Genotype–phenotype associations in WT1 glomerulopathy
2014; Elsevier BV; Volume: 85; Issue: 5 Linguagem: Inglês
10.1038/ki.2013.519
ISSN1523-1755
AutoresBeata S. Lipska‐Ziętkiewicz, Bruno Ranchin, Paraskevas Iatropoulos, Jutta Gellermann, Anette Melk, Fatih Özaltın, Gianluca Caridi, Tomáš Seeman, Kálmán Tory, Augustina Jankauskienė, Aleksandra Żurowska, Maria Szczepańska, Anna Wasilewska, Jérôme Harambat, Agnes Trautmann, Amira Peco‐Antić, Halina Borzęcka, Anna Moczulska, Bassam Saeed, Radovan Bogdanović, Mukaddes Kalyoncu, Eva Šimková, Özlem Erdoğan, Kristina Vrljičak, Ana Teixeira, Marta Azócar, Franz Schaefer,
Tópico(s)Genetic and Kidney Cyst Diseases
ResumoWT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype–phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype–phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening. WT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype–phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype–phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening. Up to 25% of steroid-resistant nephrotic syndrome (SRNS) cases in children are caused by abnormalities in genes specifically or preferentially expressed by the podocyte.1Benoit G. Machuca E. Antignac C. Hereditary nephrotic syndrome: a systematic approach for genetic testing and a review of associated podocyte gene mutations.Pediatr Nephrol. 2010; 25: 1621-1632Crossref PubMed Scopus (138) Google Scholar, 2Hinkes B.G. Mucha B. Vlangos C.N. et al.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-e919Crossref PubMed Scopus (359) Google Scholar, 3Lipska B.S. Iatropoulos P. Maranta R. et al.Genetic screening in adolescents with steroid resistant nephrotic syndrome.Kidney Int. 2013; 84: 206-213Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 4Santin S. Bullich G. Tazon-Vega B. et al.Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.Clin J Am Soc Nephrol. 2011; 6: 1139-1148Crossref PubMed Scopus (175) Google Scholar WT1 was the first gene shown to be mutated in SRNS.5Coppes M.J. Huff V. Pelletier J. Denys-Drash syndrome: relating a clinical disorder to genetic alterations in the tumor suppressor gene WT1.J Pediatr. 1993; 123: 673-678Abstract Full Text PDF PubMed Scopus (100) Google Scholar Originally identified as a Wilms tumor (WT) suppressor gene, the primary physiological role of WT1 is to control the development of the genitourinary system. In the fetal kidney, WT1 is abundantly expressed in areas of active glomerulogenesis, supporting a major role of the gene in the development and maturation of the glomerular filtration barrier.6Morrison A.A. Viney R.L. Saleem M.A. et al.New insights into the function of the Wilms tumor suppressor gene WT1 in podocytes.Am J Physiol Renal Physiol. 2008; 295: F12-F17Crossref PubMed Scopus (75) Google Scholar After completion of nephrogenesis, WT1 expression is limited to podocytes. WT1 encodes a transcription factor containing an N-terminal transactivation domain (exon 1) and four zinc-fingers at the C-terminus (exons 7–10). Germline alterations located throughout the entire coding sequence, usually truncating point mutations, predispose to WT.7Huff V. Wilms’ tumours: about tumour suppressor genes, an oncogene and a chameleon gene.Nat Rev Cancer. 2011; 11: 111-121Crossref PubMed Scopus (184) Google Scholar, 8Dome J.S. Wilms Huff V. Pagon R.A. Bird T.D. Dolan C.R. GeneReviewst (Internet). University of Washington, Seattle, WA1993Google Scholar, 9Royer-Pokora B. Beier M. Henzler M. et al.Twenty-four new cases of WT1 germline mutations and review of the literature: genotype/phenotype correlations for Wilms tumor development.Am J Med Genet A. 2004; 127A: 249-257Crossref PubMed Google Scholar A splice site at exon 9 inserts three additional amino acids between the third and the fourth zinc-finger (usually referred to as the KTS splice insert). Mutations in the KTS site result in Frasier syndrome.10Barbaux S. Niaudet P. Gubler M.C. et al.Donor splice-site mutations in WT1 are responsible for Frasier syndrome.Nat Genet. 1997; 17: 467-470Crossref PubMed Scopus (570) Google Scholar A group of mostly missense mutations in exons 8 and 9 affect the zinc-finger domains. These variably impair the DNA-binding capacity of WT1 and cause either Denys–Drash syndrome or Meacham syndrome, a condition associated with extrarenal congenital defects including diaphragmatic hernia.5Coppes M.J. Huff V. Pelletier J. Denys-Drash syndrome: relating a clinical disorder to genetic alterations in the tumor suppressor gene WT1.J Pediatr. 1993; 123: 673-678Abstract Full Text PDF PubMed Scopus (100) Google Scholar,7Huff V. Wilms’ tumours: about tumour suppressor genes, an oncogene and a chameleon gene.Nat Rev Cancer. 2011; 11: 111-121Crossref PubMed Scopus (184) Google Scholar,11Mucha B. Ozaltin F. Hinkes B.G. et al.Mutations in the Wilms tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9.Pediatr Res. 2006; 59: 325-331Crossref PubMed Scopus (111) Google Scholar Finally, large genomic rearrangements affecting chromosome 11p13 may disrupt WT1 among several genes, resulting in WAGR syndrome (Wilms Tumor, Aniridia, Genitourinary Abnormalities, and Mental Retardation).8Dome J.S. Wilms Huff V. Pagon R.A. Bird T.D. Dolan C.R. GeneReviewst (Internet). University of Washington, Seattle, WA1993Google Scholar WT1-associated disorders may include SRNS either as an initial symptom or as a later development in a patient diagnosed on the basis of extrarenal features. In addition, isolated SRNS may result from a wide range of WT1 sequence variations, predominantly but not exclusively exonic point mutations notorious for incomplete penetrance and significantly variable expressivity.8Dome J.S. Wilms Huff V. Pagon R.A. Bird T.D. Dolan C.R. GeneReviewst (Internet). University of Washington, Seattle, WA1993Google Scholar The international PodoNet registry has assembled the largest genetically screened SRNS cohort to date. In this work we used the PodoNet cohort to describe the genotypic and phenotypic spectrum of WT1-associated kidney disease. The 61 cases collected by the consortium represent the largest and best characterized cohort of WT1 nephropathy analyzed to date. We provide demographic information on the relative frequency of WT1-associated SRNS and mutation types and perform a detailed genotype–phenotype correlation study encompassing the age and symptoms at disease onset, the long-term course of kidney function, and the incidence and spectrum of extrarenal manifestations. Among the 746 consecutive SRNS cases registered in the PodoNet cohort who underwent WT1 gene screening, 46 patients (6%) from 23 clinical centers in 12 European and Middle East countries were found to be positive for a WT1 mutation. These included 2 out of 25 (8%) SRNS families with autosomal dominant inheritance reported to the Registry. In addition, information on 15 patients, including 2 additional families, diagnosed with WT1-associated SRNS before the formation of the PodoNet consortium was included in the analysis, yielding a cohort of 61 patients (Figure 1). WT1-positive patients did not cluster to any specific region, country, or era. Results of 17 (28%) patients have been previously published;3Lipska B.S. Iatropoulos P. Maranta R. et al.Genetic screening in adolescents with steroid resistant nephrotic syndrome.Kidney Int. 2013; 84: 206-213Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,12Chernin G. Vega-Warner V. Schoeb D.S. et al.Genotype/phenotype correlation in nephrotic syndrome caused by WT1 mutations.Clin J Am Soc Nephrol. 2010; 5: 1655-1662Crossref PubMed Scopus (78) Google Scholar, 13Köhler B. Biebermann H. Friedsam V. et al.Analysis of the Wilms’ tumor suppressor gene (WT1) in patients 46,XY disorders of sex development.J Clin Endocrinol Metab. 2011; 96: E1131-E1136Crossref PubMed Scopus (45) Google Scholar, 14Mestrallet G. Bertholet-Thomas A. Ranchin B. et al.Recurrence of a dysgerminoma in Frasier syndrome.Pediatr Transplant. 2011; 15: e53-e55Crossref PubMed Scopus (5) Google Scholar,15Gellermann J. Stefanidis C.J. Mitsioni A. et al.Successful treatment of steroid-resistant nephrotic syndrome associated with WT1 mutations.Pediatr Nephrol. 2010; 25: 1285-1289Crossref PubMed Scopus (56) Google Scholar, 16Wasilewska A.M. Kuroczycka-Saniutycz E. Zoch-Zwierz W. Effect of cyclosporin A on proteinuria in the course of glomerulopathy associated with WT1 mutations.Eur J Pediatr. 2011; 170: 389-391Crossref PubMed Scopus (19) Google Scholar, 17Stefanidis C.J. Querfeld U. The podocyte as a target: cyclosporin A in the management of the nephrotic syndrome caused by WT1 mutations.Eur J Pediatr. 2011; 170: 1377-1383Crossref PubMed Scopus (25) Google Scholar, 18Kaltenis P. Schumacher V. Jankauskiene A. et al.Slow progressive FSGS associated with an F392L WT1 mutation.Pediatr Nephrol. 2004; 19: 353-356Crossref PubMed Scopus (16) Google Scholar, 19Hakan N. Aydin M. Erdogan O. et al.A novel WT1 gene mutation in a newborn infant diagnosed with Denys-Drash syndrome.Genet Couns. 2012; 23: 255-261PubMed Google Scholar, 20Wasilewska A. Zoch-Zwierz W. Tenderenda E. et al.[WT1 mutation as a cause of progressive nephropathy in Frasier syndrome—case report].Pol Merkur Lekarski. 2009; 26: 642-644PubMed Google Scholar, 21Schumacher V. Thumfart J. Drechsler M. et al.A novel WT1 missense mutation presenting with Denys-Drash syndrome and cortical atrophy.Nephrol Dial Transplant. 2006; 21: 518-521Crossref PubMed Scopus (9) Google Scholar, 22Fencl F. Malina M. Stara V. et al.Discordant expression of a new WT1 gene mutation in a family with monozygotic twins presenting with congenital nephrotic syndrome.Eur J Pediatr. 2012; 171: 121-124Crossref PubMed Scopus (18) Google Scholar however, for the purposes of this study the most recent follow-up data were provided. All patients in the PodoNet cohort underwent mutational screening of exons 8 and 9 and their intronic junctions. Seven patients with clinical features highly suggestive of WT1-associated disease (concomitant WT or ovarian dysgerminoma) and no mutation in the hot spot region underwent extended screening of the entire coding sequence of WT1, yielding a causative point mutation in four cases. The remaining three patients were tested by array comparative genomic hybridization for large genomic deletions at chromosome 11p13. No deletion at the WT1 locus and/or its adjacent chromosomal region was detected. A total of 30 different WT1 mutations were detected (Figure 2), including 5 recurrent mutations: c.1288C>T, c.1372C>T, c.1384C>T, c.1432+4C>T, and c.1432+5G>A (previously referred to as R362X, R390X, R394W, IVS9+4C>T, and IVS9+5G>A, respectively). Five mutations are novel: c.1017C>A, c.1063A>T, c.1192T>C, c.1249+3A>G, and c.1337C>T (detailed evaluation of their pathogenic effect is in Supplementary Material S2 online). Download .pdf (.61 MB) Help with pdf files Supplementary Information A total of 55 (90%) patients carried mutations in the hot spot region. Mutations were categorized as truncating mutations (5 mutations in 9 subjects), missense mutations affecting amino acids in DNA-binding positions (14 mutations in 24 subjects), other missense mutations (n=7), intronic KTS mutations (3 mutations in 19 subjects), and other intronic mutations (1 mutation in 2 family members). The most common mutation, found in nine nonrelated cases, was c.1384C>T, leading to the p.Arg462Trp substitution (previously referred to as R394W). The clinical characteristics of the SRNS patients with and without WT1 mutations were compared (Table 1). Patients with WT1-associated disease were usually less symptomatic at the time of diagnosis with less marked edema and hypoalbuminemia, but more commonly presented with impaired glomerular filtration rate as compared with WT1-negative cases. The initial diagnosis was isolated SRNS in 56% of cases (n=34). In retrospect, however, only 28% (n=17) of the patients could be considered as having isolated SRNS when accounting for urogenital anomalies, sex reversal, and late-onset WTs detected subsequent to WT1 mutation. All patients with isolated SRNS were genotypic and phenotypic females, whereas the syndromic cases encompassed both male and female phenotypic patients. Patients with WT1-associated SRNS attained end-stage renal disease (ESRD) at, on average, 3 years younger age than patients with other genetic causes of SRNS (n=88; 66 NPHS2, 13 NPHS1, 5 PTPRO, 3 LAMB1, and 1 MYO1E; P=0.001) and at almost 10 years younger age than patients without detectable genetic cause (P<0.0001; Figure 3).Table 1Clinical characteristics at the time of diagnosis and prospective kidney survival rates of 61 children with SRNS related to WT1 mutations versus 700 patients screened negative in WT1WT1 SRNSNon-WT1 SRNSN61700Age at diagnosis (years)2.0 (0.7–6.8)***4.4 (1.9–10.1)Asymptomatic, incidental diagnosis27.9%**12.6%Edema (none/mild/moderate/severe)46/19/26/9%14/8/31/22%Proteinuria (small/gross)19/81%16/84%Hematuria27.3%37.5%Hypertension39.0%***15.5%eGFR (ml/min per 1.73m2)74 (60–106)**103 (71–144)Serum albumin (g/l)27 (19–35)*21(16–28)Wilms tumor37.7%***0.3%Gonadoblastoma4.9%**0.1%Sex reversal14.7%***0.1%Genital abnormalities in subjects with concordant gender31.1%***0.1%Urinary tract abnormalities11.4%***1.3%Histopathology subtype FSGS47.5%43.7% DMS34.4%***1.6% MesGN3.3%10.0% MCN1.7%15.3% Other/no data13.1%21.8%Kidney survival from diagnosis 2-Year survival rate70.1±8.2%***92.0±1.1% 5-Year survival rate55.2±7.4%***72.1±7.9% 10-Year survival rate25.0±3.5%***51.5±3.4%Abbreviations: DMS, diffuse mesangial sclerosis; eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; MCN, minimal change nephropathy; MesGN, mesangioproliferative glomerulonephritis; SRNS, steroid-resistant nephrotic syndrome; WT1, Wilms tumor 1.Data are given as median (interquartile range) or %. *P<0.05, **P<0.01, ***P<0.001. Open table in a new tab Abbreviations: DMS, diffuse mesangial sclerosis; eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; MCN, minimal change nephropathy; MesGN, mesangioproliferative glomerulonephritis; SRNS, steroid-resistant nephrotic syndrome; WT1, Wilms tumor 1. Data are given as median (interquartile range) or %. *P<0.05, **P<0.01, ***P<0.001. Grouping of mutations according to location revealed notable phenotypic differences. Patients with exonic mutations were significantly younger at diagnosis, presented with more severe proteinuria, edema, and hypertension, and progressed more rapidly to ESRD (Table 2). The average time interval from diagnosis to end-stage kidney disease was 2.7 versus 9.2 years (P<0.001). Further subgrouping by mutation type showed distinct differences between patients with missense mutations affecting DNA-binding amino acid residues and those with truncating mutations (Table 3 and Figures 1 and 4). Mutations in DNA-binding positions were associated with the earliest disease onset and the most rapid progression to ESRD (P II grade1, Horseshoe kidney; 1, kidney malrotation; 1, PUJ stenosisKidney survival from diagnosis 2 Years57.1±8.3 %***90.5±6.4% 5 Years35.8±5.7 %***85.3±8.9% 10 Years18.2±2.7 %***37.0±11.0%Abbreviations: DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis; PUJ, pelviureteric junction; SRNS, steroid-resistant nephrotic syndrome; VUR, vesicoureteral reflux; WT1, Wilms tumor 1.*P<0.05, **P<0.01, ***P<0.001. Data are given as median (interquartile range) or %.a Seven cases with prophylactic bilateral nephrectomy performed before age 5 years excluded from the analysis. Open table in a new tab Table 3Genotype–phenotype association for WT1 mutation subtypesExonicIntronicTruncatingDNA-binding siteOther missenseKTS (intron 9)Other intronicType of mutationmutationsmissense mutationsmutationsmutationsmutationsN9247192Age at SRNS onset12.3 (0.6–15.3)0.9 (0.2–1.6)2.4 (0.7–5.3)4.5 (3.1–8.1)14.2 (3.5–25)Age at 50% kidney survivalaKaplan–Meier median estimate.16.52.510.813.6NARenal histopathologybOnly successful biopsies considered. DMS2/6 (33%)17/23 (74%)4/6 (67%)1/17 (6%)0/2 FSGS4/6 (67%)4/23 (17%)2/6 (33%)15/17 (88%)2/2Isolated SRNS1/9 (11%)5/24 (21%)2/7 (29%)7/19 (37%)2/2Sex reversalcOnly patients with 46,XY karyotype considered.0/5 (0 %)3/11 (27%)0/2 (0 %)6/9 (67%)0/0Genital abnormalities in subjects with concordant gender6/9 (67%)9/21 (43%)2/7 (29%)4/13 (31%)0/2Wilms tumor7/9 (78%)13/24 (54%)2/7 (29%)1/19 (5%)0/2 Thereof bilateral4/9 (44%)1/24 (4%)———Other neoplasia0/9 (0%)2/24 (8%)0/7 (0%)3/19 (16%)0/2Urinary tract malformation2/9 (22%)1/24 (4%)1/7 (14%)3/19 (16%)0/2Abbreviations: DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis; NA, not available; SRNS, steroid-resistant nephrotic syndrome; WT1, Wilms tumor 1.Data are given as median (interquartile range) or %.a Kaplan–Meier median estimate.b Only successful biopsies considered.c Only patients with 46,XY karyotype considered. Open table in a new tab Abbreviations: DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis; PUJ, pelviureteric junction; SRNS, steroid-resistant nephrotic syndrome; VUR, vesicoureteral reflux; WT1, Wilms tumor 1. *P<0.05, **P<0.01, ***P 10 years), 5 had truncating WT1 mutations; 4 of these were long-term WT survivors. The other four late-onset patients had intronic mutations (thereof 3 KTS); these patients manifested with isolated proteinuria without extrarenal symptoms. Of the 7 peripubescent children with exonic mutations, 6 had undergone unilateral (n=5) or subtotal bilateral nephrectomy (n=1) for WT 5.3–13.6 years before the onset of SRNS. The 9 patients with the p.Arg462Trp mutation showed SRNS onset between 3 weeks and 3.1 years of age (median 9.5 months). Of the 9 subjects, 5 progressed to ESRD before their fourth birthday, 1 at age 7, and 3 still had preserved kidney function at age 5–8 years. Similar to the non-WT1-associated SRNS, the most common histopathological finding in WT1 disease was focal segmental glomerulosclerosis (FSGS; Table 1). Diffuse mesangial sclerosis (DMS), the second most common histology, was highly predictive of WT1 mutations: WT1-positive patients accounted for 60% of all DMS cases reported in the PodoNet Registry (odds ratio=21; 95% confidence interval: 9.7–50; P<0.0001). Among the WT1 patients, DMS was six times more likely to be present in children diagnosed before the age of 2 years than in older children (60% vs. 10%) and associated almost exclusively with exonic missense mutations (Tables 2 and 3). Conversely, FSGS was observed mainly in patients with first disease manifestation beyond 2 years of age (74% vs. 23%) and was present in almost 90% of patients with intronic mutations. Among the six patients with a p.Arg462Trp mutation who underwent kidney biopsy, DMS, FSGS, and membranoproliferative glomerulonephritis were reported in two cases each. Cancer was more common in patients with exonic mutations (73% vs. 19%), the highest rate of neoplasia being observed among patients with truncating mutations (78%). WTs developed in 23 patients (Tables 2 and 3). Of them, 22 were carriers of exonic WT1 mutations who were cumulatively followed-up for 199 years (that is, one case per 9 years at risk). In contrast, only a single WT occurred in a patient with a KTS mutation (incidence 1 per 343 years at risk). The median age at WT diagnosis was 1.6 (range 0.1–4.5) years. In six patients, SRNS preceded WT by a median of 1.7 years (range 2 weeks–3.9 years); 8 children first presented with SRNS at the time of WT diagnosis, and in 9 patients SRNS developed after a median of 8 years (range 4 months–13.6 years) after diagnosis and treatment of a WT. Bilateral tumors were more common in patients with truncating mutations (odds ratio=18.4; P=0.01). Gonadoblastoma, including one bilateral case, occurred in three of nine 46,XY individuals with sex reversal within a cumulative observation time of 91 years. Two patients developed Epstein–Barr virus–associated post-transplant lymphoproliferative disease (B-cell lymphoma) during adolescence. Both patients had previously undergone cyclophosphamide treatment for SRNS, were receiving ciclosporin A and mycophenolate mofetil when post-transplant lymphoproliferative disease developed, and had donor-positive/recipient-negative high-risk Epstein–Barr virus status at the time of transplantation. Male-to-female sex reversal was detected in nine subjects (1/3 of 46,XY individuals), all of whom were already diagnosed with a WT1 mutation. In two phenotypic girls with SRNS, primary amenorrhea had prompted WT1 screening. Sex reversal occurred exclusively in patients with KTS mutations and DNA-binding site mutations (Table 3). All 46,XY individuals, except one, presenting with a male phenotype had various abnormalities of the external genitalia, the most common being cryptorchidism and hypospadias, present in 83% and 67%, respectively (Table 2). Furthermore, 3 of the 34 female patients with 46,XX presented with genital abnormalities (Table 2). The median age at menarche in the 46,XX female patients was 13 (range 10–15) years. All adolescent 46,XY individuals with male-to-female sex reversal achieved menstruation on hormone replacement therapy. To date, two adult female patients have mothered children. Among four boys with the same p.Arg462Trp mutation, genital abnormalities varied and included penoscrotal hypoplasia, hypospadias, and cryptorchidism; one girl with the mutation presented with uterus bicornus. Urinary tract abnormalities were noted in seven patients (11%, Tables 2 and 3). Of them, three were female, three (phenotypic and karyotypic) were male, and one was a karyotypic male with sex reversal. Five of the patients had combined urinary tract and genital anomalies. A total of 27 patients (44%), including 4 with intronic mutations, underwent bilateral nephrectomy. Half of them, all with exonic mutations, underwent the surgery before their fifth birthday. Nephrectomy was performed electively before transplantation (n=18), because of bilateral WT (n=5) or because of suspicious sonographic findings (n=4). In 14 (52%) 46,XY individuals, both with exonic (9/18) and intronic (5/9) mutations, preemptive bilateral gonadectomy before 20 years of age was undertaken. On histopathological evaluation, mixed gonadal dysgenesis was the most common finding; however, in three patients asymptomatic gonadoblastoma was detected and in three patients no gonads were found. In this large unselected cohort, we found WT1 mutations in 6% of sporadic SRNS patients, in keeping with the prevalence figures reported in previous smaller studies ranging from 2.7 to 7%.4Santin S. Bullich G. Tazon-Vega B. et al.Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.Clin J Am Soc Nephrol. 2011; 6: 1139-1148Crossref PubMed Scopus (175) Google Scholar,11Mucha B. Ozaltin F. Hinkes B.G. et al.Mutations in the Wilms tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9.Pediatr Res. 2006; 59: 325-331Crossref PubMed Scopus (111) Google Scholar,23Denamur E. Bocquet N. Baudouin V. et al.WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis.Kidney Int. 2000; 57: 1868-1872Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,24Ruf R.G. Schultheiss M. Lichtenberger A. et al.Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome.Kidney Int. 2004; 66: 564-570Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar WT1-positive cases were equally common in all pediatric age groups, reconfirming comparable figures reported separately for congenital/infantile and adolescent SRNS.2Hinkes B.G. Mucha B. Vlangos C.N. et al.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-e919Crossref PubMed Scopus (359) Google Scholar,3Lipska B.S. Iatropoulos P. Maranta R. et al.Genetic screening in adolescents with steroid resistant nephrotic syndrome.Kidney Int. 2013; 84: 206-213Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar Compared with non-WT1-associated SRNS, patients with WT1 nephropathy were less often overtly nephrotic but more often presented with chronic kidney disease at the time of diagnosis. WT1 disease tended to progress more rapidly than SRNS from other causes, with ESRD occurring at an almost 9 years younger age on average. FSGS was the most frequent histopathological finding in WT1 nephropathy and was equally common in non-WT1-related SRNS, whereas the diagnosis of DMS was largely specific for WT1-associated disease. Furthermore, WT1 mutations were associated with a wide spectrum and expressivity of extrarenal phenotypes concerning urogenital development and the development of tumors. These were largely specific for WT1-associated disease, although a few cases of SRNS with WT or gonadoblastoma were noted in patients in whom no abnormalities in WT1 could be detected. Both renal and extrarenal phenotypes were clearly associated with the type and locatio
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