10X (c) and 100X (d)
for WT1 mutations should precede any therapeutic intervention in nephrotic patients. Our study was done in a cohort of 200 patients with nephrotic syndrome and a variable clinical phenotype from SRNS to SDNS that was also extended to adults to evaluate the incidence of WT1 in nephrotic patients over 18 years. This is, in fact, the first report on WT1 screening in an adequate number of adults with FSGS/mesangial proliferation that conclusively excludes the presence of WT1 mutations in patients above this age. Results in children confirmed the high prevalence of WT1 mutations already reported by Ruf et al. [11] and Mucha et al. [12]. Three females out of 32 with SRNS under 18 years presented, in fact, IVS9 splice mutation and one had D396N mutation at exon 9. Overall, a cohort of 300 patients under 18 years with SRNS have been screened in our own study (n=64) and in the Ruf/Mucha (n=115/121) studies that give the definitive results on incidence of WT1 mutation in patients under 18 years around 6–7%. A most remarkable finding is that in young females, this incidence is higher (10–12%), and probably becoming the most frequent inherited cause of nephrotic syndrome under 18 years in this sex cohort. Our data differ from the report by Ruf et al. [11] in respect to the pathologic variants. We describe, in fact, changes in capillary wall thickness with immunoglobulins and C3 deposition along basement membrane and images of double contours in two children who had a renal biopsy very early in the course of the disease. Lacking enough renal material for electron microscopy no definite conclusion was reached and even FSGS was excluded and
membrane-proliferative nephritis was suggested as first chance diagnosis. Renal pathology changed over the years and in one child who had a second biopsy after 5 years, glomerulosclerosis had replaced the original membrane lesions. Another main finding in the same patients was the presence of many clusters of foam cells in the interstitium. Even WT1 splice mutations are classically associated with a renal phenotype of FSGS, unusual presentations have been recently described. In 2003, Ito et al. [10] described three females with IVS9+5C>T mutations whose pathologic picture was characterized by widespread basement membrane thinning, splitting and lamellation that mimicked hereditary nephritis. Electron microscopy demonstrated membrane thickening with some overlaps with Alport syndrome. A single case of WT1 splice mutation associated with minimal change lesions and WT tumor in a child of 2 years was also reported. Therefore, our finding of early thickening changes involving capillary wall in 2 out of 3 children with classical WT1 splice mutations together with the Ito report [10] indicate that ‘non classical’ alterations probably precede FSGS and this early change should be included in the list of the classical picture of FS. Based on the findings above and depending on lack of typical renal morphology in FS, we propose a diagnostic flow chart according to which patients presenting SRNS between 1 and 18 years should be checked for NPHS2 and WT1 mutations, restricting in the later case the analysis to exons 8 and 9 that represent a sort of hot spot in FS and DDS. This diagnostic protocol should also include patients not presenting typical FSGS lesions at renal biopsy. A
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possible option would be to perform the molecular analysis as soon as steroid resistance appears evident, in order to avoid the association of steroids with potential harmful drugs in patients carrying mutations. Lack of detection of WT1 mutations in patients over 18 years and in patients with SDNS makes the molecular screening of low (if any) clinical value and may be omitted. As for genotype associated with FS, an additional case of FS in a 46,XX girl was described here. Overall, 11 cases have been already reported in the literature, five were casual reports [18–22], and five belong to the Ruf/Mucha studies [11, 12]. In summary, the results of our study on WT1 screening in patients with nephrotic syndrome confirm the alarming data on a relevant prevalence of 12% splice mutations in females with nephrotic syndrome under 18 years. This occurs in absence of a clear renal pathology picture and frequently occurs in absence of any changes in genitalia that make the diagnosis of the association difficult. In this respect, early basement membrane alterations may precede overt FSGS and complicate the diagnosis. The incidence of WT1 mutations in adults over 18 years and in children with SDNS are only anecdotal and of no clinical value. WT1 hot spot mutation analysis should be routinely done in children with SRNS. Acknowledgements This work was completed with the financial support of the Italian Ministry of Health and Fondazione Mara, Wilma e Bianca Querci (Project:‘’Nuove evoluzioni sulla multifattorialità della sindrome nefrosica’’).
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