View of Clinical Features of Patients with WT1 Hot Spot Mutations in Exon 8/9

Article

Clinical and Molecular Characterization of Patients with Heterozygous Mutations in Wilms Tumor Suppressor Gene 1

Anja Lehnhardt, Claartje Karnatz, Thurid Ahlenstiel-Grunow, Kerstin Benz, Marcus R. Benz, Klemens Budde, Anja K. Bu¨scher, Thomas Fehr, Markus Feldko¨tter, Norbert Graf, Britta Ho¨cker, Therese Jungraithmayr, Gu¨nter Klaus, Birgit Koehler, Martin Konrad, Birgitta Kranz, Carmen R. Montoya, Dominik Mu¨ller, Thomas J. Neuhaus, Jun Oh, Lars Pape, Martin Pohl, Brigitte Royer-Pokora, Uwe Querfeld, Reinhard Schneppenheim, Hagen Staude, Due to the number of Giuseppina Sparta`, Kirsten Timmermann, Frauke Wilkening, Simone Wygoda, Carsten Bergmann, and Markus J. Kemper contributing authors, the affiliations are provided in the Abstract Supplemental Background and objectives The Wilms tumor suppressor gene 1 (WT1) plays an essential role in urogenital and Material. kidney development. Genotype/phenotype correlations of WT1 mutations with renal function and proteinuria have been observed in world-wide cohorts with nephrotic syndrome or Wilms tumor (WT). This study analyzed Correspondence: mid-European patients with known constitutional heterozygous mutations in WT1, including patients without Dr. Anja Lehnhardt, Department of proteinuria or WT. Pediatric Nephrology, University Medical Design, setting, participants & measurements Retrospective analysis of genotype, phenotype, and treatment of 53 Center Hamburg- patients with WT1 mutation from all pediatric nephrology centers in Germany, Austria, and Switzerland per- Eppendorf, Martinistrasse 52, formed from 2010 to 2012. 20246 Hamburg, Germany. Email: A. Results Median age was 12.4 (interquartile range [IQR], 6–19) years. Forty-four of 53 (83%) patients had an exon [email protected] mutation (36 missense, eight truncating), and nine of 53 (17%) had an intronic lysine-threonine-serine (KTS) splice site mutation. Fifty of 53 patients (94%) had proteinuria, which occurred at an earlier age in patients with missense mutations (0.6 [IQR, 0.1–1.5] years) than in those with truncating (9.7 [IQR, 5.7–11.9]; P,0.001) and splice site (4.0 [IQR, 2.6–6.6]; P=0.004) mutations. Thirteen of 50 (26%) were treated with steroids and remained irresponsive, while three of five partially responded to cyclosporine A. Seventy-three percent of all patients required RRT, those with missense mutations significantly earlier (at 1.1 [IQR, 0.01–9.3] years) than those with truncating mutations (16.5 [IQR, 16.5–16.8]; P,0.001) and splice site mutations (12.3 [IQR, 7.9–18.2]; P=0.002). Diffuse mesangial sclerosis was restricted to patients with missense mutations, while focal segmental sclerosis occurred in all groups. WT occurred only in patients with exon mutations (n=19). Fifty of 53 (94%) patients were karyotyped: Thirty-one (62%) had XY and 19 (38%) had XX chromosomes, and 96% of male karyotypes had urogenital malformations.

Conclusions Type and location of WT1 mutations have predictive value for the development of proteinuria, renal insufﬁciency, and WT. XY karyotype was more frequent and associated with urogenital malformations in most cases. Clin J Am Soc Nephrol 10: 825–831, 2015. doi: 10.2215/CJN.10141014

Introduction WAGR syndrome (Wilms tumor [WT], aniridia, geni- The Wilms tumor 1 (WT1) gene on chromosome 11p13 tourinary malformations, and mental retardation) (6). was identified in 1990 (1,2). Its longest open reading Heterozygous germline mutations in WT1 were de- frame comprises 10 exons that code for a transcription scribed in two rare human conditions: Denys–Drash factor with four zinc-finger DNA-binding motifs. Tran- syndrome (DDS) with exon mutations in the zinc-finger scriptional function of WT1 can result in tumor sup- region (7,8) and Frasier syndrome (FS) with mutations pression or be oncogenic (3). An alternative splice site affecting the canonic donor KTS splice site of intron 9 in intron 9 allows the addition of three amino acids (9,10). DDS includes steroid-resistant nephrotic syn- (lysine-threonine-serine [KTS]) (4). WT1 plays an essen- drome rapidly progressing to ESRD, 46 XY disorder tial role in early urogenital and kidney development, in sex development (DSD) with sex reversal and a especially in the conversion of mesenchymal cells to high risk for WT (11,12). FS is defined by progressive epithelial structures (5). glomerulopathy, gonadoblastoma, and 46 XY DSD Heterozygous deletion of 11p13, resulting in altered with sex reversal (13). Over the past years, many clin- dosage of WT1 expression, is found in patients with ical pictures beyond the classic syndromes have been www.cjasn.org Vol 10 May, 2015 Copyright © 2015 by the American Society of Nephrology 825 826 Clinical Journal of the American Society of Nephrology

described in patients with heterozygous WT1 mutations, il- Mac OS X (GraphPad Software, San Diego, CA). Non- lustrating the phenotypic heterogeneity (14,15). A recent re- normally distributed variables are presented as medians and port by the international PodoNet Consortium described interquartile ranges. Differences between two or more groups genotype/phenotype correlations associated with WT1 mu- were analyzed using one-way ANOVA or nonparametric tations in 61 patients with nephrotic syndrome collected Kruskal–Wallis test as appropriate. For survival analyses, worldwide; it did not cover treatment of proteinuria (16). differences between curves were tested with a log-rank We present data on 53 patients with known heterozygous test. In general, P,0.05 was regarded as representing a sta- constitutional WT1 mutations from all major pediatric ne- tistically significant difference. In multiple comparisons, P phrology centers in Germany, Austria, and Switzerland. values were adjusted for multiplicity using Bonferroni cor- We sought to perform a representative analysis of genotype rection or Dunn multiple comparison testing. and renal phenotype of mid-European patients, including data on treatment of proteinuria and description of genitourinary abnormalities. Results WT1 Mutations At the time of analysis patients had a median age of 12.4 (IQR, 6–19) years (Table 1). Exon mutations were found in 44 Materials and Methods of 53 patients (83%); in 39 of 44 (89%) mutations were located Patients and Data Recruitment in the hot spot region comprising exons 8 and 9 (Table 2). Fifty-three patients with constitutional heterozygous WT1 Thirty-six of 44 patients (82%) had missense mutations; of mutations treated in pediatric nephrology centers in Ger- these, 28 mutations were affecting amino acids in DNA- many (n=48), Austria (n=2), and Switzerland (n=3) were in- binding positions. Truncating mutations were found in eight cluded in this retrospective study (for details, see of 44 patients (18%) (n=7 nonsense, n=1 deletion) (Figure 1). Supplemental Table 1). Approved by the ethical committee The most common mutation was a missense mutation in of the Medical Association of Hamburg, Germany, this exon 9 c.1384C.T, p.Arg462Trp (previously referred to as study was performed in cooperation with the German Soci- R394) in 14 patients (Supplemental Table 2). Intronic KTS ety for Pediatric Nephrology. After informed consent was splice site mutations, c.1432+4C.T and c.1432+5G.A, were obtained, clinical data were collected from August 2010 until found in five and four patients, respectively. All mutations August 2012 using a standardized questionnaire, in adher- found were heterozygous. Treating physicians referred to ence to the Declaration of Helsinki. their patients with exon mutations as having DDS or partial DDS and referred to their patients with intron mutations as Categories of WT1 Mutations for Genotype/Phenotype having FS, even if they did not show the full syndromal Correlation picture. Allele frequencies and prediction of pathogenicity WT1 mutations were identified by direct Sanger sequenc- of WT1-mutations are presented in Supplemental Table 3. ing technique. All mutations were matched with previous fi ndings from the literature, and, if needed, nomenclature Proteinuria was adjusted to the current recommendations of the Human Fifty of 53 (94%) patients had nephrotic-range protein- Genome Variation Society using NM_024426.4 as reference. uria, 33 (66%) of whom had clinical nephrotic syndrome. fi Patients were classi ed according to the type of mutations: Fourteen of 50 (28%), including only one male patient, had exon mutations, either missense mutations in DNA-binding isolated proteinuria. In 41 patients proteinuria was the ini- regions (reference sequence NM_024426.4: WT isoform D) or tial renal symptom. other regions or variants of truncating character (nonsense Thirteen of 50 patients (26%) were treated with steroids; mutations and out-of-frame deletions) and intronic (KTS all of them were steroid resistant. Five of 50 (10%) received splice site) mutations. cyclosporine A (CsA), which lead to partial remission in three patients with KTS splice site mutation. One patient Clinical and Histologic Data received chlorambucil without any response to treatment. The date of first diagnosis of proteinuria together with the Patients with missense mutations presented with pro- clinical picture was documented, with definitions based on teinuria significantlyearlier(median,0.6[IQR,0.1–1.5] current guidelines (17,18). If a renal biopsy was performed, years) than patients with truncating mutations (median, we recorded the underlying histologic assessment. The start 9.7 [IQR, 5.7–11.9] years; P,0.001) and those carrying of RRT was defined as start of dialysis or preemptive renal splice site mutations (median, 4.0 [IQR, 2.6–6.6] years; transplantation. The date of diagnosis of WT and its man- P=0.004) (Figure 2A). There were no statistically signifi- agement were recorded. In addition, karyotype and sex were cant differences between the two groups with missense obtained, along with information on urogenital malforma- mutations in DNA binding or other regions. Of the three tions and their management. We excluded the following patients without any proteinuria, two patients had a trun- from correlation analyses: patient 9, who initially presented cating mutation in exon 1; they both presented with bilateral with hemolytic uremic syndrome, and patient 30, who had WT and 46 XY DSD. The other patient had the p.Pro29Ser p.Pro29Ser WT1 missense variant (Supplemental Table 1). missense variant in exon 2 and no WT but did have 46 XY DSD. Statistical Analyses A custom database was created using Filemaker Pro 11 Renal Function (Filemaker Inc., Unterschleissheim, Germany). Data were Forty-seven of 53 patients (88%) had CKD stage 3–5. analyzed using GraphPad Prism software, version 6.0b for Thirty-nine of 53 (73%) required RRT at a median age of Clin J Am Soc Nephrol 10: 825–831, May, 2015 Report of the WT1-Registry of the GPN, Lehnhardt et al. 827

Table 1. Summary of patient characteristics

Type of WT1 Mutation Characteristic All Patients Missense Truncating KTS Splice Site

Patients (n)533689 Median age at follow-up (yr) 12.4 (6.0–19.0) 9.3 (4.1–15.1) 17.9 (7.5–22.9) 21.0 (12.1–33.6) Proteinuria, n (%) 50 (94) 34 (94) 6 (75) 9 (100) Median age at diagnosis of 1.2 (0.3–4.3) 0.6 (0.1–1.5) 9.7 (5.7–11.9) 4.0 (2.6–6.6) proteinuria (yr) Biopsies available, n (%) 45 (85) 31 (86) 5(63) 9 (100) Underlying histologic condition, n (%) 17 (38) 8 (26) 4 (80) 5 (56) FSGS 3(7) 0 0 3(33) Minimal-change disease 20 (44) 20 (65) 0 0 Diffuse mesangial sclerosis 5 (11) 3 (10) 1 (20) 1 (11) Others ESRD 39 (74) 30 (83) 4 (50) 4 (44) Median age at start RRT (yr) 1.7 (0.6–7.8) 1.1 (0.4–4.0) 16.5 (16.2–16.7) 12.2 (8.8–16.9) WT, n (%) 19 (36) 12 7 (88) 0 (0) Bilateral WT, n (%) 7 (37) 2 (17) 5 (71) Median age at diagnosis (yr) 1.3 (0.9–1.5) 1.3 (1.0–1.8) 1.0 (0.8–1.3) Gonadoblastoma 1 (2) 0 (0) 0 (0) 1 (11) Karyotype available, n (%) 50 (94) 34 (94) 7 (88) 9 (100) XY 31 (62) 21 (62) 6 (86) 4 (44) XY-DSD 32 (60) 21 (58) 7 (88) 4 (44) Complete gonadal dysgenesis 6(11) 3(8) 0 3(33) (sex reversal), n (%)

Median values are expressed with interquartile ranges. WT, Wilms tumor; DSD, disorder in sex development; KTS, lysine-threonine- serine.

Table 2. Overview of clinical features of patients with WT1 hot spot mutations in exon 8/9

Type of Mutation in Exon 8/9 Variable Missense Truncating

Patients (n)346 Median age at follow-up (yr) 8.9 (3.8–14.8) 20.6 (15.6–24.9) Proteinuria, n (%) 34 (100) 6 (100) Median age at diagnosis of proteinuria (yr) 0.6 (0.1–1.4) 9.7 (5.7–11.9) ESRD, n (%) 31 (91) 4 (67) Median age at start of RRT (yr) 1.1 (0.4–3.9) 16.5 (16.2–16.7) Wilms tumor, n (%) 12 (35) 5 (83) Gonadoblastoma, n (%) 0 (0) 0 (0) XY-DSD, n (%) 20 (59) 5 (83)

DSD, disorder in sex development.

1.7 (IQR, 0.6–7.8) years, 14 of 39 (36%) were diagnosed and P=0.008, respectively) (Figure 2B). There was no sta- with proteinuria and ESRD at the same time. Seventeen of tistically significant difference between the two missense 39 patients (44%) were initially treated with peritoneal di- mutation groups. alysis, 20 of 39 (51%) were treated with hemodialysis, and two of 39 (5%) received a preemptive kidney transplant. Renal Histology Twenty-nine patients received a first kidney transplant at a Data on renal histology were available in 45 of 53 (85%) median age of 4.8 (IQR, 2.7–8.9) years. No disease recurrence patients. Diffuse mesangial sclerosis (DMS) was diagnosed in allografts was reported. in 20 of 45 (44%), FSGS in 17 (38%), and minimal-change Patients with a missense mutation started RRT signifi- disease (MCD) in three (7%). In five (11%) patients, other cantly earlier than patients with a truncating or KTS splice results were found (atrophic cortex, glomerulopathy, hy- site mutation (median age, 1.1 [IQR, 0.01–9.3] versus 16.5 pertensive glomerulopathy, and complete sclerosis with [IQR, 16.5–16.8] and 12.3 [IQR, 7.9–18.2] years; P=0.002 tubular atrophy, respectively). In two patients with MCD a 828 Clinical Journal of the American Society of Nephrology

Figure 1. | Exon 8/9 is hot spot region for mutations in the WT1 gene. Gene is shown out of scale. NM_024426.4: Wilms tumor isoform D of WT1 was used as reference sequence. The bold, italic text indicates missense mutations affecting nucleotides coding for residues important for interaction with target DNA: 434–449 in exon 8 and 461–469 in exon 9 (Swiss-Prot: P19544). The lysine-threonine-serine (KTS) splice site mutations are mapped below. The number of patients affected is speciﬁed. Asterisk, translation termination (stop) codon, according to the Human Genome Variation Society.

Figure 2. | Significant influence of type of causative WT1 mutation on manifestation of nephropathy. (A) proteinuria (P=0.002) and (B) kidney survival (P,0.001). Kaplan–Meier survival analysis, log-rank test (Mantel–Cox). KTS, lysine-threonine-serine. second biopsy was performed 53 and 58 months after the KTS splice site mutations. MCD was diagnosed in three first; One confirmed MCD and the other showed FSGS. patients with KTS splice site mutation. Patients with DMS were diagnosed with proteinuria at a median age of 0.6 (IQR, 0.3–1.6) years, significantly earlier WT than patients with FSGS (2.9 [IQR, 1–9.2] years) (P=0.01). Nineteen of 53 patients (36%) developed WT at a median RRT was started at a median age of 1.2 years (IQR, 0.6–2.6 age of 1.3 (IQR, 0.9–1.5) years. Unilateral WT was diagnosed years) in 20 of 20 (100%) patients with DMS and at 8.6 in 12 (63%) and bilateral WT (stage V) in 7 (37%). All but one years (IQR, 0.3–16.5 years) in 10 of 17 (59%) patients patientwithanincidentalfinding of nephroblastomatosis and with FSGS. Histology significantly influenced progression subsequent prophylactic bilateral nephrectomies received pre- to ESRD after diagnosis of proteinuria (Figure 3). DMS operative chemotherapy (International Society of Paediatric occurred only in patients with missense mutations. Eight Oncology protocols); all survived without relapse. Patients of 17 (47%) patients with FSGS had missense mutations, with unilateral WT had ipsilateral nephrectomies. In all but four (24%) had truncating mutations, and five (29%) had one patient with a bilateral WT, enucleation of the tumors Clin J Am Soc Nephrol 10: 825–831, May, 2015 Report of the WT1-Registry of the GPN, Lehnhardt et al. 829

Table 3. Genitourinary malformations in 32 patients with 46 XY Disorder in Sex Development

Genitourinary Malformations Patients (n) in Patients with 46 XY DSD

Only cryptorchidism 5 Cryptorchidism plus hypospadia 5 Cryptorchidism, penoscrotal 10 hypospadia, micropenis with or without bifid scrotum Cryptorchidism, penoscrotal/ 4 perineal hypospadia, micropenis with or without bifid scrotum + rudimental vagina/uterus Figure 3. | Renal histology influences progression of glomerulopathy Cryptorchidism, penoscrotal 2 until start of RRT in patients with proteinuria. Kaplan–Meier analysis hypospadia, micropenis, of kidney survival from diagnosis of proteinuria; log-rank test (Mantel– urogenital sinus Cox) P,0.001. DMS, diffuse mesangial sclerosis; MC, minimal- Sex reversal (complete gonadal 6 change disease. dysgenesis) was performed. Of all patients with WT, 58% were not tested DSD, disorder in sex development. for WT1 mutation at the time of WT diagnosis but rather 11– 189 months later. In two patients, WT1 mutation status was known before WT was diagnosed. with a wide spectrum of renal abnormality, even in the Only patients with exon mutations developed WT. Twelve absence of proteinuria. Patients with truncating mutations of 36 (33%) with missense mutation and seven of eight (88%) seem to have a milder renal phenotype and develop pro- with truncating mutations developed WT; no significant – teinuria and ESRD later, although the risk for bilateral WT difference between mutations in DNA-binding or non DNA- is higher compared with missense mutations. However, binding regions was observed. Patients with missense mu- – the genotype/phenotype correlation described by us and tation were diagnosed at a median age of 1.3 (IQR, 1 1.8) other authors must be viewed with some caution because years, patients with a truncating mutation at 1 (IQR, 0.8–1.3 fi the analyses are retrospective. In addition, the effect of months) (P=0.2). Bilateral WT occurred in ve of seven (71%) treatment (especially of proteinuria) is difficult to assess patients with truncating mutations and two of 12 (17%) retrospectively and has not been addressed by all studies. patients with missense mutations (P=0.04). Extrarenal symptoms in patients with WT1 mutations are complex, and urogenital malformations resulting in multi- Prophylactic Nephrectomies ple interventions may in particular have an important ef- Of 53 patients, 13 (25%) underwent a prophylactic bi- fect on medical and psychosocial quality of life. lateral nephrectomy within the first 2 years of life. This was Our registry on mid-European patients with WT1 muta- independent of any nephrectomies for WT. A further six tions differs from previous studies that recruited patients (11%) patients had their kidney removed bilaterally at the from cohorts with nephrotic syndrome (19). Because of the start of RRT. different approach, our cohort also includes a small subset of patients without proteinuria. Some of our findings are Karyotype, Sex, Genitourinary Malformations, and in line with the previously published literature, such as the Gonadoblastoma association of age at proteinuria onset and the initiation of Fifty of 53 patients (94%) were karyotyped; 31 of these (62%) RRT, as well as frequency and bilateralism of WT with had XY and 19 (38%) had XX chromosomes. Twenty-seven of type and location of the causative mutation (20,21). 53 (51%) were phenotypic male patients; of these, 96% had a disorder in their sex development (46 XY DSD) (Table 3). Three XX female patients had relevant urogenital malforma- Genotype/Phenotype Correlations fi tions (streak ovaries, bicornuate uterus). No statistically sig- Chernin et al. (19) were the rst to describe the effect of nificant correlation with genotype was observed. Still, three exon versus intron mutations on proteinuria, but the study of four XY patients with KTS splice site mutation had a sex does not provide detailed clinical data and does not dif- reversal, whereas only three of 21 with missense and none ferentiate between missense and truncating mutations. with truncation mutation had complete gonadal dysgenesis. Compared with the data by Lipska et al. (16), the fre- Renal malformations were reported in six patients; two of quency of exon mutations is higher in our cohort (66% fi fi these had urogenital sinus. Gonadoblastoma was diagnosed versus 83%). We could not con rm a signi cant effect of in 1 patient with KTS splice site mutation and sex reversal. missense mutations residing in DNA-binding regions compared with other parts of the WT1 gene. Nevertheless, we agree that in mutations outside the hot spot region, 8/9 Discussion seem to be associated with a milder renal phenotype. Data from our representative Mid European WT1 Muta- However, patients with mutations in exon 1 and 2 may tion Registry confirm that WT1 mutations are associated also be at risk for nephropathy later in life. 830 Clinical Journal of the American Society of Nephrology

Our study confirms the more rapid progression toward WT ESRD for patients with exon mutations compared with those Thirty-six percent of our cohort developed WT, similar to with KTS splice site mutations, as described by Chernin et al. previous studies, although our series includes slightly more (19). We documented a differential effect of type of exon patients with truncating mutations (88% versus 78%) (16). mutations on onset of proteinuria and renal failure, confirm- We confirm that truncating mutations were more likely to ing the findings of Lipska et al. (16). Patients with missense lead to bilateral tumors, whereas patients with missense mu- mutations in exon 8/9 tend to have an earlier onset of ne- tations tended to have a unilateral WT (16,21). Fifty-nine phropathy than patients with truncating or KTS splice site percent had no genetic testing at diagnosis of WT. mutations. It is hypothesized that the more severe renal phe- In our cohort, 78% of patients with WT (86% with unilateral notype in these patients can be explained by the production WT) developed ESRD, which is generally uncommon among of a dominant-negative mutant WT1 protein interfering with surviving patients with WT (27). As analyzed from data of the action of wild-type protein (22). the National Wilms Tumor Study Group and the US Renal Our data include the largest subgroup analysis published Data System in 2005, the 20-year cumulative incidence of so far of 14 patients harboring the most common WT1 mu- ESRD is ,1% for former patients with unilateral WT and tation in exon 9 c.1384C.T, p.Arg462Trp (previously re- , 12% for those with bilateral disease (28). Thus, we believe ferred to as R394). All patients with this mutation had it unlikely that, in patients bearing a WT1 truncating muta- nephrotic-range proteinuria, and only one patient was able tion, glomerular hyperfiltration secondary to surgically re- to avoid RRT until age 8 years. We describe a significant duced renal mass is the key factor leading to progressive phenotypic variability caused by the same single mutation kidney disease. Screening for WT1 mutation should be rec- regarding WT development and genitourinary malforma- ommended for all patients with WT. tions. Lipska et al. (16) included nine patients with this mutation but performed only a limited subanalysis. DSDs Our data illustrate that DSDs often are the first symptom Proteinuria of WT1 mutations in male patients. However, not only XY Proteinuria was the most common symptom in our patients but also XX individuals are at risk for genitourinary mal- (94%). Isolated proteinuria was present in 27%, compared formations. We postulate that XX girls are underdiagnosed with 28% (16) and 42% (19) reported elsewhere, primarily regarding genitourinary malformations, which could pos- affecting female patients; however, our series includes sibly affect fertility, and should receive more medical at- one boy. Currently, genetic testing for mutations in WT1 is tention in this respect. Patients with missense mutations recommended in sporadic cases of congenital or steroid- display more severe gonadal dysgenesis (29). Comprehen- resistant nephrotic syndrome with onset during infancy or sive data on sex hormones and surgical outcome (erectile childhood, especially in girls (23). The fact that our cohort dysfunction and cosmetic effects in addition to reproduc- features almost see twice as many XY as XX karyotypes tive capacity) are lacking. Numbers of children tested for suggests that we are possibly missing girls with WT1 muta- WT1 mutations because of DSD may increase in the future tion. Genetic testing should be considered in proteinuric if testing is done as suggested in the literature (29). girls even if they are not nephrotic. In familial cases of late-onset proteinuria without WT or XY DSD, dominant Classic Syndromal Definitions Revisited inheritance of WT1 mutations should be considered (24,25). If patients present with congenital nephrotic syndrome, Our cohort used a variety of therapeutic interventions, WT, and complete 46 XY gonadal dysgenesis, they meet the which were not reported by other series. For instance, 26% of classic definition of DDS. In our cohort, only one patient the proteinuric patients were treated with steroids; this mainly fulfilled all these criteria. Among our patients with intronic includes patients with noncongenital manifestation who were KTS splice site mutations and delayed manifestation of diagnosed in the 1990s, when knowledge about genetic causes nephropathy, only one patient showed the full classic of nephrotic syndrome was almost nonexistent. Our series syndromal picture of a FS. DDS and FS can be regarded as also includes three patients with KTS splice site mutation (one extremes of the spectrum of disease due to WT1 gene mu- with MCD and two with FSGS) who had a partial remission tations rather than as separate diseases (30). Patients with of their nephrotic syndrome under CsA treatment, as pub- truncating mutations do not clearly fit with either DDS or lished previously (26). Whether genotype or histology is FS. Classifying patients with WT1 mutations according to more potent for predicting success of CsA treatment is un- type and location of mutations might be superior to using known. New strategies in the treatment of WT1-associated classic syndromal definitions. proteinuria (e.g., angiotensin-converting enzyme inhibitors) may delay the development of ESRD. The number of avail- Conclusion and Outlook able renal histology results is similar to that reported by Clinicians should be aware of the type and location of Lipska et al. (16). However, in our cohort DMS was present heterozygous constitutional WT1 mutation in their pa- only in patients with missense mutations, whereas Lipska tients because correlations with manifestations of renal et al. (16) also reported DMS in 33% of patients with trun- phenotype and WT do exist. XY karyotype was more fre- cating mutations and one patient with KTS splice site mu- quent in our cohort, and most had urogenital malforma- tation. Compared with Lipska et al. (16), our patients with tions. WT1 mutations and urogenital abnormalities may be KTS splice site mutation less often had FSGS (88% versus missed in girls and should be considered in female pa- 56%), which may be related to differences in the geographic tients with proteinuria. Future prospective studies should and genetic background of patients as well as timing of test the effect of early genotyping and develop strategies to biopsies and variability of pathologic assessment. reduce proteinuria and slow progression of CKD. Clin J Am Soc Nephrol 10: 825–831, May, 2015 Report of the WT1-Registry of the GPN, Lehnhardt et al. 831

Acknowledgments A, Szczepanska M, Wasilewska A, Harambat J, Trautmann A, This study was performed for the German Society of Pediatric Peco-Antic A, Borzecka H, Moczulska A, Saeed B, Bogdanovic R, Nephrology as WT1-registry and supported by the charity foun- Kalyoncu M, Simkova E, Erdogan O, Vrljicak K, Teixeira A, ‘ ’ Azocar M, Schaefer F; PodoNet Consortium: Genotype- dation step4kids . phenotype associations in WT1 glomerulopathy. Kidney Int 85: 1169–1178, 2014 Disclosures 17. Lombel RM, Hodson EM, Gipson DS; Kidney Disease: Improving C.B. is an employee of Bioscientia, Ingelheim, Germany. Global Outcomes: Treatment of steroid-resistant nephrotic syndrome in children: New guidelines from KDIGO. Pediatr Nephrol 28: 409–414, 2013 References 18. Troyanov S, Wall CA, Miller JA, Scholey JW, Cattran DC; Toronto 1. 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J Clin Endocrinol Metab 96: E1131–E1136, 2011 13. Frasier SD, Bashore RA, Mosier HD: H.D., M: Gonadoblastoma 30. McTaggart SJ, Algar E, Chow CW, Powell HR, Jones CL: Clinical associated with pure gonadal dysgenesis in monozygotic twins. spectrum of Denys-Drash and Frasier syndrome. Pediatr Nephrol J Pediatr 64: 740–745, 1964 16: 335–339, 2001 14. Melo KF, Martin RM, Costa EM, Carvalho FM, Jorge AA, Arnhold IJ, Mendonca BB: An unusual phenotype of Frasier syndrome due Received: October 12, 2014 Accepted: January 20, 2015 to IVS9 +4C.T mutation in the WT1 gene: predominantly male ambiguous genitalia and absence of gonadal dysgenesis. J Clin Published online ahead of print. Publication date available at www. Endocrinol Metab 87: 2500–2505, 2002 cjasn.org. 15. Ismaili K, Verdure V, Vandenhoute K, Janssen F, Hall M: WT1 gene mutations in three girls with nephrotic syndrome. Eur J Pediatr 167: 579–581, 2008 This article contains supplemental material online at http://cjasn. 16. Lipska BS, Ranchin B, Iatropoulos P, Gellermann J, Melk A, asnjournals.org/lookup/suppl/doi:10.2215/CJN.10141014/-/ Ozaltin F, Caridi G, Seeman T, Tory K, Jankauskiene A, Zurowska DCSupplemental.

Supplemental table 1: Overview of underlying mutations and their primary literature report, renal phenotype, Karyotype (KT) and gender

Legend: Age at onset (dx) of proteinuria and Wilms Tumour (WT) unilateral (ul) or bilateral (bl). Immunosuppressive (IS) treatment yes (Y) or no (N), all were irresponsive to steroids, *partial remission with cyclosporin A (CsA), ** no response to CsA. One patient (+) was excluded from correlation analyses as he initially presented with HUS. One patient died aged 5 months (t). In one patient @ incidental finding of nephroblastomatosis WT stage 1 when prophylactic bilateral nephrectomy was performed, no chemotherapy. One patient § with concomitant heterozygous NPHS1 mutation was included in the analysis. 6 proteinuric patients with a median follow up of 7.4 years [5.4-15.4] did have unimpaired renal function. 3 patients without proteinuria did not have impaired renal function at the age of 4.2, 5.8 and 13.1 years.

Abbreviations: FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; DMS, diffuse mesangial sclerosis ! ! Patient Exon/ Mutation Protein Age at Age at dx Renal IS Age at start eGFR WT Age at dx KT/ Primary number Intron follow-up proteinuria Histology treatment RRT WT Gender literature (years) (months) (months) (months) report Missense mutations in DNA-binding region t 1 Exon 8 c.1300C>T p.Arg434Cys 0.4 1 FSGS N 1 - XY/f 1 2 Exon 8 c.1322A>G p.His441Arg 12.7 4 DMS N 13 - XX/f this study 3 Exon 8 c.1339G>T p.Gly447Cys 17.8 3 DMS Y 3 - XX/f 2 4 Exon 9 c.1384C>T p.Arg462Trp 1.1 0.1 - N - 60 - XY/m 3 5 Exon 9 c.1384C>T p.Arg462Trp 7.6 14 DMS N 14 ul 14 XX/f 3 6 Exon 9 c.1384C>T p.Arg462Trp 8.1 10 DMS N 13 ul@ 15 XY/m 3 7 Exon 9 c.1384C>T p.Arg462Trp 18.8 59 DMS N 59 - XY/m 3 8 Exon 9 c.1384C>T p.Arg462Trp 21.9 7 Other N 7 - XY/f 3 + 9 Exon 9 c.1384C>T p.Arg462Trp 6.7 7 DMS N 7 - XY/m 3 10 Exon 9 c.1384C>T p.Arg462Trp 11.7 49 FSGS Y 52 - XX/f 3 11 Exon 9 c.1384C>T p.Arg462Trp 0.3 1 - N - 15 - XX/f 3 12 Exon 9 c.1384C>T p.Arg462Trp 6.2 17 FSGS Y - !90 ul 61 XY/m 3 13 Exon 9 c.1384C>T p.Arg462Trp 3.2 8 DMS N 8 - XX/f 3 14 Exon 9 c.1384C>T p.Arg462Trp 13.9 22 DMS N 62 ul 22 XY/m 3 15 Exon 9 c.1384C>T p.Arg462Trp 15.8 7 DMS N 7 - - /f 3 16 Exon 9 c.1384C>T p.Arg462Trp 12.8 16 FSGS Y** 94 ul 16 XX/f 3 17 Exon 9 c.1384C>T p.Arg462Trp 19.3 5 DMS N 21 ul 4 XY/f 3 18 Exon 9 c.1385G>A p.Arg462Gln 16.8 0.1 FSGS N 4 - XY/m 1 19 Exon 9 c.1385G>A p.Arg462Gln 15.3 2 DMS N 5 - XY/m 1 20 Exon 9 c.1385G>A p.Arg462Gln 4.0 0.5 Other N 0,1 - XY/m 1 21 Exon 9 c.1385G>C p.Arg462Pro 6.6 7 DMS N 7 - XX/f 4 22 Exon 9 c.1385G>C p.Arg462Pro 4.3 1 - N 1 - XY/m 4 23 Exon 9 c.1390G>A p.Asp464Asn 0.9 3 FSGS N 17 - XY/m 3 24 Exon 9 c.1390G>A p.Asp464Asn 9.2 19 DMS N 47 bl 16 XX/f 3 25 Exon 9 c.1390G>A p.Asp464Asn 1.0 4 DMS N 4 - XY/m 3 26 Exon 9 c.1390G>C p.Asp464His 0.9 3 DMS N 4 bl 3 XY/m 5 27 Exon 9 c.1391A>G p.Asp464Gly 2.4 4 - N 12 - ul 12 XX/f 3 Patient Exon/ Mutation Protein Age at Age at dx Renal IS Age at start eGFR WT Age at dx KT/ Primary number Intron follow-up proteinuria Histology treatment RRT if no WT Gender literature (years) (months) (months) RRT (months) report 28 Exon 9 c.1394A>C p.His465Pro 11.9 63 DMS N 63 - ul 18 XX/f 6 29 Exon 9 c.1394A>C p.His465Pro 12.1 15 FSGS Y 112 - - XX/f 6 Missense mutations in other regions 30 Exon 2 c.745C>T p.Pro249Ser 13.2 - - N - !90 - - XY/m 7 31 Exon 7 c.1220A>G p.His407Arg 17.7 198 FSGS N - 60 - - - /m this study 32 Exon 8 c.1008 G>T p.Gln369His 14.6 0.1 DMS N 35 - - - XY/m this study 33 Exon 8 c.1289G>C p.Arg430Pro 8.5 21 DMS Y** 21 - - - XY/m this study 34 Exon 9 c.1357T>C p.Cys453Arg 4.3 7 DMS N 15 - - - XY/m 8 35 Exon 9 c.1357T>C p.Cys453Arg 25.4 35 Other N 50 - ul 35 XY/m 8 36 Exon 9 c.1366T>C p.Cys456Arg 9.3 1 DMS N 20 - ul 18 XX/f 6 Truncating mutations 37 Exon 1 c.369_385del p.Ala123fs*89 4.3 - - N - !90 bl 8 XY/m this study 38 Exon 1 c.525T>A p.Cys175* 5.8 - - N - !90 bl 12 XY/m this study 39 Exon 8 c.1288C>T p.Arg430* 23.1 142 FSGS N 201 - bl 11 XY/m 9 40 Exon 8 c.1288C>T p.Arg430* 19.1 147 FSGS N 197 - bl 14 XX/f 9 41 Exon 9 c.1372C>T p.Arg458* 16.6 94 - N 199 - bl 10 XY/m 7 42 Exon 9 c.1372C>T p.Arg458* 30.4 138 FSGS N - 58 - - XY/m 7 43 Exon 9 c.1372C>T p.Arg458* 22.1 38 FSGS N 193 - ul 22 - /m 7 44 Exon 9 c.1372C>T p.Arg458* 12.4 79 Other N - 51 ul 16 XY/m 7 Splice site mutations 45 Intron 9 IVS9+4C>T (c.1432+4C>T) 26.1 116 Other N 137 - - - XY/f 10 46 Intron 9 IVS9+4C>T (c.1432+4C>T) 21.8 47 MCD Y* - !90 - - XX/f 10 47 Intron 9 IVS9+4C>T (c.1432+4C>T) 9.2 8 FSGS Y* - 50 - - XY/f 10 48 Intron 9 IVS9+4C>T (c.1432+4C>T) 8.5 75 FSGS N - !90 - - XY/m 10 49 Intron 9 IVS9+4C>T (c.1432+4C>T) 43.1 83 FSGS Y 156 - - - XX/f 10 50 Intron 9 IVS9+5G>A (c.1432+5G>A) 21.0 35 FSGS Y* 218 - - - XX/f 4 51 Intron 9 IVS9+5G>A (c.1432+5G>A) 15.0 48 MCD Y - 46 - - XY/f 4 52 Intron 9 IVS9+5G>A (c.1432+5G>A) 21.0 66 MCD Y - 50 - - XX/f 4 § 53 Intron 9 IVS9+5G>A (c.1432+5G>A) 41.0 27 FSGS Y 95 - - - XX/f 4 !"#"$"%&"'(#)$(*+,,-"."%/0-(/01-"(2( ( (1) Jeanpierre, C. et al. Identification of constitutional WT1 mutations, in patients with isolated diffuse mesangial sclerosis, and analysis of genotype/phenotype correlations by use of a computerized mutation database. Am. J. Hum. Genet. 62, 824–833 (1998). (2) Schumacher, V. et al.Spectrum of early onset nephrotic syndrome associated with WT1 missense mutations. Kidney Int. 53, 1594–1600 (1998). (3) Pelletier, J. et al. Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys- Drash syndrome.Cell 67, 437–447 (1991). (4) Bruening, W. et al. Germline intronic and exonic mutations in the Wilms’ tumour gene (WT1) affecting urogenital development. Nat. Genet. 1, 144–148 (1992). (5) Hakan, N. et al. A novel WT1 gene mutation in a newborn infant diagnosed with Denys-Drash syndrome. Genet. Couns.23, 255–261 (2012). (6) Ruf, R. G. et al. Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome. Kidney Int. 66,564–570 (2004). (7) Schumacher, V. et al.Correlation of germ-line mutations and two-hit inactivation of the WT1 gene with Wilms tumors of stromal- predominant histology. Proc. Natl. Acad. Sci. U.S.A. 94, 3972–3977 (1997). (8) Kikuchi, H. et al. Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome? J. Med. Genet. 35, 45–48 (1998). (9) Clarkson, P. A. et al.Mutational screening of the Wilms’s tumour gene, WT1, in males with genital abnormalities. J. Med. Genet. 30, 767– 772 (1993). (10) Barbaux, S. et al. Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nat. Genet. 17,467–470 (1997). ( Supplemental table 2: Subgroup analysis of 14 patients with missense mutation c.1384C>T leading to p.Arg462Trp ! Age at follow up 9.9 (5.5-16.6) years

Nephrotic range proteinuria 14/14 (100%)

Age at diagnosis of proteinuria 9 (6.5-18.3) months

Renal replacement treatment 11/14 (79%)

Age at start of RRT 14 (7-59) months

Renal histology DMS 7/14 (50%)

FSGS 3/14 (22%)

Wilms Tumour (unilateral) 6/14 (43%)

Age at diagnosis of WT 15.5 (12-32) months

Karyotype/gender XY/m 6/14 (43%)

XY/f 2/14 (14%)

XX/f 5/14 (36%)

?/f 1/14 (7%)

XY DSD

Cryptorchidism 1/8 (12.5%)

Cryptorchidism, glandular 1/8 (12.5%) hypospadia 4/8 (50%) Cryptorchidism, penoscrotal hypospadias, +/-micropenis 2/8 (25%9 Sex Reversal (complete gonadal dysgenesis)

XX females with streak ovaries 1/5 (20%)

! Supplemental table 3: Allele frequencies and prediction of pathogenicity of WT1 mutations

SIFT PolyP.-2 PolyP.-2 FATHMM FATHMM VEST CADD Patient number Genomic Position CDC Protein TGP AF ESP AF CG69 AF NCI-60 AF GoNL AF ExAC AF SIFT Pred. VEST Pred. CADD Pred. Score HumVar Score HumVar Pred. Score Pred. Score Score

Missense mutations in DNA-binding region Probably 132414251c.1300C>Tp.Arg434Cys0.00 %0.00 %0.00 %0.00 % 0.00 % 0.00 % 0.01 Damaging 1 1.26 Tolerated 0.969 Damaging 27.4 Pathogenic Damaging Probably 232414229c.1322A>Gp.His441Arg0.00 %0.00 %0.00 %0.00 % 0.00 % 0.00 % 0 Damaging 0.999 -2.18 Damaging 0.963 Damaging 25.9 Pathogenic Damaging Probably 332414212c.1339G>Tp.Gly447Cys0.00 %0.00 %0.00 %0.00 % 0.00 % 0.00 % NA NA 0.997 1.72 Tolerated 0.923 Damaging 33 Pathogenic Damaging 4 ; 5 ; 6 ; 7 ; 8 ; 9 ; 10 ; 11 ; Probably 32413566 c.1384C>T p.Arg462Trp 0.00 % 0.00 % 0.00 % 0.00 %0.00 %0.00 %0.01Damaging1 1.95Tolerated0.953Damaging23Pathogenic 12 ; 13 ; 14 ; 15 ; 16 ; 17 Damaging Probably 18 ; 19 ; 20 32413565 c.1385G>A p.Arg462Gln 0.00 % 0.00 %0.00 %0.00 %0.00 %0.00 %0.29Tolerated0.998 1.982Tolerated 0.859 Tolerated 36 Pathogenic Damaging Probably 21 ; 22 32413565 c.1385G>C p.Arg462Pro 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0 Damaging 1 1.95 Tolerated 0.983 Damaging 34 Pathogenic Damaging Probably 23 ; 24 ; 25 32413560 c.1390G>A p.Asp464Asn 0.00 % 0.00 %0.00 %0.00 %0.00 %0.00 %0.02Damaging0.944 1.992Tolerated 0.102 Tolerated 36 Pathogenic Damaging Probably 26 32413560 c.1390G>C p.Asp464His 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.02 Damaging 0.993 2.338 Tolerated 0.961 Damaging 31 Pathogenic Damaging 27 32413559 c.1391A>G p.Asp464Gly 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.17 Tolerated 0.198 Benign 2 Tolerated 0.979 Damaging 26.3 Pathogenic Probably 28 ; 29 32413556 c.1394A>C p.His465Pro 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0 Damaging 0.999 1.61 Tolerated 0.973Damaging25.7Pathogenic Damaging

Missense mutations in other regions

30 32450067 c.745C>T p.Pro249Ser 0.04 % 0.04 % 0.00 % 0.00 % 0.10 % 0.04 % 0.32 Tolerated 0.016 Benign -1.84 Damaging 0.729 Tolerated 20.9 Pathogenic Possibly 31 32417832 c.1220A>G p.His407Arg 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.02 Damaging 0.786 1.32 Tolerated 0.851 Tolerated 32 Pathogenic Damaging 32 32421584 c.1008C>A p.Ser336Arg 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.42 Tolerated 0.065 Benign -1.94 Damaging0.304Tolerated13.34 Nonpathogenic Probably 33 32414262 c.1289G>C p.Arg430Pro 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0 Damaging 0.954 2.12 Tolerated 0.974 Damaging 32 Pathogenic Damaging Probably 34 ; 35 32413593 c.1357T>C p.Cys453Arg 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0 Damaging 1 -1.96 Damaging 0.995 Damaging 24.7 Pathogenic Damaging Probably 36 32413584 c.1366T>C p.Cys456Arg 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0 Damaging 0.999 -2.05 Damaging 0.992 Damaging 26.6 Pathogenic Damaging

Truncating mutations c.369_385delGCGGAG 37 32456507 p.Ala123fs 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % NA NA NANANANANANANANA CCGGTGGCGGC 38 32456367 c.525T>A p.Cys175* 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 1 Tolerated NA NA NA NA NA NA 39 Pathogenic

39 ; 40 32414263 c.1288C>T p.Arg430* 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 1 Tolerated NA NA NA NA NA NA 40 Pathogenic

41 ; 42 ; 43 ; 44 32413578 c.1372C>T p.Arg458* 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 0.00 % 1 Tolerated NA NA NA NA NA NA 40Pathogenic All cDNA and protein positions are relative to transcript NM_024426.4 TGP AF = 1000 genomes project allele frequency ESP AF = Exome Sequencing Project allele frequency CG69 AF = Complete Genomics sequencing project allele frequency NCI-60 AF = National Cancer Institute sequencing project allele frequency GoNL AF = Genomes of the Netherlands sequencing project allele frequency ExAC AF = Exome Aggregation Consortium allele frequency

SIFT = Functional consequence prediction tool, Damaging: [0 ! Score ! 0.05]; Tolerated [0.06 ! Score ! 1] PolyPhen-2 = Functional consequence prediction tool, Probably Damaging: [0.909 ! Score ! 1]; Possibly Damaging: [0.447 ! Score ! 0.908]; Benign: [0 ! Score ! 0.446] FATHMM = Functional consequence prediction tool. Damaging: [-18.09 ! Score < -1.5]; Tolerated: [-1.15 ! Score ! 11.00] VEST = Functional consequence prediction tool. [0 ! Score ! 1]. CADD = Functional consequence prediction tool. Non-pathogenic: [0 ! Score ! 15]; Pathogenic: [15 < Score ! 99] Anja Lehnhardt 1, Claartje Karnatz 1, Thurid Ahlenstiel-Grunow2, Kerstin Benz3, Marcus R. Benz4, Klemens Budde5, Anja K. Büscher6, Thomas Fehr7, Markus Feldkötter8, Norbert Graf9, Britta Höcker10, Therese Jungraithmayr11, Günter Klaus12, Birgit Koehler13, Martin Konrad14, Birgitta Kranz14, Carmen R. Montoya15, Dominik Müller16, Thomas J. Neuhaus17, Jun Oh1, Lars Pape2, Martin Pohl18, Brigitte Royer-Pokora19, Uwe Querfeld16, Reinhard Schneppenheim20, Hagen Staude21, Giuseppina Spartà22, Kirsten Timmermann23, Frauke Wilkening24, Simone Wygoda25, Carsten Bergmann26,27, Markus J. Kemper1

1Pediatric Nephrology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Pediatric Nephrology, Hannover Medical School, Hannover, Germany; 3Pediatric Nephrology, Universitätsklinikum Erlangen, Erlangen, Germany; 4 Pediatric Nephrology, Dr. von Haunersches Kinderspital, University Children’s Hospital, Ludwig-Maximilians Universität München, Munich, Germany; 5Department of Nephrology, Charité Universitätsmedizin Berlin, Berlin, Germany; 6,University Hospital Essen, Essen, Germany; 7Division of Nephrology, University Hospital Zürich, Zurich, Switzerland; 8Pediatric Nephrology, Universität zu Köln, Cologne, Germany; 9Department of Pediatric Oncology and Hematology, Saarland University, Homburg, Germany. 10Department of Pediatrics I, University Childrens Hospital, Heidelberg, Germany; 11Pediatric Nephrology, Innsbruck Medical University, Innsbruck, Austria; 12KfH Pediatric Kidney Centre, Marburg, Germany; 13Pediatric Endocrinology, University Childrens Hospital, Charitè, Humbold University, Berlin Germany; 14Department of General Pediatrics, University Childrens’s Hospital, Münster, Germany; 15Pediatric Nephrology, Klinikum München-Schwabing, Munich, Germany; 16Department of Pediatric Nephrology, Charité, Berlin, Germany; 17Department of Pediatrics, Kinderspital Luzern, Luzern, Switzerland; 18Pediatric Nephrology, Department of Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg, Germany; 19Institute of Human Genetics and Anthropology, Heinrich- Heine University of Düsseldorf, Düsseldorf, Germany; 20Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 21Pediatric Nephrology, University of Rostock, Rostock, Germany; 22Pediatric Nephrology, University Children’s Hospital, Zürich, Switzerland; 23Pediatric Nephrology, Olgahospital Stuttgart, Stuttgart, Germany; 24Pediatric Nephrology, Helios Kliniken Schwerin, Schwerin, Germany; 25Pediatric Nephrology, St. Georg Hospital, Leipzig, Germany; 26Bioscientia Center for Human Genetics, Ingelheim, Germany; 27Department of Nephrology&Center for Clinical Research, University Hospital Freiburg, Germany