Article

Reverse Phenotyping after Whole-Exome Sequencing in Steroid-Resistant Nephrotic Syndrome

Samuela Landini,1,2,3 Benedetta Mazzinghi ,4 Francesca Becherucci ,4 Marco Allinovi,2,3 Aldesia Provenzano ,1,3 Viviana Palazzo,1 Fiammetta Ravaglia,4 Rosangela Artuso ,1 Emanuele Bosi ,2 Stefano Stagi ,5 Giulia Sansavini,4 Francesco Guzzi ,2,3,4 Luigi Cirillo ,4 Augusto Vaglio ,2,3,4 Luisa Murer ,6 Licia Peruzzi ,7 Andrea Pasini ,8 Marco Materassi,4 Rosa Maria Roperto,4 Hans-Joachim Anders ,9 Mario Rotondi ,10 Sabrina Rita Giglio ,1,2,3 and Paola Romagnani 2,3,4 Due to the number of contributing authors, Abstract the affiliations are Background and objectives Nephrotic syndrome is a typical presentation of genetic podocytopathies but listed at the end of occasionally other genetic nephropathies can present as clinically indistinguishable phenocopies. We hypoth- this article. esized that extended genetic testing followed by reverse phenotyping would increase the diagnostic rate for these Correspondence: patients. Prof. Paola Romagnani or Prof. Design, setting, participants, & measurements All patients diagnosed with nephrotic syndrome and referred to our Sabrina Giglio, center between 2000 and 2018 were assessed in this retrospective study. When indicated, whole-exome sequencing Department of fi Clinical and and in silico ltering of 298 related to CKD were combined with subsequent reverse phenotyping in patients Experimental and families. Pathogenic variants were defined according to current guidelines of the American College of Medical Biomedical Sciences Genetics. “Mario Serio”, University of Results A total of 111 patients (64 steroid-resistant and 47 steroid-sensitive) were included in the study. Not a Florence, Viale Pieraccini 6, 50139, single pathogenic variant was detected in the steroid-sensitive group. Overall, 30% (19 out of 64) of steroid- Florence, Italy. resistant patients had pathogenic variants in podocytopathy genes, whereas a substantial number of variants E-mail:paola. were identified in other genes, not commonly associated with isolated nephrotic syndrome. Reverse romagnani@unifi.it or phenotyping, on the basis of a personalized diagnostic workflow, permitted to identify previously unrecognized sabrina.giglio@ meyer.it clinical signs of an unexpected underlying genetic nephropathy in a further 28% (18 out of 64) of patients. These patients showed similar multidrug resistance, but different long-term outcome, when compared with genetic podocytopathies.

Conclusions Reverse phenotyping increased the diagnostic accuracy in patients referred with the diagnosis of steroid-resistant nephrotic syndrome. CJASN 15: 89–100, 2020. doi: https://doi.org/10.2215/CJN.06060519

Introduction nephropathies outside the podocytopathy spectrum Isolated nephrotic syndrome is classified, according (e.g., Alport syndrome, Dent disease, or Fabry to the response to steroids, as steroid-sensitive or disease) are usually recognized upon standard diag- steroid-resistant nephrotic syndrome (1,2). Although nostic work-up. However, steroid-resistant nephrotic steroid-sensitive nephrotic syndrome usually has a syndrome can rarely be the only evident clinical sign, favorable prognosis, steroid-resistant nephrotic at disease onset or even later (14–18). When this syndrome can progress to ESKD (3–7). Indeed, happens, these genetic nephropathies are often mis- despite some patients’ response to immunosuppres- classified and treated as isolated steroid-resistant sion in terms of proteinuria reduction, many are nephrotic syndrome when genetic testing for com- multidrug resistant (6). Genetic testing using ex- monly reported disease-causing genes (i.e., podocyt- tended panels of podocytopathy genes has become a opathy genes) proves to be negative. These conditions valuable diagnostic tool to identify monogenic are referred to as phenocopies of monogenic podocyto- podocytopathies, which account for about 30% of pathies (19–25). A phenocopy is defined as “a pheno- patients affected by steroid-resistant nephrotic syn- typic trait or disease that resembles the trait expressed drome (5,8–12). In addition, a recent report sugges- by a particular genotype, but in an individual who is ted that steroid-sensitive nephrotic syndrome may not a carrier of that genotype” (26). Recently, by also occasionally be of genetic origin (13). All other applying whole-exome sequencing, Warejko et al.(27) cases of isolated nephrotic syndrome are usually reported the diagnosis of a phenocopy in 5% of patients assumed to be of nongenetic cause. Genetic with steroid-resistant nephrotic syndrome. We www.cjasn.org Vol 15 January, 2020 Copyright © 2020 by the American Society of Nephrology 89 90 CJASN

252 patients diagnosed with nephrotic syndrome and referred to our center between January 2000 and December 2018

47 patients presenting with: - clinical, laboratory or biopsy signs of an immune-mediated disease, n=17 - macroscopic hematuria, predominant tubular proteinuria, n=2 - syndromic nephrotic syndrome and/or extra-renal involvement, n=4 - known family history of nephrotic syndrome, n=10 - parents DNA untraceable, n=14

205 evaluated patients

141 steroid-sensitive nephrotic syndrome

94 patients affected by unfrequently relapsing and not steroid-dependent nephrotic syndrome

64 47 steroid-resistant steroid-sensitive nephrotic syndrome nephrotic syndrome

111 patients sent to genetic analysis

Figure 1. | Flowchart for the selection of 111 patients included in the study. hypothesized that establishing standardized criteria to define nephrologists and from medical records. Inclusion criteria podocytopathy versus phenocopy genes, and adding re-eval- were onset of symptoms before 30 years of age and a uation of patients and their family (i.e., “reverse phenotyp- clinical diagnosis of nephrotic syndrome (e.g.,nephrotic ing”) after genetic testing, could correctly segregate the range proteinuria, hypoalbuminemia, edema) or nephrotic identified genetic variants to previously unrecognized range proteinuria with kidney histology of FSGS, minimal clinical symptoms and increase the sensitivity of the genetic change disease, or diffuse mesangial sclerosis. Exclusion analysis. criteria were (1) evidence of clinical, laboratory, or kidney biopsy signs of an immune-mediated disease; (2)macro- scopic hematuria or predominantly tubular proteinuria Materials and Methods (low-molecular-weight .50% according to urine Patients electrophoresis) (28); (3) syndromic nephrotic syn- All consecutive patients diagnosed with nephrotic syn- drome and/or presence of extrarenal signs or symptoms drome and referred to the Nephrology Unit of the Meyer (e.g., sensorineural hearing loss, ocular abnormalities); (4) Children’s University Hospital of Florence from January known family history of nephrotic syndrome; and (5) 2000 to December 2018 were assessed for inclusion in this untraceable parents’ DNA (Figure 1). The remaining pa- retrospective study (Figure 1, Supplemental Table 1). tients were then subclassified in steroid-resistant or steroid- Participants were followed from the day of referral until sensitive nephrotic syndrome. The majority of patients 31 December 2018, with no loss to follow-up. Demographic, with steroid-resistant nephrotic syndrome were diagnosed clinical, and laboratory data were retrospectively collected in another nephrology center and subsequently referred to from direct interview of patients, families, and referring our hospital, therefore detailed laboratory information at CJASN 15: 89–100, January, 2020 Reverse Phenotyping in Nephrotic Syndrome, Landini et al. 91

disease onset was not always available. Those affected count approach as detailed in Supplemental Appendix 1. by congenital nephrotic syndrome, or with a histologic Synonymous variants and intronic variants that were not diagnosis of diffuse mesangial sclerosis, or advanced kidney located within splice site regions were excluded. Vari- failure at diagnosis were considered comparable with ants were confirmed in the patients’ and families’ DNA patients who were steroid-resistant, although not treated by Sanger sequencing. with steroids. Among patients who were steroid-sensitive, only frequently relapsing or steroid-dependent patients Definition of Podocytopathy and Phenocopy Genes were selected for genetic testing and inclusion in the study. We systematically defined podocytopathy versus phe- First-degree relatives were either included before the study nocopy genes on the basis of Online Mendelian Inheri- fi or asked to participate after the identi cation of potentially tance in Man (OMIM; https://www.omim.org). causative variants in the patient. The local Ethics Accordingly, genes identified in OMIM as causing “ne- ’ Committee of the Meyer Children s University Hospital of phrotic syndrome” or “FSGS” were considered “podo- Florence approved the study. The study was conducted cytopathy genes.” In contrast, genes identified in OMIM according to the Declaration of Helsinki. A clinical geneticist to cause a syndromic disorder, with nephrotic syndrome counseled all patients and their families regarding the beingonlyoneamongmanyotherclinicalsignsoreven whole-exome sequencing procedure, and all participants not mentioned at all, were considered “phenocopy genes.” or their legal guardians gave written informed consent. Recently published literature indicating a causative path- ogenic role for a gene in nephrotic syndrome was also Sequencing and Bioinformatic Analysis considered to stratify cases as podocytopathy versus pheno- We evaluated whole-exome sequencing data for causa- copy when not yet reported in OMIM (37). Patients tive in 298 genes reported as even rare cause of carrying pathogenic variants in phenocopy genes and steroid-resistant nephrotic syndrome or CKD (Supplemen- their families underwent reverse phenotyping. tal Table 2). Genomic DNA was isolated from blood lymphocytes and sequenced with the platform NextSeq500 Reverse Phenotyping (Illumina Inc., San Diego, CA); reads were aligned with the To perform reverse phenotyping, we established a mul- – human reference hg19 genome using Burrows Wheeler tidisciplinary team composed of at least one geneticist, one Aligner (29), mapped and analyzed with the Integrative nephrologist and one nephropathologist, discussing clini- Genome Viewer software (2013 Broad Institute) (30). cal cases of patients undergoing whole-exome sequencing Downstream alignment processing (i.e.,alignmentsorting, during regularly scheduled meetings. Patients with muta- indexing, deduplication, and base quality score recalibra- tions in phenocopy genes and their families were thor- tion) was performed with the Genome Analysis Toolkit oughly revised, also with the help of external consultants, fi Uni ed Genotyper Module (GATK) (31), SAMtools (32), according to the clinical suspicion raised by the genetic and Picard Tools (http://picard.sourceforge.net/). The test. As a general procedure, the geneticist illustrated the fi GATK Uni ed Genotyper was used to obtain a set of suspicious variant(s) and the related existing literature. The single nucleotide variants and indel calls. We performed a nephrologist proposed a list of clinical exams and specialist fi hard ltering of called variants on the basis of four criteria: consultations to detect overlooked signs or symptoms of fi (1) the variant con dence (QUAL), that is, the Phred-scaled the syndromic genetic disorder suggested by DNA anal- posterior probability of the variant to be homozygous ysis. In addition, the nephropathologist proposed if and reference; (2) the quality by depth (QD), that is, the QUAL how to re-evaluate the kidney biopsy looking for specific fi divided by the un ltered depth (DP); (3) the DP, that is, the signs of the suspected genetic nephropathy, eventually number of reads spanning the variant site; and (4)the including further staining. strand bias (FS), measured as the Phred-scaled P value, using Fisher exact test to detect FS (the variation being seen on only either the forward or the reverse strand) in the Statistical Analyses Statistical analysis was performed using SPSS software reads. We excluded the variant calls for which at least one (SPSS, Inc., Evanston, IL). All continuous data are ex- of these filters was true: QD,5.0, DP,5, FS.60.0, pressed as median and interquartile range. QUAL,30.0. Variants were annotated using Annovar Kaplan–Meier estimates were used to generate an overall tool (33) to obtain information such as variants frequency survival curve for the development of ESKD and differ- in different populations. Variants were bioinformatically ences among groups were assessed by log-rank test. A P prioritized by using the diagnostic workflow shown in value ,0.05 was considered statistically significant. Length Figure 2 and detailed in Supplemental Appendix 1, in of follow-up was compared between groups using one-way accordance with the American College of Medical Ge- ANOVA. netics and Genomics guidelines (34). Probable effect on the function of the encoded protein was assessed using Polyphen-2, SIFT, Taster, Mutation Assessor, Results FATHMM, and FATHMM MKL. Mutations were desig- Whole-Exome Sequencing Identifies Heterogeneous nated as pathogenic on the basis of the criteria reported Genotypes in Patients with Idiopathic Steroid-Resistant in Figure 2 and Supplemental Table 3. We used manual Nephrotic Syndrome inspection for the P.[Arg229Gln] variant in NPHS2 when A total of 252 consecutive patients were assessed segregating with variants localized in exon 7 and 8 (35) for inclusion. A total of 111 patients affected by isolated and for APOL1 G1 and G2 risk alleles (36). To estimate nephrotic syndrome were selected for final analysis (Figure copy number variations we used a normalized read 1, Supplemental Table 1, Table 1). The baseline characteristics 92 CJASN

Whole-exome sequencing in 111 nephrotic syndrome patients and in silico analysis of 298 genes

Criteria for selection of variants: • never reported or with a frequency ≤0.01 for autosomal recessive- and with a frequency ≤0.001 for autosomal dominant-transmitted genes in: - population databases - in-house exome control cohort (300 exomes, 600 alleles) • segregation within the family or de novo • at least three software predicting pathogenicity

78 selected variants 18 variants of unknown significance

60 selected variants: • 60 variants in steroid-resistant nephrotic syndrome patients • 0 variants in steroid-sensitive nephrotic syndrome patients

Variants in podocyte genes giving Variants in syndromic genes rarely presenting with isolated isolated steroid-resistant nephrotic steroid-resistant nephrotic syndrome and/or apparently not syndrome segregating in the family segregating in the family

32 pathogenic variants in 19 steroid- 28 variants in 18 steroid-resistant nephrotic syndrome patients resistant nephrotic syndrome patients

Multi-disciplinary board discussion Diagnosis of (geneticist, nephrologist and nephropathologist monogenic and consultants when appropriate) podocytopathies

Reverse phenotyping of patients and family members:

Clinical monitoring of steroid- • search for clinical findings according to genetic results resistant nephrotic syndrome - sensorineural hearing loss and ocular abnormalities and associated risk conditions - kidney imaging (ultrasound and scintigraphy) in the patient - assays, X-rays, etc. • kidney biopsy re-evaluation • parents and first-degree relatives’ comprehensive assessment

Diagnosis of phenocopies of monogenic podocytopathies

Clinical monitoring of steroid- resistant nephrotic syndrome and of the underlying syndrome in the patient and the family

Figure 2. | Reverse phenotyping after genetic testing doubled diagnostic rate in steroid-resistant nephrotic syndrome patients. of 64 patients with steroid-resistant nephrotic syndrome patients with steroid-resistant nephrotic syndrome in “podo- and 47 patients with steroid-sensitive nephrotic syndrome cytopathy genes,” identified in OMIM as causing “nephrotic are shown in Table 1 and detailed in Supplemental Table 1. syndrome” or “FSGS.” In addition, 28 variants in genes that After the diagnostic workflow shown in Figure 2, we did not apparently fit the clinical phenotype were identified in identified 32 pathogenic variants in 19 out of 64 (30%) 18 out of 64 (28%) patients. These genes included those CJASN 15: 89–100, January, 2020 Reverse Phenotyping in Nephrotic Syndrome, Landini et al. 93

Table 1. Baseline characteristics of the study population

Steroid-Sensitive Steroid-Resistant Characteristics Total (n=111) Nephrotic Syndrome (n=47) Nephrotic Syndrome (n=64)

Age at onset, yr 4 [3–8] 5 [3–9] 5 [3–8] Sex (male) 30/47 (64%) 32/64 (50%) 62/111 (56%) Ethnicity (European) 34/47 (72%) 60/64 (94%) 94/111 (85%) Clinical onset Nephrotic-range proteinuria 45/45 (100%) 53/53 (100%) 98/98 (100%) Serum albumin, g/dl 2.0 [1.6–2.4] 2.2 [1.9–2.7] 2.1 [1.8–2.6] Hypoalbuminemia, #3g/dl 41/45 (91%) 42/48 (87%) 83/93 (89%) Dyslipidemia 28/30 (93%) 36/44 (81%) 64/74 (86%) Edema 31/36 (86%) 29/54 (54%) 60/90 (67%) Histopathological findings FSGS 1/5 (20%) 43/59 (73%) 44/64 (69%) Minimal change disease 4/5 (80%) 14/59 (24%) 18/64 (28%) Diffuse mesangial sclerosis 0/5 (0%) 2/59 (3%) 2/64 (3%) Length of follow-up, yr 7 [5–11] 7 [4–11] 7 [5–11]

Continuous variables are presented as median [interquartile range] and categorical variables are presented as n (%). Dyslipidemia was defined as increased cholesterol and/or triglycerides levels according to age-adjusted reference values of each laboratory.

already indicated as “phenocopy genes” by Warejko et al. (27), (pediatric) nephrology unit distributed all over Europe before as well as all other genes identified in OMIM with the name of referral to our hospital. All 18 out of 64 phenocopy patients the underlying disease, but where nephrotic syndrome is showed specific extrarenal involvement in the patient (cases rare and/or represents only one of the many possible 1, 5, and 8–18, Table 2) or in first-degree relatives (cases 1–8 symptoms of a syndromic disorder. Moreover, ten out of and 17, Table 2), thus confirming the diagnosis upon reverse 64 (16%) patients with steroid-resistant nephrotic syndrome phenotyping (Figure 3B). FSGS was the most common histo- carried variants of unknown significance (Supplemental logic pattern in all groups (Supplemental Table 1), making Table 4). Because this group potentially included unrecog- kidney biopsy unremarkable in distinguishing patients be- nized genetic patients, it was considered separately being longing to different genetic groups. referred as “undefined.” Only 17 out of 64 (26%) patients with steroid-resistant nephrotic syndrome were negative upon Genetic Patients Are Multidrug Resistant and do Not Relapse genetic analysis. Finally, none of the patients with steroid- after Transplantation sensitive nephrotic syndrome showed pathogenic variants at We next analyzed how treatment response and long- fi genetic analysis. Genetic pro les of patients showing path- term kidney outcome related to the genetic diagnosis in ogenic variants are detailed in Supplemental Table 5. patients with steroid-resistant nephrotic syndrome. Strik- ingly, complete remission occurred exclusively in nega- Reverse Phenotyping tive patients (eight out of 17, 47%; Table 4) and had been Reverse phenotyping was performed after obtaining the induced by calcineurin inhibitors in five out of ten negative genetic testing results, which was on average 561years patients treated (50%; Table 4). By contrast, 13 out of 14 after disease onset. Clinical reassessment of the 18 patients (93%) phenocopies and podocytopathies were resistant to with unexpected genetic findings and their families led to calcineurin inhibitor treatment, and only one patient the identification of minor or overlooked signs referable to showed partial response (Table 4). At Kaplan–Meier anal- the genetic diseases suggested by whole-exome sequenc- ysis, patients affected by monogenic podocytopathies had a ing, as summarized in Table 2. Those signs and symptoms 10-year kidney survival rate of 38% (Figure 4). Undefined were either present at the time of referral or appeared later patients also showed a poor outcome, with a 10-year kidney on, during follow-up. Reverse phenotyping allowed correct survival rate of only 20%. By contrast, negative patients and identification of six patients with Alport syndrome, three phenocopy patients showed a 10-year survival rate of 66% patients with Dent disease, two patients with Papillo-renal and 75%, respectively (Figure 4). Finally, one major problem syndrome, one patient with cystinosis, one patient with of patients with steroid-resistant nephrotic syndrome is Fabry disease, and five patients with other rare monogenic relapse of the disease after kidney transplantation (38). In diseases (Table 2) who had been misdiagnosed and treated our cohort, none of the patients with a clear genetic diagnosis for isolated steroid-resistant nephrotic syndrome. Clinical developed disease recurrence after transplantation (zero out reassessment of all 19 patients with pathogenic variants in of 11 patients, zero out of 15 grafts). By contrast, approxi- podocytopathy genes was unremarkable instead, as expected mately 40% of negative and undefined patients developed (Supplemental Tables 3 and 5, Table 3). disease recurrence (four out of ten patients, five out of 11 Plotting age, proteinuria, and blood albumin levels at the grafts). Together, phenocopy patients, as well as podocyto- time of referral to our hospital resulted in a large overlap pathies, do not benefit from immunosuppressive treatment. that made single patients indistinguishable (Figure 3A). Overall, 84% of patients with steroid-resistant nephrotic syndrome had been diagnosed and treated in at least another 94 CJASN

Table 2. Summary of the results of whole-exome sequencing and reverse phenotyping in phenocopy patients

Pre Whole-Exome Post Whole-Exome Phenotype Reverse Phenotyping Patient Sex Ethnicity Sequencing Gene Variant Sequencing OMIM Diagnosis Diagnosis/ Diagnosis Number (#) Biopsy Results

Case 1 F Senegalese SRNS/FSGS COL4A4 APOL1 Hom Comp het - SNHL not present at Alport syndrome 203780 onset and diagnosed by rechecking WES results - Father with mild proteinuria and microscopic hematuria - Mother with microscopic hematuria Confirmed Alport syndrome Case 2 F European SRNS/FSGS COL4A4 Comp het - Parents with microscopic Alport syndrome 203780 hematuria Confirmed Alport syndrome Case 3 F European SRNS/MCD COL4A3 Comp het - Parents with microscopic Alport syndrome 203780 hematuria Confirmed Alport syndrome Case 4 F European SRNS/FSGS COL4A5 Het - Mother affected by SNHL Alport syndrome 301050 Confirmed Alport syndrome Case 5 M European SRNS/MCD COL4A5 Hem - SNHL not present at Alport syndrome 301050 onset and diagnosed by rechecking WES results - Mother with microscopic hematuria Confirmed Alport syndrome Case 6 F European SRNS/FSGS COL4A5 Het - Mother developed Alport syndrome 301050 nephrotic syndrome during pregnancy (regressed after delivery) Confirmed Alport syndrome Case 7 M European SRNS/FSGS LAMB2 Comp het - Sister with chronic Pierson syndrome 609049 glomerulopathy of unknown etiology Confirmed Pierson syndrome Case 8 F European SRNS/FSGS GLA Het - Podocyte vacuolization Fabry disease 301500 and microscopic lamellar bodies on EM - Father affected by SNHL Confirmed Fabry disease Case 9 M European SRNS/FSGS FAT1 Hom - Cluster headache FAT1-related — - Tubular atrophy glomerulotubular Confirmed FAT1-related nephropathy glomerulotubular nephropathy Case 10 F European SRNS/- FAT4 Comp het - Left kidney hypodysplasia Van Maldergem 615546 at kidney scintigraphy syndrome 2 (normal US scanning) Confirmed kidney involvement in Van Maldergem syndrome 2 Case 11 F European SRNS/FSGS PAX2 Het - No ocular abnormalities Papillo-renal 120330 Confirmed FSGS in syndrome/FSGS Papillo-renal syndrome Case 12 F Latino SRNS/- PAX2 Het - No ocular abnormalities Papillo-renal 120330 Confirmed FSGS in syndrome/FSGS Papillo-renal syndrome Case 13 M European SRNS/FSGS CLCN5 Hem - Growth failure Dent disease 300009 Confirmed Dent disease Case 14 M European SRNS/MCD CLCN5 Hem - Late-onset hypercalciuria Dent disease 300009 Confirmed Dent disease Case 15 M European SRNS/FSGS CLCN5 Hem - Microlithiasis Dent disease 300009 Confirmed Dent disease Case 16 M European SRNS/MCD CTNS Comp het - Cornealcrystalsatslit-lamp Cystinosis 219800 examination - Multinucleated podocytes on EM on kidney biopsy - Intraleukocytes cystin levels 2 nmol/mg protein (normal ,0.3) Confirmed cystinosis CJASN 15: 89–100, January, 2020 Reverse Phenotyping in Nephrotic Syndrome, Landini et al. 95

Table 2. (Continued)

Pre Whole-Exome Post Whole-Exome Phenotype Reverse Phenotyping Patient Sex Ethnicity Sequencing Gene Variant Sequencing OMIM Diagnosis Diagnosis/ Diagnosis Number (#) Biopsy Results

Case 17 M Tunisian SRNS/FSGS LMX1B Het - Lack ofossificationnuclei Nail-patella 161200 of the radium (bilateral) syndrome - Severe growth failure - Absence of thumbs lunulae - Father with microscopic hematuria and mild proteinuria Confirmed Nail-patella syndrome Case 18 M European SRNS/MCD KANK1 Comp het - Multidrug resistant Cerebral palsy 612900 epilepsy Confirmed cerebral palsy

All postwhole-exome sequencing diagnoses are defined according to OMIM nomenclature (https://www.omim.org) with the exception of FAT1-related glomer- ulotubular nephropathy (37). OMIM, Online Mendelian Inheritance in Man; F, female; SRNS, steroid-resistant nephrotic syndrome; Hom, homozygous; Comp het, compound heterozygous; SNHL, sensorineural hearing loss; MCD, minimal change disease; Het, heterozygous; M, male; Hem, hemizygous; WES, whole-exome sequencing; EM, electron microscopy; -, biopsy not performed; US, ultrasound.

Discussion subgroup within patients with steroid-resistant nephrotic We hypothesized that among patients with steroid- syndrome, involving subtle phenotypes of syndromic ge- resistant nephrotic syndrome, particularly those who are netic disorders; and (3) phenocopies are usually multidrug multidrug resistant, phenocopies of monogenic podocyto- resistant. Recent studies have applied genetic screening pathies may be more frequent than previously thought and using whole-exome sequencing and bioinformatic filtering that whole-exome sequencing for an extended panel of of gene panels to patients with different CKDs (39–41) nephropathy-related genes plus reverse phenotyping may including nephrotic syndrome (11,27). By using a filtering help identifying them. In this study, we show that (1)by strategy on the basis of 140 genes, Bullich et al. (40), combining a whole-exome sequencing–based diagnostic increased the rate of diagnosis in previously undiagnosed workflow for an extended panel of nephropathy-related patients but could rescue wrong diagnoses in only 2% of genes with reverse phenotyping of the patients and their patients with inherited glomerular disorders. By adding families, we double the current diagnostic rate for genetic the further step of reverse phenotyping, we increased the diagnosis underlying steroid-resistant nephrotic syndrome rate of rescued diagnoses to 28% thanks to the from 30% to 60%; (2) phenocopies comprise a significant

Table 3. Summary of the results of whole-exome sequencing in podocytopathy patients

Pre Whole-Exome Post Whole-Exome Phenotype Patient Sex Ethnicity Sequencing Gene Variant Sequencing OMIM Diagnosis/Biopsy Results Diagnosis Number (#)

Case 19 M European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 20 M European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 21 F European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 22 M European SRNS/MCD NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 23 M European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 24 M European SRNS/FSGS NPHS2 Hom Nephrotic syndrome, type 2 600995 Case 25 F European SRNS/- NPHS2 Hom Nephrotic syndrome, type 2 600995 Case 26 M European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 27 F European SRNS/FSGS NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 28 M European SRNS/MCD NPHS2 Comp het Nephrotic syndrome, type 2 600995 Case 29 F European SRNS/- NPHS1 Comp het Nephrotic syndrome, type 1 256300 Case 30 M European SRNS/FSGS ANLN Het FSGS 8 616032 Case 31 M European SRNS/FSGS PLCE1 Comp het Nephrotic syndrome, type 3 610725 Case 32 F European SRNS/DMS PLCE1 Comp het Nephrotic syndrome, type 3 610725 Case 33 F European SRNS/FSGS ACTN4 Het Glomerulosclerosis, focal segmental, 1 603278 Case 34 F European SRNS/FSGS ACTN4 Het Glomerulosclerosis, focal segmental, 1 603278 Case 35 F European SRNS/FSGS WT1 Het Nephrotic syndrome, type 4 256370 Case 36 F European SRNS/- WT1 Het Nephrotic syndrome, type 4 256370 Case 37 M European SRNS/FSGS WT1 Het Nephrotic syndrome, type 4 256370

All postwhole-exome sequencing diagnoses are defined according to OMIM nomenclature (https://www.omim.org). OMIM, Online Mendelian Inheritance in Man; M, male; SRNS, steroid-resistant nephrotic syndrome; Comp het, compound heterozygous; F, female; MCD, minimal change disease; Hom, homozygous; -, biopsy not performed; Het, heterozygous; DMS, diffuse mesangial sclerosis. 96 CJASN

A B

50

45

40

35 30 30 28 25 20 16 15 26

10

Urinary protein/Creatinine ratio (mg/mg) ratio protein/Creatinine Urinary 5 35 0 30 0.0 25 0.5 20 Podocytopathies Undefined 1.0 15 Serum albumin1.5 (g/dl) 10 2.0 Phenocopies Negative 2.5 5 3.0 0 3.5 Age at phenotyping (years)

Podocytopathies Undefined SSNS Phenocopies Negative

Figure 3. | Clinical and genetic findings of the patients included in the study. (A) Age, urinary protein-to-creatinine ratio, and serum albumin levels at referral forall of the patients enrolled in the study. Eachpoint represents a single patient. (B) Conclusivediagnosis in patientswith steroid- resistant nephrotic syndrome: podocytopathies (10 NPHS2,1NPHS1,1ANLN,2PLCE1,2ACTN4,3WT1) and phenocopies (2 COL4A4,1 COL4A3,3COL4A5,1LAMB2,1GLA,1FAT1,1FAT4,2PAX2,3CLCN5,1CTNS, 1 LMX1B,1KANK1). SSNS, steroid-sensitive nephrotic syndrome. identification of other genetic disorders appearing as clinical phenotype and biopsy results. Indeed, when phenocopies of monogenic podocytopathies. specific signs of the underlying disease are absent at onset, To optimize the identification of phenocopies, we only whole-exome sequencing may suggest the correct systematically considered genes identified in OMIM as diagnosis. However, even with whole-exome sequencing causing “nephrotic syndrome” or “FSGS” as “podocytop- the diagnosis can be missed, especially when (1) filtering athy genes” and genes identified as causing a syndromic criteria for in silico analysis of variants are set very tight to disorder as “phenocopy genes” (see Materials and Meth- increase specificity at the expense of sensitivity; (2)in- ods). On the basis of this, we performed reverse phenotyp- terpretation of whole-exome sequencing results does not ing every time that phenocopy genes were identified by include analysis of segregation within the family, which is whole-exome sequencing. In none of the patients we essential to validate the presence and significance of describe, the true diagnosis had been suspected from pathogenic variants occurring in unexpected genes; and

Table 4. Summary of the clinical and pathologic characteristics of the patients with steroid-resistant nephrotic syndrome included in the study, divided by genetic group

Podocytopathies Phenocopies Negative Undefined Characteristics (n=19) (n=18) (n=17) (n=10)

Age at onset, yr 4 [1–7] 5 [3–10] 5 [3–8] 5 [3–13] Sex (male) 10/19 (53%) 9/18 (50%) 10/17 (59%) 3/10 (30%) Ethnicity (European) 19/19 (100%) 15/18 (83%) 17/17 (100%) 9/10 (90%) Histopathological findings (FSGS) 13/16 (81%) 11/16 (69%) 11/17 (65%) 8/10 (80%) Remission Complete 0/19 (0%) 0/18 (0%) 8/17 (47%) 0/0 (0%) Partial 1/19 (5%) 11/18 (61%) 5/17 (29%) 0/0 (0%) None 18/19 (95%) 7/18 (39%) 4/17 (24%) 10/10 (0%) Response to CNIs 1/8 (13%) 0/6 (0%) 5/10 (50%) 0/6 (0%) Response to RASi 1/15 (7%) 10/18 (56%) 7/15 (47%) 0/8 (0%) ESKD at 10 yr 11/15 (73%) 3/10 (30%) 3/6 (50%) 8/10 (80%) Post-transplant recurrence 0/11 (0%) 0/0 (0%) 1/3 (33%) 3/7 (43%) Length of follow-up, yr 8 [5–12] 7 [2–10] 6 [5–10] 8 [4–12]

Continuous variables are presented as median [interquartile range] and categorical variables are presented as n (%). CNIs, calcineurin inhibitors; RASi, inhibitors of the renin-angiotensin-aldosterone system. CJASN 15: 89–100, January, 2020 Reverse Phenotyping in Nephrotic Syndrome, Landini et al. 97

100

80

60

40

Cumulative survival (%) Phenocopies 20 Podocytopathies Negative 0 Undefined

0246810 Time to ESKD (years)

Phenocopies 18 15 10 10 7 7 Podocytopathies 19 12 10 5 5 4 Negative 17 17 14 6 5 3 Undefined 1096532

Total 64 53 40 26 20 16

Figure 4. | Kidney survival of patients with steroid-resistant nephrotic syndrome according to the genetic diagnosis. Kaplan–Meier kidney survival analysis over a period of 10 years of follow-up. Patients with steroid-resistant nephrotic syndrome are stratified according to whole- exome sequencing results. Length of follow-up was similar between groups (ANOVA P=0.74). (+) Censored observations. Survival rates were compared between groups by log-rank test (significant values): podocytopathies versus phenocopies (log-rank 5.47; P=0.02), podocytopathies versus negative (log-rank 5.77; P=0.02), undefined versus phenocopies (log-rank 6.15; P=0.01), and undefined versus negative (log-rank 6.12; P=0.01).

(3) genetic testing is performed without the opportunity of treatments and inform treatment, in particular clinical re-evaluation of the patients and the family mem- when a specific therapy is available (e.g., cystinosis). In bers for subtle and previously unrecognized clinical signs, addition, a correct identification of phenocopies would especially when unexpected findings occur. This is particularly make it possible to accurately enroll them in clinical trials in relevant for patients referred for genetic testing from larger cohorts and better establish their prognosis on a case- peripheral centers, when only the DNA is shipped and by-case basis, determined by the underlying disorder. More- there is no independent clinical re-evaluation. over, our study suggests that phenocopies show a better The results of this study demonstrate the power of outcome compared with genetic podocytopathies. This may “reverse phenotyping” in the interpretation of whole-exome reflect the fact that although in monogenic podocytopathies sequencing data directly in the clinical setting with the the podocyte is primarily affected, in phenocopies podocyte awareness that, for certain genetic diseases, syndromic damage may be, at least partially, secondary to nephron loss, features may be nonpenetrant in the patient as well as in justifying the longer time needed for kidney function loss. the family or may become evident only with age. In these The high number of analyzed genes led to the identi- patients, extrarenal involvement and syndromic features fication of many patients carrying variants of unknown may be subtle and need to be specifically assessed to clinical significance. These “undefined” patients had a reach conclusive diagnoses. For these reasons, we sug- significantly worse outcome compared with negative ones, gest that for phenocopy genes, reverse phenotyping of likely because they included unrecognized genetic causes, as the patient and relatives should always be part of the well as disorders possibly related to permeability factor(s) diagnostic workup with geneticist, nephrologist, and (42,43). Consistently, these patients also showed a high risk nephropathologist working closely to avoid misdiagno- of nephrotic syndrome recurrence after kidney transplant sis. These results suggest that phenocopies of monogenic (43,44). By contrast, patients with genetic forms showed a podocytopathies may be underestimated. Indeed, cases negligible risk of post-transplantation disease recurrence, liketheoneswedescribeinthisstudyaredifficult to in agreement with previous studies (11,12). Finally, in case recognize in registry-based studies, suggesting the lim- of a misdiagnosed genetic phenocopy, kidney donation itations of this kind of analyses in moving forward our may be detrimental not only for the recipient, but also for daily clinical practice. Identification of phenocopies per- the affected parent. mits to avoid unnecessary multiple immunosuppressive 98 CJASN

The generalizability of these results is limited by the low 5. Bu¨scher AK, Beck BB, Melk A, Hoefele J, Kranz B, Bamborschke number of patients with a long follow-up that could be D, Baig S, Lange-Sperandio B, Jungraithmayr T,Weber LT,Kemper MJ, Tonshoff B, Hoyer PF, Konrad M, Weber S; German Pediatric analyzed. For this reason, independent replications and ¨ Nephrology Association (GPN): Rapid response to cyclosporin a meta-analysis efforts are needed and can be addressed in and favorable renal outcome in nongenetic versus genetic steroid- future research. resistant nephrotic syndrome. Clin J Am Soc Nephrol 11: 245– Taken together, our results suggest that in steroid- 253, 2016 resistant nephrotic syndrome a workflow including anal- 6. Trautmann A, Schnaidt S, Lipska-Zie˛tkiewicz BS, Bodria M, ysis of an extended panel of genes, multidisciplinary Ozaltin F, Emma F, Anarat A, Melk A, Azocar M, Oh J, Saeed B, Gheisari A, Caliskan S, Gellermann J, Higuita LMS, Jankauskiene meetings and reverse phenotyping increased the diagnostic A, Drozdz D, Mir S, Balat A, Szczepanska M, Paripovic D, rate from 30% to 60%. Relevant clinical implications deriving Zurowska A, Bogdanovic R, Yilmaz A, Ranchin B, Baskin E, from this approach include: (1)theidentification of the un- Erdogan O, Remuzzi G, Firszt-Adamczyk A, Kuzma- derlying cause of the disease; (2)thedefinition of appropriate Mroczkowska E, Litwin M, Murer L, Tkaczyk M, Jardim H, diagnostic work-up, with referral and screening for previously Wasilewska A, Printza N, Fidan K, Simkova E, Borzecka H, Staude H, Hees K, Schaefer F; PodoNet Consortium: Long-term outcome unrecognized extrarenal features; (3) the possibility to guide of steroid-resistant nephrotic syndrome in children. JAmSoc therapy and identify potential tailored treatments; (4)amore Nephrol 28: 3055–3065, 2017 accurate prognostic evaluation; and (5) genetic counseling, 7. Romagnani P, Remuzzi G, Glassock R, Levin A, Jager KJ, Tonelli cascade screening of at-risk relatives, and donor selection for M, Massy Z, Wanner C, Anders HJ: Chronic kidney disease. Nat transplantation. Rev Dis Primers 3: 17088, 2017 8. Giglio S, Provenzano A, Mazzinghi B, Becherucci F, Giunti L, Sansavini G, Ravaglia F, Roperto RM, Farsetti S, Benetti E, Rotondi Disclosures M, Murer L, Lazzeri E, Lasagni L, Materassi M, Romagnani P: The authors have nothing to disclose. Heterogeneous genetic alterations in sporadic nephrotic syn- drome associate with resistance to immunosuppression. JAmSoc Funding Nephrol 26: 230–236, 2015 This article is supported by the European Research Council 9. Sadowski CE, Lovric S, Ashraf S, Pabst WL, Gee HY, Kohl S, under the European Union’s Horizon 2020 Research and In- Engelmann S, Vega-Warner V,Fang H, Halbritter J, Somers MJ, Tan W, Shril S, Fessi I, Lifton RP, Bockenhauer D, El-Desoky S, Kari JA, novationProgramme (grantagreement648274),theTuscanRegion Zenker M, Kemper MJ, Mueller D, Fathy HM, Soliman NA, ’ “ to the Meyer Children sHospital Programma attuativo regionale Hildebrandt F; SRNS Study Group: A single-gene cause in 29.5% Fas-FSC,” and from the Tuscan Association for Childhood Renal of cases of steroid-resistant nephrotic syndrome. JAmSoc Diseases. Nephrol 26: 1279–1289, 2015 10. Wang F, Zhang Y, Mao J, Yu Z, Yi Z, Yu L, Sun J, Wei X, Ding F, Supplemental Material Zhang H, Xiao H, Yao Y, Tan W, Lovric S, Ding J, Hildebrandt F: Spectrum of mutations in Chinese children with steroid-resistant This article contains the following supplemental material online nephrotic syndrome. Pediatr Nephrol 32: 1181–1192, 2017 at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/ 11. Bierzynska A, McCarthy HJ, Soderquest K, Sen ES, Colby E, Ding CJN.06060519/-/DCSupplemental. WY, Nabhan MM, Kerecuk L, Hegde S, Hughes D, Marks S, Supplemental Table 1. Clinical profile of all of the patients in- Feather S, Jones C, Webb NJ, Ognjanovic M, Christian M, Gilbert cluded in the study. RD, Sinha MD, Lord GM, Simpson M, Koziell AB, Welsh GI, Saleem MA: Genomic and clinical profiling of a national ne- Supplemental Table 2. Panel of 298 genes analyzed with whole- phrotic syndrome cohort advocates a precision medicine ap- exome sequencing. proach to disease management. Kidney Int 91: 937–947, 2017 Supplemental Table 3. List of pathogenic variants on the basis of 12. Trautmann A, Bodria M, Ozaltin F, Gheisari A, Melk A, Azocar M, the American College of Medical Genetics and Genomics score. Anarat A, Caliskan S, Emma F, Gellermann J, Oh J, Baskin E, Supplemental Table 4. List of variants of unknown significance on Ksiazek J, Remuzzi G, Erdogan O, Akman S, Dusek J, Davitaia T, O¨ zkaya O, Papachristou F, Firszt-Adamczyk A, Urasinski T, Testa the basis of the ACMG score. ¨ fi S, Krmar RT, Hyla-Klekot L, Pasini A, Ozcakar ZB, Sallay P, Cakar Supplemental Table 5. Genetic pro le of patients showing path- N, Galanti M, Terzic J, Aoun B, Caldas Afonso A, Szymanik- ogenic variants. Grzelak H, Lipska BS, Schnaidt S, Schaefer F; PodoNet Consor- Supplemental Appendix 1. DNA extraction, DNA library prep- tium: Spectrum of steroid-resistant and congenital nephrotic aration, assembly, variant calling, variant interpretation strategy, syndrome in children: The PodoNet registry cohort. Clin J Am Soc Nephrol 10: 592–600, 2015 statistical analysis, web resources, case reports, and references. 13. Ashraf S, Kudo H, Rao J, Kikuchi A, Widmeier E, Lawson JA, Tan W, Hermle T, Warejko JK, Shril S, Airik M, Jobst-Schwan T, Lovric S, References Braun DA, Gee HY, Schapiro D, Majmundar AJ, Sadowski CE, 1. Kidney Disease Improving global outcomes (KDIGO) Pabst WL, Daga A, van der Ven AT, Schmidt JM, Low BC, Gupta AB, Tripathi BK, Wong J, Campbell K, Metcalfe K, Schanze D, Glomerulonephritis Work Group: KDIGO clinical practice Niihori T,Kaito H, Nozu K, Tsukaguchi H, Tanaka R, Hamahira K, guidelines for glomerulonephritis. Kidney Int Suppl (2011) 2: Kobayashi Y, Takizawa T, Funayama R, Nakayama K, Aoki Y, 139–274, 2012 Kumagai N, Iijima K, FehrenbachH, Kari JA, El Desoky S,Jalalah S, 2. Noone DG, Iijima K, Parekh R: Idiopathic nephrotic syndrome in Bogdanovic R, Stajic N, Zappel H, Rakhmetova A, Wassmer SR, children. Lancet 392: 61–74, 2018 Jungraithmayr T, Strehlau J, Kumar AS, Bagga A, Soliman NA, 3. 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Gallon L, Leventhal J, Skaro A, Kanwar Y, Alvarado A: Resolution Wheeler transform. Bioinformatics 25: 1754–1760, 2009 of recurrent focal segmental glomerulosclerosis after retrans- 30. Thorvaldsdo´ttir H, Robinson JT, Mesirov JP: Integrative Genomics plantation. N Engl J Med 366: 1648–1649, 2012 Viewer (IGV): High-performance genomics data visualization 44. Francis A, Trnka P, McTaggart SJ: Long-term outcome of kidney and exploration. Brief Bioinform 14: 178–192, 2013 transplantation in recipients with focal segmental glomerulo- 31. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, sclerosis. Clin J Am Soc Nephrol 11: 2041–2046, 2016 Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: A MapReduce Received: May 17, 2019 Accepted: October 8, 2019 100 CJASN

S.L., B.M., and F.B. contributed equally to this work. See related editorial, ““It’s In Your Genes”: Exome Sequencing Enables Precision Diagnostics in Proteinuric Kidney Diseases,” on Published online ahead of print. Publication date available at pages 10–12. www.cjasn.org.

AFFILIATIONS

1Medical Genetics Unit, Meyer Children’s University Hospital, Florence, Italy; 2Department of Clinical and Experimental Biomedical Sciences “Mario Serio,” 3Excellence Centre for Research, Transfer and High Education for the development of DE NOVO Therapies (DENOTHE), and 5Department of Health Sciences, University of Florence, Florence, Italy; 4Nephrology and Dialysis Unit, Meyer Children’s University Hospital, Florence, Italy; 6Pediatric Nephrology Dialysis and Transplant Unit, Department of Pediatrics, University of Padua, Padua, Italy; 7Pediatric Nephrology Unit, Regina Margherita Children’s Hospital, Citta` della Salute e della Scienza di Torino, Turin, Italy; 8Nephrology and Dialysis Unit, Department of Pediatrics, Azienda Ospedaliero Universitaria, Policlinico Sant’Orsola-Malpighi, Bologna, Italy; 9Medizinische Klinik and Poliklinik IV, Klinikum der Ludwig Maximilians University (LMU) Mu¨nchen, Mu¨nchen, Germany; and 10Unit of Internal Medicine and Endocrinology, Laboratory for Endocrine Disruptors, Istituti Clinici Scientifici Maugeri Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), University of Pavia, Pavia, Italy

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Material Table of Contents

Supplemental Table 1: Clinical profile of all the patients included in the study.

Supplemental Table 2: Panel of 298 genes analyzed with whole exome sequencing.

Supplemental Table 3: List of pathogenic variants based on the ACMG score.

Supplemental Table 4: List of variants of unknown significance based on the ACMG score.

Supplemental Table 5: Genetic profile of patients showing pathogenic variants.

Supplemental Methods: DNA extraction, DNA library preparation, Assembly, Variant

Calling, Variant interpretation strategy, Statistical analysis, Web resources

Case Reports

References

1

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Table 1. Clinical profile of all the patients included in the study.

Age at Clinical onset CKD stage Post- Age at Response Response Patient Gender Ethnicity onset Histology Remission at last transplant phenotyping to CNIs to RASi Albumine (years) Proteinuria Dyslipidemia Oedema follow-up recurrence (years) g/dl

Case 1 F Senegalese 3.4 FSGS 4.2 mg/mg 2.6 No No - Yes P ESKD - 10

Case 2 F European 26.2 FSGS 5 mg/mg 3 Yes No - Yes P III - 27.2

Case 3 F European 11.8 MCD 2.5 mg/mg 3 NA NA No Yes P I - 20.7

Case 4 F European 3.6 FSGS 60 mg/m2/h NA NA No - No N I - 16

Case 5 M European 4.7 MCD 3.8 mg/mg 3.3 No No - No N I - 15.7

Case 6 F European 6 FSGS NA NA NA NA - Yes P ESKD - 20.9

Case 7 M European 0.8 FSGS 111 mg/m2/h NA NA No - Yes P I - 4.8

Case 8 F European 9.7 FSGS 6.5 mg/mg 1.7 Yes Yes No No N IV - 10.8

2

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 9 M European 22 FSGS NA NA NA No - Yes P III - 23.4

Case 10 F European 9 NP 48 mg/m2/h NA NA NA - Yes P III - 14

Case 11 F European 16.5 FSGS NA NA NA NA - Yes P II - 17.8

Case 12 F Latino 1.9 NP 8 mg/mg NA No No - No N ESKD - 3.8

Case 13 M European 1.6 FSGS 10 mg/mg 3.1 No No No No N II - 6

Case 14 M European 2.9 MCD NA NA NA NA - No N I - 4

Case 15 M European 1.6 FSGS 292 mg/m2/h 1.6 Yes No No No N II - 6.6

Case 16 M European 5 MCD 61.3 mg/m2/h NA No No - Yes P II - 7.5

Case 17 M Tunisian 7.5 FSGS 22.3 mg/mg 1.9 Yes Yes No Yes P ESKD - 9.5

Case 18 M European 3.9 MCD 5 mg/mg 1.8 Yes Yes No No P I - 5

3

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 19 M European 1.2 FSGS 45 mg/mg 2 Yes Yes - No N ESKD No 1.2

Case 20 M European 6.4 FSGS 7.6 mg/mg 1.8 Yes Yes - No N ESKD No 7

Case 21 F European 5 FSGS 5.2 mg/mg 2 Yes No No No N ESKD - 15

Case 22 M European 0.2 MCD 8 mg/mg 2 NA No No No N ESKD - 13

Case 23 M European 3 FSGS 450 mg/m2/h 2.1 NA Yes No No N ESKD No 21

Case 24 M European 0.02 FSGS NA NA NA Yes - No N ESKD No 22

36 mg/mg Case 25 F European 0.4 NP 2 Yes Yes - No N ESKD No 1

Case 26 M European 29 FSGS 12 mg/mg 2.6 Yes NA - - N III - 33

Case 27 F European 11.3 FSGS 5 mg/mg 3.4 NA No No No N ESKD - 15

4

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 28 M European 2.8 MCD 97 mg/m2/h 2.5 Yes No No No N I - 10

Case 29 F European 4 NP 7 mg/mg 2.2 Yes No Yes Yes P I - 4

Case 30 M European 6 FSGS NA NA NA No - - N ESKD - 18

Case 31 M European 13 FSGS 3.4 mg/mg 2.8 Yes Yes - No N ESKD No 18

Case 32 F European 0.3 DMS NA NA NA Yes - No N ESKD No 14

Case 33 F European 7.7 FSGS 15 mg/mg 1.4 Yes Yes No No N ESKD No 12

Case 34 F European 3.8 FSGS 10 mg/mg 2.4 Yes Yes No No N ESKD No 4

Case 35 F European 1.6 FSGS 8 mg/mg 2.3 Yes Yes - - N ESKD No 2

Case 36 F European 1 NP 35.2 mg/mg 1.3 Yes No - No N ESKD No 1

5

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 37 M European 8.6 FSGS 4.7 mg/mg 3.4 Yes No - - N III - 9

Case 38 M European 2.5 MCD 5.3 mg/mg 2.7 No Yes - Yes C I - 3.5

Case 39 M European 0.4 FSGS 41 mg/mg 2.3 Yes Yes - Yes C I - 1

Case 40 F European 3.4 FSGS 3 mg/mg 1.8 Yes Yes - Yes C I - 8

Case 41 F European 2.6 FSGS 9.3 mg/mg 2.4 Yes Yes No Yes C I - 4

Case 42 M European 2.6 FSGS 3 mg/mg 1.9 Yes Yes Yes Yes C I - 3

Case 43 F European 2.8 FSGS 2.7 mg/mg 3.4 Yes No - Yes P I - 3

Case 44 M European 2.7 FSGS 40 mg/mg 1.2 Yes Yes Yes - C I - 3

Case 45 M European 2.6 FSGS 4.8 mg/mg 2.2 NA Yes No No P II - 13

6

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 46 F European 7.9 MCD 20.1 mg/mg 1.7 Yes Yes Yes No C I - 8

Case 47 M European 5 MCD NA NA NA NA Yes - C - - 11

Case 48 M European 5.11 MCD 2 mg/mg 2.7 Yes No - No P I - 7

Case 49 M European 13 FSGS 12.4 mg/mg 2.1 Yes Yes - No N I - 13.5

Case 50 F European 6 MCD 4 mg/mg 2.3 Yes Yes - No P I - 10

Case 51 M European 6 FSGS 4 mg/mg 1.9 Yes Yes Yes Yes P ESKD No 10

Case 52 F European 13 MCD 3 mg/mg 2.7 Yes No No No N III - 13

Case 53 F European 13 FSGS 4.4 mg/mg 3 Yes No No No N ESKD No 20

7

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 54 M European 12 FSGS 8 mg/mg 1.5 Yes Yes No No N ESKD Yes 21

Case 55 F Maroccan 0.8 DMS 3.5 mg/mg 2.2 Yes Yes - No N ESKD No 5

Case 56 F European 2 FSGS NA NA NA NA - No N III - 18

Case 57 F European 9 FSGS 3.3 mg/mg 2.1 NA Yes No No N III - 16

Case 58 F European 3.5 FSGS 5 mg/mg 1.1 Yes Yes No No N ESKD Yes 16

Case 59 F European 6 MCD 3 mg/mg 1.8 No No No No N ESKD Yes 8

Case 60 M European 2.3 FSGS 7 mg/mg 2.1 Yes Yes No No N ESKD Yes 10

Case 61 M European 29.6 FSGS NA NA NA NA - - N ESKD - 32

Case 62 M European 26 FSGS NA NA NA NA - - N ESKD No 27

Case 63 F European 4.6 FSGS 9.8 mg/mg 3 Yes No No No N ESKD No 9

8

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 64 F European 14.2 FSGS 2.8 mg/mg 4.3 No No No No N ESKD No 21

Case 65 M European 5 NP 8 mg/mg 1.9 Yes Yes - - C I - 5

Case 66 M European 6.1 MCD 6.1 mg/mg 1.2 Yes Yes Yes Yes C I - 6.1

Case 67 F European 1.5 NP NA NA NA NA - - C I - 1.5

Case 68 M European 2.9 NP 4 mg/mg 2.2 Yes NA - - C I - 2.9

Case 69 M European 1.9 NP 31.5 mg/mg 2.3 Yes Yes No - C I - 1.9

Case 70 M European 3.2 NP 5.9 mg/mg 2.6 NA NA - - C I - 3.2

Case 71 M European 3 NP 13.8 mg/mg 2.2 Yes Yes Yes - C I - 3

Case 72 M Tunisian 9.2 NP 7 mg/mg 1.3 Yes No - - C I - 9.2

Case 73 M European 10 NP 4 mg/mg 2.4 Yes Yes - - C I - 10

Case 74 F European 2.9 NP 12.9 mg/mg 2.4 Yes NA Yes - C I - 2.9

9

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 75 F European 3.2 MCD 11 mg/mg 1.4 Yes Yes Yes - C I - 3.2

Case 76 M European 3.9 NP 25 mg/mg 2.3 No No - - C I - 3.9

Case 77 F European 3.3 NP 13.6 mg/mg 1.9 Yes Yes Yes - C I - 3.3

Case 78 M European 3.4 NP 13 mg/mg 1.9 Yes Yes - - C I - 3.4

Case 79 M European 9.2 NP 6 mg/mg 3.1 NA Yes Yes - C I - 9.2

Case 80 M European 7.7 NP 6 mg/mg 1.5 NA Yes - - C I - 7.7

Case 81 M European 2.5 NP 19.4 mg/mg 2.6 NA NA Yes - C I - 2.5

Case 82 M European 5.7 NP 5 mg/mg 2.4 Yes Yes - - C I - 5.7

Case 83 M Moroccan 4 NP 34.1 mg/mg 2.4 Yes NA Yes - C I - 4

Case 84 F Senegalese 7.5 NP 44.2 mg/mg 2.4 No No - - C I - 7.5

Case 85 F Chinese 3 NP 29.1 mg/mg 0.9 NA Yes Yes - C I - 3

10

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 86 M Chinese 10.3 MCD 12.6 mg/mg 1.5 Yes NA No - C I - 10.3

Case 87 M European 7.8 NP 23.5 mg/mg 2 NA NA Yes - C I - 7.8

Case 88 M European 3 NP 2.1 mg/mg 1.7 Yes Yes - - C I - 3

Case 89 M European 3.4 NP 28.2 mg/mg 2 Yes Yes Yes - C I - 3.4

Case 90 M European 6.5 NP 23.2 mg/mg 1.1 NA Yes Yes - C I - 6.5

Case 91 F European 3.6 NP 16.8 mg/mg 1.9 Yes Yes Yes - C I - 3.6

Case 92 M European 11.6 NP 12.4 mg/mg 1.7 Yes Yes No - C I - 11.6

Case 93 M European 3.5 NP 3 mg/mg 3.4 NA NA Yes - C I - 3.5

Case 94 F Moroccan 3.2 NP 42 mg/mg 0.9 Yes Yes Yes - C I - 3.2

Cape Case 95 F 11 MCD 3.5 mg/mg 3.4 NA NA - - C I - 11 Verdean

Case 96 F European 4.5 NP 26.3 mg/mg 2.7 NA NA Yes - C I - 4.5

11

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 97 M European 10.6 NP NA NA NA No - - C I - 10.6

Case 98 M European 5.2 NP 8 mg/mg 1.7 Yes Yes - - C I - 5.2

Case 99 F European 2 NP 7.2 mg/mg 2.6 Yes Yes Yes - C I - 2

Case 100 F European 2.9 NP 13.4 mg/mg 2 NA Yes - - C I - 2.9

Case 101 F Senegalese 7.3 NP 6 mg/mg 2.2 NA Yes Yes Yes C I - 7.3

Case 102 F Senegalese 8.2 NP 5 mg/mg 2.4 NA Yes Yes - C I - 8.2

Case 103 F European 3.5 NP 9.2 mg/mg 1.6 Yes Yes - - C I - 3.5

Case 104 F European 2.1 NP 46.1 mg/mg 2.1 Yes Yes Yes - C I - 2.1

Case 105 M European 4.1 NP 20.5 mg/mg 1 Yes Yes - - C I - 4.1

Case 106 M European 2.1 NP 22.4 mg/mg 1.6 Yes Yes - - C I - 2.1

Case 107 M European 6 NP 2.2 mg/mg 1.2 Yes Yes - - C I - 6

12

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 108 F European 6.6 FSGS 18.5 mg/mg 2.2 Yes No - - C I - 6.6

Case 109 M Albanian 8.8 NP 18.9 mg/mg 2.1 NA Yes Yes - C I - 8.8

Case 110 M Chinese 11.1 NP 5.4 mg/mg 3.4 NA Yes Yes - C I - 11.1

Case 111 M Chinese 4.5 NP 3.3 mg/mg 2 Yes Yes Yes - C I - 4.5

F, female; M, male; FSGS, focal segmental glomerulosclerosis; MCD, minimal change disease; DMS, diffuse mesangial sclerosis; NP, not performed; -, therapy not performed; C, complete remission; N, no remission; P, partial remission; CNIs, calcineurin inhibitors; RASi, inhibitors of the renin-angiotensin-aldosterone system; CKD, chronic kidney disease; ESKD, end stage kidney disease.

13

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Table 2. Panel of 298 genes analyzed with whole-exome sequencing.

Gene Locus OMIM Reference FOXI1 chr5: 169532901-169536727 * 601093 17

ACE chr17:61554422-61575741 + 106180 1 FOXP1 chr3:71003844-71633140 * 605515 18

AGT chr1:230838272-230850336 + 106150 1 FRAS1 chr4:78978724-79465423 * 607830 1

AGTR1 chr3:148415658-148460790 * 106165 1 FREM1 chr9: 14734664-14910993 * 608944 1

AGTR2 chrX:115301958-115306225 * 300034 1 FREM2 chr13:39261173-39461267 * 608945 1

AIFM3 chr22: 21319396-21335649 * 617298 2 GANAB chr11: 62392298-62414104 * 104160 4

ALG1 chr16: 5083703-5137380 * 605907 3 GATA3 chr10:8096667-8117164 * 131320 5 ALG8 chr11: 77811982-77850706 * 608103 4 GDNF chr5: 37812779-37839788 * 600837 19 BMP4 chr14:54416455-54421270 * 112262 1 GLI3 chr7:42000548-42276618 * 165240 20 BMP7 chr20:55743809-55841707 * 112267 5 GRIP1 chr12:66741211-67072925 * 604597 1 CDC5L chr6:44355251-44418161 * 602868 5

HNF1B chr17:36046434-36105096 * 189907 1 CHD1L chr1:146714291-146767447 1 * 613039

HPSE2 chr10:100216834-100995632 * 613469 21 CHD7 chr8: 61591337-61779465 * 608892 6

ITGA8 chr10: 15555948-15762124 * 604063 22 CHRM3 chr1: 239549865-240078750 * 118494 1

ITGB1 chr10: 33189247-33294720 * 135630 23 CRKL chr22: 21271714-21308037 * 602007 2

LRIG2 chr1:113615831-113667342 * 608869 24 DCHS1 chr11: 6642556-6677085 * 603057 7

LRP4 chr11:46878419-46940193 * 604270 8 DCHS2 chr4: 155153399-155412930 * 612486 8

LRP5 chr11: 68080077-68216743 * 603506 4 DNAJB11 chr3: 186285192-186315061 * 611341 9

MUC1 chr1:155158300-155162706 * 158340 1 DSTYK chr1:205111631-205180727 * 612666 1

NRIP1 chr21: 16333556-16437321 * 602490 25 DZIP1L chr3: 137780832-137834660 * 617570 10

E2F3 chr6:20402398-20493941 * 600427 11 OSR1 chr2:19551246-19558372 * 608891 26

EXT1 chr8: 118806729-119124092 * 608177 12 PAX2 chr10:102505468-102589697 * 167409 1

EYA1 chr8:72109668-72268979 * 601653 13 PBX1 chr1: 164524821-164868533 * 176310 27

FAT1 chr4: 187508937-187647876 * 600976 14 PKD1 chr16: 2138711-2185899 * 601313 4

FAT3 chr11: 92085262-92629618 * 612483 8 PKD2 chr4: 88928820-88998929 * 173910 4

FAT4 chr4: 126237554-126414087 * 612411 15 PRKCSH chr19: 11546109-11561783 * 177060 4

FGF20 chr8:16850334-16859674 * 605558 1 REN chr1:204123944-204135465 * 179820 1

FGFR1 chr8:38268656-38325363 * 136350 8 RET chr10:43572517-43625797 * 164761 1

FGFR2 chr10:123237844-123357972 * 176943 16 ROBO2 chr3:77089294-77699114 * 602431 1

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Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

SALL1 chr16:51169886-51185183 * 602218 1 COQ2 chr4:84184979-84205964 * 609825 1

SALL4 chr20:50400583-50419048 * 607343 28 COQ6 chr14:74416955-74429813 * 614647 1

SEC61B chr9: 101984346-101992897 * 609214 4 CRB2 chr9:126118448-126141032 * 609720 1

SEC63 Chr6: 108188960-108279393 * 608648 4 DGKE chr17:54911460-54946036 * 601440 1

SHH chr7:155595558-155604967 * 600725 29 DLC1 chr8: 12940870-13373167 * 604258 32

SIX1 chr14:61111417-61116155 * 601205 1 EMP2 chr16:10622279-10674539 * 602334 1

SIX2 chr2:45232324-45236542 * 604994 1 GPC3 chrX:132669776-133119673 * 300037 33

SIX5 chr19:46268043-46272497 * 600963 1 GPC5 chr13:92050935-93519487 * 602446 34

SLIT2 chr4:20255235-20620788 * 603746 30 INF2 chr14:105155943-105185947 * 610982 35

SNAP29 chr22:21213271-21245506 * 604202 2 ITGA3 chr17:48133340-48167848 * 605025 36

SOX17 chr8:55370495-55373456 * 610928 1 ITGB4 chr17:73717516-73753899 * 147557 36

SOX9 chr17:70117161-70122560 * 608160 31 ITSN1 chr21: 35014706-35272165 * 602442 32

SPRY2 chr13: 80910111-80915086 * 602466 1 ITSN2 chr2: 24425733-24583583 * 604464 32

TBX18 chr6: 85397069-85474237 * 604613 1 KANK2 chr19: 11274943-11308467 * 614610 1

TBX3 chr12:115108059-115121969 * 601621 1 KANK4 chr1: 62702651-62785085 * 614612 1

TNXB chr6: 32008931-32083111 * 600985 1 MAGI2 chr7: 77646393-79082890 * 606382 37

TRAP1 chr16:3708038-3767598 * 606219 1 MYH9 chr22:36677324-36784063 * 160775 38

UMOD chr16:20344373-20364037 * 191845 1 MYO1E chr15:59428564-59665071 * 601479 1

UPK3A chr22:45680868-45691755 * 611559 1 NPHS1 chr19:36316274-36342739 * 602716 1

USF2 chr19:35759896-35770718 * 600390 1 NPHS2 chr1:179519677-179545084 * 604766 1

ACTN4 chr19:39138327-39221170 * 604638 1 NUP107 chr12:69080514-69136785 * 607617 1

ADCK4 chr19:41197434-41222790 * 615567 1 NUP205 chr7:135242667-135333505 * 614352 1

ANLN chr7:36429432-36493400 * 616027 1 NUP93 chr16: 56764017-56878797 * 614351 1

APOL1 chr22:36649117-36663577 * 603743 1 NXF5 chrX:101087085-101112549 * 300319 39

ARHGAP24 chr4:86396284-86923823 * 610586 1 PDSS2 chr6:107473761-107780779 * 610564 1

ARHGDIA chr17:79825597-79829282 * 601925 1 PLCE1 chr10:95753746-96088146 * 608414 1

CD151 chr11:832952-838834 * 602243 1 PODXL chr7:131185023-131241376 * 602632 40

CD2AP chr6:47445525-47594994 * 604241 1 PTPRO chr12:15475487-15750335 * 600579 1

CDK20 chr9: 90581356-90589668 * 610076 32 SCARB2 chr4:77079894-77135035 * 602257 1

SGPL1 chr10: 72575717-72640930 * 603729 41

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Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

SMARCAL1 chr2:217277473-217347772 * 606622 1 JAG1 chr20:10618332-10654694 * 601920 55

SYNPO chr5:150020220-150038792 * 608155 11 KAL1 chr6:20534688-21232634 * 300836 1

TENC1 chr12:53440753-53458156 * 607717 32 KANK1 chr9: 470291-746105 * 607704 1

TRPC6 chr11:101322296-101454659 * 603652 1 KCNJ10 chr1: 160007257-160040038 * 602208 56

WT1 chr11:32409325-32457087 * 607102 1 LMX1B chr9:129376748-129463311 * 602575 1

XPO5 chr6: 43490072-43543812 * 607845 1 NARS2 chr11: 78147007-78285919 * 612803 57

ADAMTS13 chr9:136287120-136324508 * 604134 42 NEU1 chr6: 31825436-31830683 * 608272 58

C3 chr19:6677846-6720662 * 120700 1 NOTCH2 chr1:120454176-120612317 * 600275 59

CD46 chr1:207925383-207968861 * 120920 1 OCRL chrX: 128673826-128726538 * 300535 13

CFB chr6: 31895475-31919861 * 138470 43 PEX1 chr7: 92116334-92157845 * 602136 60

CFH chr1:196621008-196716634 * 134370 1 PMM2 chr16:8891670-8943194 * 601785 61

CFHR1 chr1:196743930-196763203 * 134371 43 ROR2 chr9: 94325373-94712444 * 602337 62

CFHR2 chr1: 196788898-196928356 * 600889 43 TFAP2A chr6: 10393419-10419892 * 107580 63

CFHR3 chr1:196743930-196763203 * 605336 43 TSC1 chr9: 135766735-135820020 * 605284 64

CFHR4 chr1: 196819371-196888102 * 605337 43 TSC2 chr16: 2097466-2138716 * 191092 64

CFHR5 chr1: 196946667-196978804 * 608593 43 VIPAS39 chr14: 77893018-77924295 * 613401 65

CFI chr4:110661848-110723335 * 217030 1 VPS33B chr15: 91541646-91565833 * 608552 66

PLG chr6: 161123270-161174347 * 173350 44 WDR73 chr15:85186012-85197521 * 616144 1

THBD chr20:23026270-23030301 * 188040 45 WNT4 chr1:22443798-22469519 * 603490 1

AGXT chr2: 241807896-241819919 * 604285 1 WNT5A chr3: 55499743-55523973 * 164975 67

COL4A1 chr13: 110801318-110959496 * 120130 46 VHL chr3: 10182692-10193904 * 608537 68

CREBBP chr16:3775056-3930121 * 600140 47 ZMPSTE24 chr1:40723779-40759856 * 606480 14

DHCR7 chr11:71145457-71159477 * 602858 48 AHI1 chr6: 135604670-135818914 * 608894 1

DIS3L2 chr2:232826293-233201908 * 614184 49 ALDOB chr9:104182860-104198105 * 612724 69

DLL4 chr15:41221531-41231258 * 605185 50 ALMS1 chr2: 73612886-73837920 * 606844 70

FGF10 chr5: 44303646-44389808 * 602115 51 ANKS6 chr9: 101493611-101559247 * 615370 1

GLA chrX: 100652791-100662913 * 300644 52 ARL13B chr3: 93698983-93774512 * 608922 1

GRHPR chr9: 37422663-37436987 * 604296 53 ARL6 chr3: 97483365-97519953 * 608845 1

HOGA1 chr10: 99344080-99372559 * 613597 54 ATXN10 chr22: 46067678-46241187 * 611150 1

16

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

B9D1 chr17: 19240867-19281495 * 614144 1 LZTFL1 chr3: 45864808-45957534 * 606568 1

B9D2 chr19: 41860326-41870078 * 611951 1 MKKS chr20: 10381657-10414870 * 604896 1

BBS1 chr11: 66278077-66301098 * 209901 1 MKS1 chr17: 56282803-56296966 * 609883 1

BBS2 chr16: 56500748-56554195 * 606151 1 NEK1 chr4: 170314426-170533780 * 604588 13

BBS4 chr15: 72978527-73030817 * 600374 1 NEK8 chr17: 27052915-27070473 * 609799 13

BBS5 chr2: 170335688-170382432 * 603650 1 NPHP1 chr2: 110879888-110962643 * 607100 13

BBS7 chr4: 122745595-122791652 * 607590 1 NPHP3 chr3: 132276986-132441303 * 608002 13

C14ORF179/ chr14: 76368479-76550928 * 614068 1 NPHP4 chr1: 5922871-6052533 * 607215 13 IFT43

C4ORF24/ chr4: 123653857-123666097 * 610683 1 OFD1 chrX:13752832-13787480 * 300170 13 BBS12

C5ORF42 chr5: 37106330-37249530 * 614571 1 PDE6D chr2: 232597135-232650982 * 602676 1

CC2D2A chr4: 15471489-15603180 * 612013 1 PKHD1 chr6: 51480098-51952423 * 606702 1

CEP164 chr11: 117185273-117283984 * 614848 1 POC1A chr3: 52109269-52188706 * 614783 1

CEP290 chr12: 88442793-88535993 * 610142 1 PTHB1/BBS9 chr7: 33168856-33645680 * 607968 1

CEP41 chr7: 130033612-130082274 * 610523 1 RPGRIP1L chr16: 53631595-53737850 * 610937 1

CEP83/ chr12: 94700225-94853764 * 615847 71 SCLT1 chr4: 129786076-130014764 * 611399 1 CCDC41

DYNC2H1 chr11: 102980160-103350591 * 603297 1 SDCCAG8 chr1: 243419320-243663394 * 613524 1

EVC chr4: 5712924-5830772 * 604831 1 TBC1D32 chr6: 121400640-121655891 * 615867 1

EVC2 chr4: 5544499-5711275 * 607261 1 TCTN1 chr12: 111051832-111087235 * 609863 1

FAN1 chr15: 31196055-31235311 * 613534 72 TCTN2 chr12: 124155660-124192948 * 613846 1

GLIS2 chr16: 4364762-4389598 * 608539 73 TMEM138 chr11: 61129473-61136981 * 614459 1

IFT122 chr3: 129158968-129239198 * 606045 1 TMEM216 chr11: 61159159-61166335 * 613277 1

IFT140 chr16: 1560428-1662111 * 614620 1 TMEM231 chr16: 75572015-75590184 * 614949 1

IFT172 chr2: 27667238-27712656 * 607386 1 TMEM237 chr2: 202484907-202508293 * 614423 1

IFT80 chr3: 159974774-160117668 * 611177 1 TMEM67 chr8: 94767072-94831462 * 609884 1

INPP5E chr9: 139323071-139334274 * 613037 1 TRIM32 chr9: 119449581-119463579 * 602290 1

INVS chr9: 102861538-103063282 * 243305 73 TTC21B chr2:166729872-166810348 * 612014 1

IQCB1 chr3: 121488610-121553926 * 609237 74 TTC8 chr14: 89290497-89344335 * 608132 1

KIF14 chr1: 200520628-200589862 * 611279 1 WDPCP chr2: 63348518-64054977 * 613580 1

KIF7 chr15: 90152020-90198682 * 611254 1 WDR19 chr4: 39184024-39287430 * 608151 1

17

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

FXYD6- * 606683 WDR34 chr9: 131395940-131419066 * 613363 1 chr11: 117690878-117747382 82 FXYD2 * 601814

WDR35 chr2: 20110021-20189892 * 613602 1 G6PC chr17: 41052814-41065386 * 613742 83

WDR60 chr7: 158649269-158749438 * 615462 1 HNF4A chr20: 42984340-43061485 * 600281 84

XPNPEP3 chr22: 41253081-41363838 * 613553 1 KCNJ1 chr11:128706210128737268 * 600359 1

ZNF423 chr16: 49521435-49891830 * 604557 1 KLHL3 chr5: 136953189-137071779 * 605775 13

AQP2 chr12: 50344524-50352664 * 107777 13 MRPS22 chr3: 138724648-139076065 * 605810 85

AQP6 chr12: 50360977-50370922 * 601383 74 NR3C2 chr4: 148999913-149365850 * 600983 86

ATP6V0A4 chr7:138391040-138484305 * 605239 1 OSGEP chr14: 20914570-20923264 * 610107 87

ATP6V1B1 chr2:71163012-71192536 * 192132 1 RRM2B chr8: 103216730-103251346 * 604712 88

ATP6V1C2 chr2:10861775-10925236 * 618070 75 SCNN1A chr12: 6456009-6486896 * 600228 13

AVP chr20: 3063202-3065370 * 192340 13 SCNN1B chr16: 23289552-23392620 * 600760 14

AVPR2 chrX: 153167985-153172620 * 300538 13 SCNN1G chr16: 23194036-23228204 * 600761 13

BCS1L chr2: 219523487-219528166 * 603647 76 SLC12A1 chr15: 48483861-48596275 * 600839 13

BSND chr1: 55464606-55476556 * 606412 13 SLC12A3 chr16: 56899119-56949762 * 600968 13

CA2 chr8: 86376081-86393722 * 611492 13 SLC2A2 chr3: 170714137-170744539 * 138160 13

CASR chr3: 121902530-122005342 * 601199 13 SLC34A1 chr5: 176806236-176825849 * 182309 13

CLCN5 chrX: 49687225-49863892 * 300008 13 SLC36A2 chr5: 150694539-150727151 * 608331 89

CLCNKA chr1: 16345370-16360545 * 602024 13 SLC3A1 chr2: 44502599-44548633 * 104614 13

CLCNKB chr1: 16370272-16383803 * 602023 13 SLC4A1 chr17: 42325753-42345509 + 109270 13

CLDN16 chr3: 190040330-190129932 * 603959 13 SLC4A4 chr4: 72053003-72437804 * 603345 13

CLDN19 chr1: 43198764-43205925 * 610036 13 SLC4A5 chr2: 74443369-74570541 * 606757 13

CNNM2 chr10:104678050-104849978 * 607803 13 SLC4A8 chr12: 51785101-51902980 * 605024 13

COG6 chr13: 40229764-40365802 * 606977 77 SLC4A9 chr5: 139739787-139754728 * 610207 13

COQ9 chr16: 57481337-57495187 * 612837 78 SLC5A2 chr16: 31494323-31502181 * 182381 13

CTNS chr17: 3,539,762-3,564,836 * 606272 79 SLC6A18 chr5: 1225470-1246304 * 610300 89

CUBN chr10:16865965-17171816 * 602997 80 SLC6A19 chr5: 1201710-1225232 * 608893 89

CUL3 chr2: 225334867-225450110 * 603136 13 SLC6A20 chr3: 45796942-45838027 * 605616 89

EGF chr4: 110834040-110933422 * 131530 13 SLC7A7 chr14:23242431-23299029 * 603593 13

EHHADH chr3: 184908412-184999778 * 607037 81 SLC7A9 chr19: 33321415-33360672 * 604144 13

TP53RK chr20: 45313004-45318418 * 608679 90

18

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

TRPM6 chr9:77337411-77503010 * 607009 13

WNK1 chr12: 861759-1020618 * 605232 13

WNK4 chr17: 40932696-40948954 * 601844 13

COL4A3 chr2:228029281-228179508 * 120070 1

COL4A4 chr2:227867427-228029275 * 120131 1

COL4A5 chrX:107683074-107940775 * 303630 1

LAMB2 chr3:49158548-49170599 * 150325 1

FN1 chr2: 216225163-216300895 * 135600 91

19

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Supplemental Table 3. List of pathogenic variants based on the ACMG score.

Variant Patient Gene MOI Nucleotide change Amino acid change Zygosity PVS1 PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 Class

c.1445C>G Likely p.Pro482Arg Hom N N N N Y N N Y N N N N N N Y N COL4A4 AR Pathogenic

c.1024A>G Case 1 p.Ser342Gly Het N N N Y N Y N Y N N N N N Y Y Y c.1152T>G Pathogenic p.Ile384Met Het N N N Y N Y N Y N N N N N Y Y Y APOL1 AR c.1164_1169delTTA Pathogenic p.Asn388_Tyr389del Het N N N Y N Y N Y Y N N N N Y Y Y TAA Pathogenic

c.3289+1G>A / Het Y N N Y Y N Y Y N N N N N Y Y Y Pathogenic Case 2 COL4A4 AR c.2590G>A p.Gly864Arg Het N N N N Y Y Y Y N Y N N N Y Y Y Pathogenic

c.4421T>C p.Leu1474Pro Het N N N N N N Y Y N N N N N Y Y Y Pathogenic Case 3 COL4A3 AR c.1831G>A p.Gly611Arg Het N N N N Y Y Y Y N N N N N Y Y N Pathogenic

Case 4 COL4A5 XLD c.1912G>A p.Gly638Ser Het N N N N Y Y Y N N Y N Y N Y Y N Pathogenic

Case 5 COL4A5 XLD c.991G>A p.Gly331Ser Hem N N N N Y Y Y N N Y N Y N Y Y N Pathogenic

Case 6 COL4A5 XLD c.3197G>A p.Gly1066Asp Het N N N N Y Y Y N N Y N Y N Y Y N Pathogenic

c.4868G>A p.Gly1623Asp Het N N N N Y N Y Y N N N N N Y Y N Pathogenic Case 7 LAMB2 AR c.1931G>A p.Arg644His Het N N N N Y N Y Y N N N N N Y Y N Pathogenic

Likely Case 8 GLA XL c.937G>T p.Asp313Tyr Het N N N N Y N N N N Y N Y Y N Y N Pathogenic

Case 9 FAT1 AR c.6823G>A p.Asp2275Asn Hom N N N N Y N Y Y N N N N N Y Y N Pathogenic

Case c.11265A>T p.Glu3755Asp Het N N N N Y N Y Y N N N N N Y Y N Pathogenic FAT4 AR 10 c.11953T>A p.Tyr3985Asn Het N N N N Y N Y Y N N N N N Y Y N Pathogenic

Case PAX2 AD c.239C>T p.Pro80Leu Het N N Y N Y N Y N N N N N N Y Y N Pathogenic 11

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Case PAX2 AD c.410+1G>A Het Y N Y N Y N Y N N N N N N Y Y N Pathogenic 12

Case CLCN5 XLR c.749G>A p.Gly250Asp Hem N N N N Y N Y N N Y N N N Y Y N Pathogenic 13

Case CLCN5 XLR c.608C>G p.Ser203Trp Hem N N N N Y N Y N N Y N N N Y Y N Pathogenic 14

Case CLCN5 XLR c.781G>A p.Gly261Arg Hem N N N N Y N Y N N Y N N N Y Y N Pathogenic 15 c.198_218del p.Ile67_Pro73del Het N N N N Y N Y Y Y N N N N Y Y Y Case arr[GRCh37] Pathogenic CTNS AR 16 17p13.2 Pathogenic (3505485_3560005) Het Y N N N Y N Y Y N N N N N Y Y N x1 Case LMX1b AD c.764C>T p.Ala255Val Het N N N Y Y N Y N N N N Y N Y Y N Pathogenic 17 Pathogenic Case c.971A>G p.Tyr324Cysp.Arg67G Het N N Y N Y N Y Y N N N N N Y Y N KANK1 AR Likely 18 c.200G>A ln Het N N N N Y N N Y N N N N N Y Y N Pathogenic Case c.413G>A p.Arg138Gln Het N N N Y N N Y Y N Y N N N Y Y Y Pathogenic NPHS2 AR 19 c.467_468dupT p.Leu156PhefsTer10 Het Y N N N N N Y Y Y N N N N Y Y Y Pathogenic Case c.104dupG p.Arg36ProfsTer33 Het Y N N N Y N Y Y N N N N N Y Y N Pathogenic NPHS2 AR 20 c.1143delC p.Met382CysfsTer? Het Y N N N Y N Y Y N N N N N Y Y N Pathogenic Case c.686G>A p.Arg229Gln Het N N N Y N Y N Y N Y N N N Y Y Y Pathogenic 21 NPHS2 AR c.911C>T p.Ser304Phe Het N N N N Y N Y Y N N N N N Y Y N Pathogenic

Case c.479A>G p.Asp160Gly Het N N N N Y N Y Y N Y N N N Y Y Y Pathogenic NPHS2 AR 22 c.855_856delAA p.Arg286ThrfsTer16 Het Y Y N N N N Y Y N N N N N Y Y Y Pathogenic

Case c.365G>C p.Trp122Ser Het N N N N Y N Y Y N Y N N N Y Y Y Pathogenic NPHS2 AR 23 c.416T>C p.Leu139Pro Het N N N N Y N Y Y N Y N N N Y Y N Pathogenic

Case NPHS2 AR c.538G>A p.Val180Met PatUPD Y N N N Y N Y N N N N N N Y Y Y Pathogenic 24 Case NPHS2 AR c.419delG p.Gly140AspfsTer41 Hom Y N N N N N Y Y N N N N N Y Y Y Pathogenic 25 Case c.686G>A p.Arg229Gln Het N N N Y N Y N Y N Y N N N Y Y Y Pathogenic NPHS2 AR 26 c.883G>A p.Ala295Thr Het N N N N Y N Y Y N N N N N Y Y N Pathogenic

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Case c.946C>T p.Pro316Ser Het N N N N Y N Y Y N N N N N Y Y N Pathogenic NPHS2 AR 27 c.686G>A p.Arg229Gln Het N N N Y N Y N Y N Y N N N Y Y Y Pathogenic Case c.467T>C p.Leu156Ser Het N N N N Y N Y Y N N N N N Y Y N Pathogenic NPHS2 AR 28 c.855_856delAA p.Arg286ThrfsTer16 Het Y Y N N N N Y Y N N N N N Y Y Y Pathogenic Case c.2928G>T p.Arg976Ser Het N N N N Y N Y Y N N N N N Y Y Y Pathogenic 29 NPHS1 AR c.2299C>T p.Pro767Ser Het N N N N Y N Y Y N N N N N Y Y N Pathogenic

Case Likely 30 ANLN AD c.745A>G p.Asn249Asp Het N N N N Y N Y N N N N Y N Y Y N Pathogenic

Case c.325_326delAT p.Leu110GlufsTer13 Het Y N N N Y N Y Y N N N Y N Y Y N Pathogenic 31 PLCE1 AR c.4570_4572delAT p.Met1524del Het N N N N Y N Y Y Y N N Y N Y Y N Pathogenic G Case c.4327G>A p.Gly1443Arg Het N N N N Y N Y Y N N N N N Y Y N Pathogenic 32 PLCE1 AR c.2038C>T p.Gln680Ter Het Y N N N Y N Y Y N N N N N Y Y N Pathogenic

Case 33 ACTN4 AD c.782T>A p.Val261Glu Het N N Y N Y N Y N N N N N N Y Y N Pathogenic

Case ACTN4 AD c.464T>C p.Ile155Thr Het N N Y N Y N Y N N N N N N Y Y N Pathogenic 34 Case Pathogenic WT1 AD c.1388C>T p.Ser463Phe Het N N Y N Y Y Y N N N N N N Y Y N 35 Case Pathogenic WT1 AD c.1384C>T p.Arg462Trp Het N N Y N Y Y Y N N N N N N Y Y N 36 Case Pathogenic WT1 AD c.1300C>T p.Arg434Cys Het N N Y N Y Y Y N N N N N N Y Y N 37

ACMG, American College of Medical Genetics and Genomics; MOI, model of inheritance; AR, autosomal recessive; AD, autosomal dominant; XLR, X-linked recessive;

XLD, X-linked dominant; PatUPD, paternal uniparental isodisomy; Hom, homozygous; Het, heterozygous; Hem, hemizygous; N, no; Y, yes.

PVS1: Null variant (nonsense, frameshift, canonical +/− 1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a

known mechanism of disease.

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PS1: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

PS2: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

PS4: The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls.

PM1: Located in a mutational hot spot and/or critical and well-established functional domain.

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC.

PM3: For recessive disorders, detected in trans with a pathogenic variant.

PM4: Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.

PM5: Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

PM6: Assumed de novo, but without confirmation of paternity and maternity.

PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

PP2: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc).

PP4: Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

PP5: Reputable source recently reports variant as pathogenic but the evidence is not available to the laboratory to perform an independent evaluation.

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Supplemental Table 4. List of variants of Unknown significance based on the ACMG score.

Nucleotide Amino acid PVS Variant Gene MOI Zygosity PS1 PS2 PS3 PS4 PM1 PM2 PM3 PM4 PM5 PM6 PP1 PP2 PP3 PP4 PP5 BA1 BS1 BS2 BS3 BS4 BP1 BP2 BP3 BP4 BP5 BP6 BP7 change change 1 Class

Case SIX5 AD c.811G>A p.Asp271Asn Het N N N N Y N N N N N Y N N Y Y N N Y Y N N N N N Y N Y N VUS 55

Case SOX17 AD c.181C>G p.Arg61Gly Het N N N N Y N Y N N N Y N N Y Y N N N N N N N N N N N N N P 56

Case c.3604G>A p.Ala1202Thr Het N N N N Y N Y N N N N N N Y Y N N N N N N N N N N N N N LP CUBN AR 57 c.8203G>T p.Asp2735Tyr Het N N N N Y N Y N N N N N N Y Y N N N N N N N N N N N N N LP

Case TRPC6 AD c.172C>T p.Arg58Trp Het N N N N Y N Y N N N Y N N N Y N N Y N N N N N N Y N Y N VUS 58

Case c.2230G>A p.Val744Met Het N N N N Y N Y N N N N N N Y Y N N N N N N N N N N N N N LP NPHP4 AR 59 c.3329C>T p.Ala1110Val Het N N N N Y N Y N N N N N N Y Y N N N Y N N N N N N N Y N VUS

Case CUBN AR c.5911C>A p.Pro1971Thr Hom N N N N Y N Y N N N N N N N Y N N N N N N N N N Y N Y N VUS 60

Case c.2276C>T p.Pro759Leu Het N N N N Y N Y N N N N N N Y Y N N N Y N N N N N N N Y N VUS COL4A4 AR 61 c.3871C>G p.Pro1291Ala Het N N N N Y N Y N N N N N N Y Y N N N Y N N N N N N N Y N VUS

Case c.64A>T p.Arg22Term Het Y N N N N N N Y Y N N N N N Y N Y Y Y N N N N N Y N Y N VUS COQ2 AR 62 c.879G>C p.Trp293Cys Het N N N N Y N Y Y N N N N N Y Y N N N N N N N N N N N N N P

CEP290 AR c.6401T>C p.Ile2134Thr Het N N N N Y N N N N N N N N Y Y Y N Y Y N N N N N N N N N VUS Case c.1991A>G p.Asp664Gly Het N N N N Y N N Y N N N N N Y Y N N Y Y N N N N N N N N N VUS 63 NPHP4 AR c.1852G>A p.Glu618Lys Het N N N N Y N N N N N N N N Y Y N N Y Y N N N N N N N N N VUS

FRAS1 AR c.160G>C p.Asp54His Het N N N N Y N N Y N N N N N N Y N N N Y N N N N N Y N Y N VUS Case c.6623T>C p.Leu2208Pro Het N N N N Y N Y Y N N N N N Y Y N N N N N N N N N N N N N P 64 FREM2 AR c.685C>T p.Arg229Cys Het N N N N Y N Y Y N N N N N Y Y N N N N N N N N N N N N N P

ACMG, American College of Medical Genetics and Genomics; MOI, model of inheritance; AR, autosomal recessive; AD, autosomal dominant; Hom, homozygous; Het, heterozygous; N, no; Y, yes; LP, likely Pathogenic, P, pathogenic; VUS; variant of uncertain significance.

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PVS1: Null variant (nonsense, frameshift, canonical +/− 1 or 2 splice sites, initiation codon, single or multi-exon deletion) in a gene where loss of function (LOF) is a known mechanism of disease.

PS1: Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

PS2: De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

PS3: Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

PS4: Prevalence of the variant in affected individuals significantly increased compared to the prevalence in controls.

PM1: Located in a mutational hot spot and/or critical and well-established functional domain.

PM2: Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or ExAC.

PM3: For recessive disorders, detected in trans with a pathogenic variant.

PM4: Protein length changes due to in-frame deletions/insertions in a non-repeat region or stop-loss variants.

PM5: Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

PM6: Assumed de novo, but without confirmation of paternity and maternity.

PP1: Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

PP2: Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

PP3: Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc).

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PP4: Patient’s phenotype or family history highly specific for a disease with a single genetic etiology.

PP5: Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

BA1: Allele frequency is above 5% in Exome Sequencing Project, 1000 Genomes, or ExAC.

BS1: Allele frequency is greater than expected for disorder.

BS2: Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder with full penetrance expected at an early age.

BS3: Well-established in vitro or in vivo functional studies showing no damaging effect on protein function or splicing.

BS4: Lack of segregation in affected members of a family.

BP1: Missense variant in a gene for which primarily truncating variants are known to cause disease.

BP2: Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

BP3: In-frame deletions/insertions in a repetitive region without a known function.

BP4: Multiple lines of computational evidence suggesting no impact on gene or gene product (conservation, evolutionary, splicing impact, etc).

BP5: Variant found in a case with an alternate molecular basis for disease.

BP6: Reputable source recently reports variant as benign, but the evidence not available to the laboratory to perform an independent evaluation.

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BP7: A synonymous (silent) variant for which splicing prediction algorithms predict no impact to the splice consensus sequence nor the creation of a new splice site

AND the nucleotide is not highly conserved. Variants should be classified as of Uncertain Significance if other criteria are unmet or the criteria for benign and pathogenic are contradictory

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Supplemental Table 5. Genetic profile of patients included in the study.

PATHOGENIC VARIANTS

Patient Gender Ethnicity In-house ACMG gnomAD control score25 Gene MOI OMIM Nucleotide change Amino acid change Inheritance allele Reference cohort (Variant frequency (300 WES) class) c.1445C>G p.Pro482Arg mat Likely NP NP -ND COL4A4 AR c.1445C>G p.Pro482Arg pat Pathogenic * 120131

Case 1 F Senegalese 0.22630 NP -92 c.1024A>G p.Ser342Gly mat Pathogenic * 603743 0.22600 NP -92 APOL1 AR c.1152T>G p.Ile384Met mat Pathogenic 0.14040 NP -92 c.1164_1169delTTATAA p.Asn388_Tyr389del pat Pathogenic c.3289+1G>A mat NP NP Pathogenic -93 Case 2 F European COL4A4 AR * 120131 c.2590G>A p.Gly864Arg pat NP NP Pathogenic -94

c.4421T>C p.Leu1474Pro mat 0.00489 NP Pathogenic -95 Case 3 F European COL4A3 AR * 120070 c.1831G>A p.Gly611Arg pat NP NP Pathogenic -ND

Case 4 F European COL4A5 XLD * 303630 c.1912G>A p.Gly638Ser mat 0.00000 NP Pathogenic -96

Case 5 M European COL4A5 XLD * 303630 c.991G>A p.Gly331Ser mat NP NP Pathogenic -ND

Case 6 F European COL4A5 XLD * 303630 c.3197G>A p.Gly1066Asp mat NP NP Pathogenic -ND

c.4868G>A p.Gly1623Asp mat NP NP Pathogenic -ND Case 7 M European LAMB2 AR * 150325 c.1931G>A p.Arg644His pat 0.00023 NP Pathogenic -ND

Likely Case 8 F European GLA XLR/XLD * 300644 c.937G>T p.Asp313Tyr pat 0.004458 NP -97 Pathogenic

c.6823G>A p.Asp2275Asn mat Case 9 M European FAT1 AR * 600976 0.00003 NP Pathogenic -ND c.6823G>A p.Asp2275Asn pat

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c.11265A>T p.Glu3755Asp pat NP NP Pathogenic -ND Case 10 F European FAT4 AR * 612411 c.11953T>A p.Tyr3985Asn mat NP NP Pathogenic -ND

Case 11 F European PAX2 AD * 167409 c.239C>T p.Pro80Leu de novo NP NP Pathogenic -98

Case 12 F Latino PAX2 AD * 167409 c.410+1G>A de novo NP NP Pathogenic -ND

Case 13 M European CLCN5 XLR * 300008 c.749G>A p.Gly250Asp mat NP NP Pathogenic -ND

Case 14 M European CLCN5 XLR * 300008 c.608C>G p.Ser203Trp NA NP NP Pathogenic -ND

Case 15 M European CLCN5 XLR * 300008 c.781G>A p.Gly261Arg mat NP NP Pathogenic -99

c.198_218del p.Ile67_Pro73del mat NP 0.00003 -79 Case 16 M European CTNS AR * 606272 Pathogenic arr[GRCh37] 17p13.2 (CTNS included in -ND pat NP (3505485_3560005)x1 deleted region, protein absent)

Case 17 M Tunisian LMX1b AD * 602575 c.764C>T p.Ala255Val pat NP NP Pathogenic -100

Pathogenic c.971A>G p.Tyr324Cys de novo 0.00002 NP -ND Case 18 M European KANK1 AR * 607704 Likely c.200G>A p.Arg67Gln mat 0.00628 NP -ND Pathogenic

c.413G>A p.Arg138Gln mat 0.00115 NP Pathogenic -101 Case 19 M European NPHS2 AR * 604766 c.467_468dupT p.Leu156PhefsTer10 pat 0.00083 NP Pathogenic -102

c.104dupG p.Arg36ProfsTer33 mat NP NP Pathogenic -103 Case 20 M European NPHS2 AR * 604766 c.1143delC p.Met382CysfsTer? pat NP NP Pathogenic -100

c.686G>A p.Arg229Gln mat 0.03591 NP Pathogenic -104 Case 21 F European NPHS2 AR * 604766 c.911C>T p.Ser304Phe pat NP NP Pathogenic -100

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c.479A>G p.Asp160Gly mat 0.00000 NP Pathogenic -103 Case 22 M European NPHS2 AR * 604766 c.855_856delAA p.Arg286ThrfsTer16 pat 0.00014 NP Pathogenic -103

c.365G>C p.Trp122Ser mat 0.00000 NP Pathogenic -105 Case 23 M European NPHS2 AR * 604766 c.416T>C p.Leu139Pro pat NP NP Pathogenic -ND

c.538G>A p.Val180Met Case 24 M European NPHS2 AR * 604766 patUPD 0.00000 NP Pathogenic -103 c.538G>A p.Val180Met c.419delG p.Gly140AspfsTer40 mat Case 25 F European NPHS2 AR * 604766 NP NP Pathogenic -103 c.419delG p.Gly140AspfsTer40 pat

c.686G>A p.Arg229Gln mat 0.03591 NP Pathogenic -104 Case 26 M European NPHS2 AR * 604766 c.883G>A p.Ala295Thr pat NP NP Pathogenic -106

c.946C>T p.Pro316Ser pat NP NP Pathogenic -36 Case 27 F European NPHS2 AR * 604766 c.686G>A p.Arg229Gln mat 0.03591 NP Pathogenic -104

c.467T>C p.Leu156Ser pat NP NP Pathogenic -ND Case 28 M European NPHS2 AR * 604766 c.855_856delAA p.Arg286ThrfsTer16 mat 0.00014 NP Pathogenic -103

c.2928G>T p.Arg976Ser pat 0.00007 NP Pathogenic -107 Case 29 F European NPHS1 AR * 602716 c.2299C>T p.Pro767Ser mat 0.00001 NP Pathogenic -108

Likely Case 30 M European ANLN AD * 616027 c.745A>G p.Asn249Asp pat NP NP -ND Pathogenic

c.325_326delAT p.Leu110GlufsTer13 pat 0.00000 NP Pathogenic -ND Case 31 M European PLCE1 AR * 608414 c.4570_4572delATG p.Met1524del mat NP NP Pathogenic -ND

c.4327G>A p.Gly1443Arg pat NP NP Pathogenic Case 32 F European PLCE1 AR * 608414 -100 c.2038C>T p.Gln680Ter mat NP NP Pathogenic

Case 33 F European ACTN4 AD * 604638 c.782T>A p.Val261Glu de novo NP NP Pathogenic -100

Case 34 F European ACTN4 AD * 604638 c.464T>C p.Ile155Thr de novo NP NP Pathogenic -100

Case 35 F European WT1 AD * 607102 c.1388C>T p.Ser463Phe de novo NP NP Pathogenic -ND

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Case 36 F European WT1 AD * 607102 c.1384C>T p.Arg462Trp de novo 0.00000 NP Pathogenic -109

Case 37 M European WT1 AD * 607102 c.1300C>T p.Arg434Cys de novo 0.00000 NP Pathogenic -109

M, male; F, female; mat, maternal; pat, paternal; NP, not present; ND, not described; MOI, model of inheritance; AR, autosomal recessive; AD,

autosomal dominant; XLR, X-linked recessive; XLD, X-linked dominant; patUPD, paternal uniparental isodisomy; WES, whole-exome sequencing,

ACMG, American College of Medical Genetics and Genomics. Sequence variant nomenclature follow the last update of HGVS (110).

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Supplemental Methods

DNA extraction

Peripheral blood DNA was extracted using QIAamp Mini Kit (QIAGEN®, Hilden,

Germany), according to manufacturer’s instructions, and quantified by NanoDROP 2000

Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

DNA library preparation

To construct DNA libraries we used a strategy based on enzymatic fragmentation to

produce dsDNA fragments followed by End repair, A-tailing, adapter ligation and

library amplification (Kapa Biosystems, Wilmington, MA). Libraries were hybridized

with the protocol SeqCap EZ Exome v3 (Nimblegen, Roche, Basel, Switzerland) and

sequenced with NextSeq500 (Illumina Inc., San Diego, CA).

Assembly, Variant Calling

The reads were aligned with the human reference hg19 genome using Burrows-Wheeler

Aligner (BWA) (111), mapped and analyzed with the IGV software (Integrative Genome

Viewer, 2013 Broad Institute) (112). Downstream alignment processing (i.e. alignment

sorting, indexing, deduplication and base quality score recalibration) was performed

with the Genome Analysis Toolkit Unified Genotyper Module (GATK) (113) SAMtools

(114) and Picard Tools (http://picard.sourceforge.net/). The GATK Unified Genotyper

was used to obtain a set of single nucleotide variants (SNV) and indel calls. We

performed a hard filtering of called variants based on four criteria: i) the variant

confidence (QUAL), that is the Phred-scaled posterior probability of the variant to be

homozygous reference; ii) the Quality by Depth (QD), that is the variant confidence

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divided by the unfiltered depth; iii) the unfiltered depth (DP), that is the number of reads

spanning the variant site, and iiii) the strand bias (FS), measured as the Phred-scaled p-

value using Fisher’s Exact Test to detect strand bias (the variation being seen on only

either the forward or the reverse strand) in the reads. We excluded the variant calls for

which at least one of these filters was true: QD < 5.0, DP < 5, FS > 60.0, QUAL < 30.0.

Variants were annotated using Annovar tool (115) to obtain information such as variant frequency in different populations and the predictions of the variant effect using different methods (SIFT, Polyphen2, MutationTaster, MutationAssessor, FATHMM and

FATHMM MKL). A variant effect consensus was obtained by categorizing the variant effects obtained for each method in deleterious and non-deleterious, then counting the number of deleterious effects to prioritize variants.

In order to estimate copy number variants (CNVs) we used a normalized read count

approach implemented in-house: in detail, for each patient we estimated the average

depth of coverage (aDOC) of every considered region using the program samtools depth

(114). The output has been passed to an awk command to compute the average coverage

for the region, as well as the percentage of base pairs with a depth of coverage of at least

1, 5, 10, 20 or 30. The bash function to perform this task is reported below:

function trancheCov

{

bam=$1

coord=$2

samtools depth -r $coord -a -a $bam |\

awk -v coord=$coord -F "\t" '{

if($3>=30){thirty++;twenty++;ten++;five++;one++}

else if($3>=20){twenty++;ten++;five++;one++}

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else if($3>=10){ten++;five++;one++} else if($3>=5){five++;one++} else if($3>=1){one++} mysum+=$3; tot++}

END{printf

"%s\t%s\t%s\t%s\t%s\t%s\t%s\n",coord,mysum/tot,100*one/tot,100*five/tot,100*ten/t ot,100*twenty/tot,100*thirty/tot}'

}

Where $coord is the coordinate string as reported in the bed file, and $bam is the indexed bam file of the corresponding patient.

Then we derived, for each patient, a normalized depth of coverage (nDOC), obtained by dividing the aDOC values of each region for the sum of aDOC over the considered regions.

In order to detect CNVs, finally, we compared for each region the nDOC values of every patients, computing the Median Absolute Deviation (MAD) to identify outliers in patients having low/high nDOC values.

The ratio between the nDOC values of the outliers and the median (R) indicates the type of CNVs: double deletion (R=0), single-copy deletion (R=0.5) and duplication (R>=1.5).

Variant interpretation strategy

Variants were filtered in silico for a panel of 298 genes (Supplemental Table 2) known to be associated with chronic kidney disease (CKD). A team of nephrologists, clinician scientists and geneticists performed variants prioritization.

34

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Variants were classified and scored in agreement with the interpretation guidelines of

the American College of Medical genetics and Genomics (ACMG) (Supplemental Table

3) (116).

In details, we selected only non-synonymous, short insertion/deletion or splice-site variants (30 bp splice acceptor, 30 bp splice donor) with the following characteristics

(Figure 2):

- Variants not present or with a minor allele frequency ≤ 0.01 for autosomal recessive

(AR) and with a minor allele frequency ≤0.001 for autosomal dominant (AD)- transmitted genes in population database “1000 Genomes Project”, “Exome Variant

Server”, dbSNP147, ExAC, gnomAD. We used manual inspection for the p.[Arg229Gln] variant in NPHS2 when segregating with variants localized in exon 7 and

8, as previously reported (104) and for APOL1 G1 and G2 variants (92).

- Variants not present or with a minor allele frequency ≤ 0.01 for AR and with a minor allele frequency ≤ 0.001 for AD-transmitted genes in “in-house” exome control cohort

(300 exomes) of unrelated subjects analyzed for non-renal diseases referred to the

Medical Genetic Unit of the Meyer Children’s Hospital (Florence, Italy).

- Variants predicted as damaging by at least 3 in silico tools (Polyphen-2, SIFT, Mutation

Taster, Mutation Assessor, FATHMM, FATHMM MKL).

- Variants correctly segregating within the family or representing de novo variants.

Variants were classified as pathogenic if they fitted all the criteria and were consistent with the clinical phenotype of the patient and family carriers after reverse phenotyping

(Figure 2). After the interpretation, the candidate variants were validated by Sanger sequencing. If no pathogenic variant was identified filtering for the 298 genes associated with CKD, we extended the analysis to all the exome.

35

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Statistical analysis

Statistical analysis was performed using SPSS statistical software (SPSS, Inc., Evanston,

IL). Kaplan-Meier estimates for SRNS patients were used to generate an overall 10 years

kidney survival curve, starting point disease onset and ending point end-stage kidney

disease development or censoring. Differences among groups were assessed by log-rank

test. Between-group comparisons were performed using one-way ANOVA for normally

distributed variables. A p-value <0.05 was considered statistically significant.

Web resources

1000 Genomes Project, http:// www.1000genomes.org

Exome Variant Server of the NHLBI Exome Sequencing Project (ESP),

http://evs.gs.washington.edu/ews

dbSNP147, https://www.ncbi.nlm.nih.gov/projects/SNP/

ExAC, http://exac.broadinstitute.org gnomAD, http://gnomad.broadinstitute.org/

Human Gene Mutation Database (HGMD), http://www.hgmd.cf.ac.uk/ac/index.php

Clinvar database, http://www.ncbi.nlm.nih.gov/clinvar/

Polyphen-2, http://genetics.bwh.harvard.edu/pph2/

SIFT, http://sift.jcvi.org/

Mutation Taster, http://www.mutationtaster.org/

Mutation Assessor, http://www.ngrl.org.uk/Manchester/page/mutation-assessor

FATHMM, http://fathmm.biocompute.org.uk/

FATHMM MKL, http://fathmm.biocompute.org.uk/fathmmMKL.ht

36

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case Reports

Case 1-3. These three patients showed steroid-resistant nephrotic syndrome, diagnosed

and treated accordingly in 2 (cases 1 and 2) or 3 (case 3) different European nephrology

centers and referred to our hospital on an average of 6 years after the clinical onset.

Kidney biopsies had shown focal segmental glomerulosclerosis (cases 1 and 2), or minimal change disease (case 3) without raising the suspect of Alport syndrome, even after performing electron microscopy. In addition, all 3 patients had been checked for sensorineural hearing loss at diagnosis of nephrotic syndrome with normal findings. Case 1 developed sensorineural hearing loss later on, when rechecked upon whole exome sequencing assessment eight years after onset of nephrotic syndrome, while cases 2 and 3 still showed no sensorineural hearing loss ten years after onset of nephrotic syndrome. Case 3 was treated with steroids, cyclosporine, tacrolimus, and mycophenolate mofetil, without any response to treatment. Whole exome sequencing identified pathogenic variants in

COL4A4 in cases 1-2, and in COL4A3 in case 3, as described in a previous report with distinct mutations (117, 118). Heterozygous parents showed microscopic hematuria without or with a mild proteinuria (300-500 mg/day) on reverse phenotyping. Parents

37

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of case 1 were not consanguineous but came from an inbred population of an African

village. Case 1 also carried APOL1 G1/G2 risk alleles (92) explaining her rapid

progression to end-stage kidney disease (7 years after onset).

Case 4-6. These patients showed steroid-resistant nephrotic syndrome and no signs of

Alport syndrome at biopsy, being diagnosed with focal segmental glomerulosclerosis or (cases 4 and 6) or minimal change disease (case 5) without raising the suspect of Alport syndrome, even after performing electron microscopy. Steroid-resistant nephrotic syndrome diagnosis was confirmed in 3 different

European nephrology centers for cases 4 and 6, who were referred to our hospital on average 13 years after onset of nephrotic syndrome. Whole exome sequencing identified pathogenic variants in COL4A5

inherited from the respective mothers. Reverse

phenotyping revealed that the mother of case 4 had no

renal involvement but had sensorineural hearing loss

while the mother of case 5 had microscopic hematuria.

In case 6, the mother had developed during pregnancy a nephrotic syndrome that

regressed after delivery with a mild residual proteinuria.

Case 7. This patient was diagnosed with steroid-resistant nephrotic syndrome in another pediatric nephrology center and referred to our Hospital for a second opinion on cyclosporine treatment. The biopsy showed focal segmental glomerulosclerosis. Whole

38

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

exome sequencing revealed compound heterozygous pathogenic missense variants in

LAMB2. LAMB2 deletion causes

Pierson syndrome, a condition that includes steroid-resistant nephrotic syndrome, but missense mutations are associated with isolated kidney involvement (119). The patient had an older sister previously treated for mesangioproliferative glomerulonephritis in another adult nephrology center that carried the same LAMB2 variants.

Case 8. This patient showed a severe isolated steroid-resistant nephrotic syndrome diagnosed in another Italian pediatric nephrology center, with focal segmental glomerulosclerosis at biopsy. After steroid treatment, she received cyclosporine, mycophenolate mofetil, tacrolimus, rituximab and even plasmapheresis without any response.

She was referred to our hospital for a second opinion one year after the onset of nephrotic syndrome. Our clinical assessment confirmed idiopathic steroid-resistant nephrotic syndrome. Whole exome sequencing revealed the

“D313Y” variant in GLA (97, 120), a pseudodeficiency allele causative for organ- specific Fabry disease, often with distinct organ involvement in different members of the same family (97). Reverse phenotyping showed lysosomal accumulation and vacuolization in podocytes with evidence of few very small concentric lamellar bodies in the podocytes in the kidney biopsy (97). The variant was inherited from the apparently healthy father who showed decreased α-Gal A activity and was diagnosed

39

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

with sensorineural hearing loss only upon reverse phenotyping. sensorineural hearing loss is reported in 60% of subjects with Fabry disease, particularly hemizygous males

(121).

Case 9. This patient was diagnosed with nephrotic syndrome in another nephrology center and referred to our hospital for a second opinion one year after. He had history of cluster headache. Kidney biopsy showed focal segmental glomerulosclerosis with tubular atrophy. Genetic testing revealed homozygous pathogenic variants in FAT1, inherited by non-consanguineous healthy parents from an inbred Italian village, consistently with a recent report (14).

Case 10. This patient showed nephrotic proteinuria (3-4 g/day). Renal ultrasound scanning at onset was unremarkable.

She showed compound heterozygous pathogenic variants in FAT4, a well- known gene of congenital abnormalities of the kidney and urinary tract (CAKUT), causing kidney hypodysplasia.

Reverse phenotyping with renal scintigraphy revealed a left renal hypodysplasia (15).

Case 11-12. These patients were diagnosed with idiopathic nephrotic syndrome in another nephrology center and referred to our hospital for a second opinion 2 years after the diagnosis of nephrotic syndrome. Case 11 showed focal segmental

40

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

glomerulosclerosis at biopsy. In both cases, whole exome sequencing revealed de novo

pathogenic variant in PAX2 (98). Reverse phenotyping

confirmed that they were affected by isolated focal

segmental glomerulosclerosis. Consistently, deletions of

PAX2 are associated with the renal-coloboma syndrome, while missense mutations, are associated with isolated focal segmental glomerulosclerosis and steroid-resistant

nephrotic syndrome (122).

Cases 13-15. These patients showed nephrotic syndrome and focal segmental

glomerulosclerosis (cases 13 and 15) or minimal change

disease (case 14) at biopsy. Kidney function was normal.

Nephrocalcinosis was not observed. Patients 14 and 15

were diagnosed with steroid-resistant nephrotic syndrome

in at least two different nephrology centers and treated with

calcineurin inhibitors with no response before referral to

our hospital on average 3 years after the onset of nephrotic

syndrome. In all these patients whole exome sequencing

showed a pathogenic variant in CLCN5. No other signs of

tubular dysfunction (e.g. glycosuria, hypercalciuria)

appeared over time in two patients, while the other one

developed hypercalciuria later on, in agreement with a

recent report showing that 50% of patients affected by Dent

disease present with NS in absence of hypercalciuria (123).

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Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

Case 16. This patient showed isolated nephrotic syndrome with a severe albuminuria

and was diagnosed with steroid-resistant nephrotic syndrome in another nephrology

center. Polyuria, hematuria, low-molecular weight proteinuria or other signs of tubular

dysfunction were absent. Kidney ultrasounds and scintigraphy were normal. Kidney

biopsy showed minimal change disease. He was referred to our center for a second

opinion two years after the clinical onset. Whole exome sequencing showed two

pathogenic variants in CTNS inherited from each of the parents. Upon reverse

phenotyping, slit-lamp examination revealed symmetric corneal crystals. The half- cysteine levels in peripheral blood polymorphonuclear cells were elevated

(2.0 nmol/mg of protein, normal values <0.3). Finally, biopsy re-evaluation showed several multinucleated podocytes on electron microscopy. Previous studies described how nephropathic cystinosis may clinically appear as a podocytopathy, with nephrotic syndrome (124-126), sometimes in absence of tubular dysfunction or extra-renal signs

(127).

Case 17. This patient developed severe nephrotic syndrome resistant to steroids and calcineurin inhibitors. The biopsy showed focal segmental glomerulosclerosis.

Although whole exome sequencing identified a pathogenic variant in LMX1B inherited from the apparently healthy father, no signs of Nail-

Patella syndrome were evident apart from NS,

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Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

consistently with previous reports (128). Reverse phenotyping by X-rays showed a bilateral lack of the ossification nuclei of the radium, the only bone abnormality related to the syndrome. Upon re-evaluation we observed normal nails, but absence of the thumbs lunulae. Reverse phenotyping of the father showed microscopic hematuria and mild proteinuria (300-500 mg/day) (100).

Case 18. This patient showed isolated nephrotic syndrome with a severe albuminuria and was diagnosed with steroid-resistant nephrotic syndrome in another nephrology center.

Genetic testing revealed compound heterozygous pathogenic variants in KANK1, one inherited by the mother and the other appeared de novo.

Kidney biopsy showed a minimal change disease. The patient was also followed in the neurology department for an epilepsy that turned out to be multi-drug resistant that upon reverse phenotyping was also attributed to the KANK1 mutation (129).

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Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material.

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