CJASN ePress. Published on November 10, 2017 as doi: 10.2215/CJN.04120417 Article

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome

Jillian K. Warejko, Weizhen Tan, Ankana Daga, David Schapiro, Jennifer A. Lawson, Shirlee Shril, Svjetlana Lovric, Shazia Ashraf, Jia Rao, Tobias Hermle, Tilman Jobst-Schwan, Eugen Widmeier, Amar J. Majmundar, Ronen Schneider, Heon Yung Gee, J. Magdalena Schmidt, Asaf Vivante, Amelie T. van der Ven, Hadas Ityel, Jing Chen, Carolin E. Sadowski, Stefan Kohl, Werner L. Pabst, Makiko Nakayama, Michael J.G. Somers, Nancy M. Rodig, Ghaleb Daouk, Michelle Baum, Deborah R. Stein, Michael A. Ferguson, Avram Z. Traum, Neveen A. Soliman, Jameela A. Kari, Sherif El Desoky, Hanan Fathy, Martin Zenker, Sevcan A. Bakkaloglu, Dominik Mu¨ller, Aytul Noyan, Fatih Ozaltin, Due to the number of Melissa A. Cadnapaphornchai, Seema Hashmi, Jeffrey Hopcian, Jeffrey B. Kopp, Nadine Benador, Detlef Bockenhauer, contributing authors, the affiliations are Radovan Bogdanovic, Natasˇa Stajic, Gil Chernin, Robert Ettenger, Henry Fehrenbach, Markus Kemper, provided in the Reyner Loza Munarriz, Ludmila Podracka, Rainer Bu¨scher, Erkin Serdaroglu, Velibor Tasic, Shrikant Mane, Supplemental Richard P. Lifton, Daniela A. Braun, and Friedhelm Hildebrandt Material.

Correspondence: Abstract Dr. Friedhelm Background and objectives Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More Hildebrandt, Division than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously of Nephrology, detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic Department of Medicine, Boston syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We Children’s Hospital, employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an 300 Longwood international cohort of 300 families. Avenue, Boston, MA 02115. Email: Design, setting, participants, & measurements Three hundred thirty-five individuals with steroid-resistant friedhelm. hildebrandt@ nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted childrens.harvard.edu to ,25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes.

Results In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid- resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2,andSMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome.

Conclusions Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid- resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 13: ccc–ccc, 2018. doi: https://doi.org/10.2215/CJN.04120417

Introduction ESRD. At this time, there is no effective therapy to curtail Nephrotic syndrome in childhood is characterized by the relentless progression to ESRD. proteinuria (.40 mg/m2 per hour), hypoalbuminemia, The most frequent kidney histologic feature of ste- edema, and hyperlipidemia. It can cause hypertension, roid-resistant nephrotic syndrome is FSGS. In patients severe infections, and thrombotic events. Patients are with FSGS, the risk of recurrence after kidney trans- classified by their response to steroid therapy. In children plantation is estimated to be approximately 33% (2–4). and young adults, about 80% of patients respond to FSGS constitutes the third most prevalent cause of standard steroid therapy and are termed steroid sensitive ESRD in the first two decades of life (5). To date, .30 (1). In contrast, individuals with steroid-resistant ne- monogenic causes of steroid-resistant nephrotic syn- phrotic syndrome overwhelmingly progress to CKD and drome have been identified (6), many of which www.cjasn.org Vol 13 January, 2018 Copyright © 2018 by the American Society of Nephrology 1 2 Clinical Journal of the American Society of Nephrology

implicate the glomerular podocyte and slit membrane as the nondeleterious variants as previously described (8,11). Vari- primary sites where the pathogenesis of steroid-resistant ants with minor allele frequencies .1% in the dbSNP (version nephrotic syndrome unfolds (7). The majority of genes 142) or in the 1000 Genomes Project (1094 patients of various known to cause steroid-resistant nephrotic syndrome are ethnicities; May 2011 data release) databases were excluded recessively inherited. Patients with mutations in these genes because they were unlikely to be deleterious. We used manual manifest with steroid-resistant nephrotic syndrome in child- inspection for the p.Arg229Gln mutation in the NPHS2 gene hood and adolescence, whereas dominant steroid-resistant which is reported to be deleterious with other variants, which nephrotic syndrome genes often manifest later in life. would be filtered out using this method (14). Synonymous We showed previously by targeted panel sequencing of 27 variants and intronic variants that were not located within known steroid-resistant nephrotic syndrome genes that in 30% splice site regions were excluded. Remaining variants included of steroid-resistant nephrotic syndrome cases with onset before nonsynonymous variants and splice site variants. 25 years, a causative mutation can be detected (8). However, panel sequencing by multiplex PCR is limited by requiring Mutation Calling in Known Steroid-Resistant Nephrotic large numbers of Sanger sequencing to confirm individual Syndrome Genes genetic variants (8). Additionally, evaluation of genes by panel We evaluated whole exome sequencing data for causative sequencing is limited to approximately 30 genes. With the mutations in 33 steroid-resistant nephrotic syndrome genes growing number of genes available, we sought a mechanism known at the time (Supplemental Table 2). Mutation calling by which we could evaluate a patient for a high number of was applied as stated above, followed by filtering remaining steroid-resistant nephrotic syndrome genes, as well as detect fi variants for changes in the regions of the 33 genes. Remaining novel causes of nephrosis should no known gene be identi ed. variants were ranked on the basis of their probable effect on In a cohort of patients with CKD and the phenotype of fi the function of the encoded protein considering evolutionary increased kidney echogenicity, we identi ed a causative conservation among orthologs using ENSEMBL Genome mutation in 63% using whole exome sequencing (9). We Browser and assembled using Clustal Omega, as well as evaluated here the utility of whole exome sequencing in an PolyPhen-2 (15), SIFT (16), and MutationTaster (17). Muta- international cohort with steroid-resistant nephrotic syn- tions were designated as likely pathogenic on the basis of drome. To date, this cohort is the largest to undergo whole criteria given by Supplemental Table 3. Mutation calling was exome sequencing (10). Given the very high rate of fi performed by clinician scientists/geneticists, with knowledge establishing an etiologic diagnosis and the signi cant of the clinical phenotypes and pedigree structure, as well as implications for clinical management and pretransplant experience with homozygosity mapping and whole exome and post-transplant care, whole exome sequencing should sequencing evaluation. Remaining variants were confirmed be considered in all individuals with steroid-resistant in patient DNA by Sanger sequencing as previously de- nephrotic syndrome diagnosed before age 25 years. scribed (8). Whenever parental DNA was available, segre- gation analysis was performed. If no causative mutation was identified, we evaluated for Materials and Methods mutations in genes that may represent phenocopies of Human Participants steroid-resistant nephrotic syndrome (Supplemental Table The study was approved by the institutional review 2). Variants were evaluated as above. Correlation of ge- board of the University of Michigan and Boston Children’s notype and phenotype was examined and, if matching, the Hospital. From April of 1998 to June of 2016, patients were genetic variant was deemed a causative mutation. enrolled after obtaining informed consent. Inclusion crite- ria were: onset of symptoms before 25 years and a clinical diagnosis of steroid-resistant nephrotic syndrome (e.g., Mutation Calling to Identify Novel Causes of Steroid- proteinuria, hypoalbuminemia, edema) or nephrotic range Resistant Nephrotic Syndrome proteinuria with kidney histology of FSGS or diffuse Ifnocausativemutationwasfoundinaknownsteroid- mesangial sclerosis (Supplemental Table 1). Three hundred resistant nephrotic syndrome gene and a family had homo- thirty-five individuals from 300 families were enrolled. zygosity (.100 Mbp) detected after homozygosity mapping, Before December of 2013, enrolled individuals were we then evaluated whole exome sequencing data for homo- screened for mutations in WT1 and NPHS2. Those that zygous variants. Single heterozygous variants were excluded. screened positive were not included in this study. We applied homozygosity mapping in consanguineous fam- ilies or linkage analysis in sibling cases to filter variants (18). Remaining variants were ranked as described above. Variants Whole Exome Sequencing and Variant Calling fi Whole exome sequencing and variant burden analysis were were con rmed as described above. performed as previously described (11–13). Genomic DNA was isolated from blood lymphocyte or saliva samples and Homozygosity Mapping and Linkage Analysis subjected to exome capture using Agilent SureSelect human Before 2014, for genome-wide homozygosity mapping, the exome capture arrays (Life technologies) followed by next GeneChip Human Mapping 250k d Array (Affymetrix) was generation sequencing on the Illumina HighSeq sequencing used. Alternatively, homozygosity mapping data were gen- platform. Sequence reads were mapped to the human refer- erated from whole exome sequencing data. Nonparametric ence genome assembly (NCBI build 37/hg19) using CLC logarithm (base 10) of odds scores were calculated using a Genomics Workbench (version 6.5.2) software (CLC bio, modified version of the program GENEHUNTER2.1 (19,20) Aarhus, Denmark). After alignment to the human reference through stepwise use of a sliding window with sets of 110 genome (GRCh37/hg19), variants were filtered for most likely SNPs and the program ALLEGRO (21) in order to identify Clin J Am Soc Nephrol 13: ccc–ccc, January, 2018 Exome Sequencing for Steroid-Resistant Nephrotic Syndrome, Warejko et al. 3

Table 1. Number and proportion of 300 families with steroid-resistant nephrotic syndrome, in whom causative mutations in one of 23 known monogenic causes of steroid-resistant nephrotic syndrome were detected

Number of Families Percentage of Families Number of Mutations Number of Novel Gene with Causative with Gene, % Known from Biobasea Mutations Mutation

SRNS genes NPHS1 13 18 10 1 PLCE1 11 15 8 2 NPHS2 81191 SMARCAL1 81151 LAMB2 6833 NUP93 4521 MYO1E 2311 SGPL1 34— 2 WDR73 343— ITGA3 232— LMX1B 231— NUP205 232— TTC21B 2311 WT1 231— COQ2 1111 DGKE 111— INF2 11— 1 KANK4 111— PDSS2 111— TRPC6 111— Total 74 100 53 15 Phenocopy genes COL4A5 3272— AGXT 21802 CLCN5 19.1 — 1 COL4A3 19.1 — 1 CTNS 19.11— FN1 19.1 — 1 GLA 19.11— WDR19 19.11— Total 11 100.0 5 5

Fifty-three of the mutations detected have previously been reported in BioBase, and 15 are novel (respective genes are under the table subheading ‘SRNS genes’). The most common genes to have a mutation detected in steroid-resistant nephrotic syndrome families were NPHS1, PLCE1, NPHS2,andSMARCAL1 (51% of all steroid-resistant nephrotic syndrome gene mutations detected). In an additional 11 families, mutations were detected in eight genes that cause a kidney disease that is a phenocopy of steroid-resistant nephrotic syndrome (respective genes are under the table subheading ‘Phenocopy genes’). Five of the mutations identified have previously been reported in BioBase, and five are novel. SRNS, steroid-resistant nephrotic syndrome; —, no mutations detected. aBiobase: https://portal.biobase-international.com/hgmd/pro. regions of homozygosity as described (18,22) using a disease Exome Variant Server, http://evs.gs.washington.edu/EVS; allele frequency of 0.0001 and white marker allele frequencies. Exome Aggregation Consortium, exac.broadinstitute.org; To generate homozygosity mapping after 2014, downstream HGMD Professional 2016.3, https://portal.biobase- processing of aligned binary alignment map files was done international.com/hgmd; using Picard and SAMtools4 (23). Single nucleotide variants Online Mendelian Inheritance in Man (OMIM), http://www. calling was performed using Genome Analysis Tool Kit (24) omim.org; and the generated variant call format file was subsequently Polyphen2, http://genetics.bwh.harvard.edu/pph2; used in Homozygosity Mapper (25). Sorting Intolerant From Tolerant (SIFT), http://sift.jcvi.org; MutationTaster, http://www.mutationtaster.org.

Web Resources Results UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/ Identification of Causative Mutations in One of 20 hgGateway; Steroid-Resistant Nephrotic Syndrome Genes in 25% of 1000 Genomes Browser, http://browser.1000genomes.org; Steroid-Resistant Nephrotic Syndrome Cases Clustal Omega, http://www.ebi.ac.uk/Tools/msa/clustal; Whole exome sequencing was performed in 335 in- Ensembl Genome Browser, http://www.ensembl.org; dividuals from 300 families and evaluated for mutations 4 Clinical Journal of the American Society of Nephrology

in the 33 steroid-resistant nephrotic syndrome genes known at the time (Table 1). In 74 families (25%), a caus- ative mutation in one of 20 known steroid-resistant ne- phrotic syndrome genes was detected (Figure 1, Table 1). NPHS1 (13 families), PLCE1 (11 families), NPHS2 (eight families), and SMARCAL1 (eight families) were the most common genes in which mutations were identified, comprising 51% of all mutations identified (Figure 1, Table 1). Ninety-four of the 300 families studied by whole exome sequencing have been previously studied using Fluidigm panel sequencing. The overlap between cohorts is given in Supplemental Table 4. We found that, whereas in 20 of 74 (27%) families the causative mutation was previously detected using panel sequencing, 9 of 74 (12%) had a diagnosis made by whole exome sequencing and not by panel sequencing. In an additional 28% of families, we detected a likely causative mutation in one or more potential novel steroid-resistant nephrotic syndrome genes Figure 1. | In 74 of 300 (25%) families with steroid-resistant ne- (Figure 1), given in Supplemental Table 5. phrotic syndrome, a causative mutation was detected in one of 20 genes known to cause steroid-resistant nephrotic syndrome (shades of blue). In 3.7% of families, a mutation was found in genes causing a Novel Mutations Detected in Known Steroid-Resistant kidney disease that may represent phenocopies of steroid-resistant Nephrotic Syndrome Genes nephrotic syndrome (orange). In 28% of families, one or more po- We detected 68 different mutations in 20 of 33 known tential novel candidate genes were identified (red). In 44% of families, steroid-resistant nephrotic syndrome genes, 53 of which no causative mutations or candidate genes were detected. SRNS, had previously been reported in the literature (Table 1). steroid-resistant nephrotic syndrome. Fifteen novel mutations have not been reported pre- viously (Table 1). Individual families in whom a causa- tive mutation was detected are described in Supplemental drome gene (Figure 4, Supplemental Table 7). In 56 of 147 Table 9. families with .100 Mbp of homozygosity on mapping (38%), a causative mutation was detected in a steroid- Whole Exome Sequencing Identifies Phenocopies in 11 of 90 resistant nephrotic syndrome gene. In contrast, in 17 of 135 Families with a Causative Mutation Detected (13%) nonconsanguineous families and 18 of 153 (12%) We detected a causative mutation in 11 of 300 families families with ,100 Mbp of homozygosity (nonhomozy- with steroid-resistant nephrotic syndrome (3.7%) (Figure 1, gous) on mapping, a causative mutation was detected in a Table 1). Mutations were found in eight phenocopy genes, fi steroid-resistant nephrotic syndrome gene (Figure 4). The speci cally COL4A5, COL4A3, CLCN5, GLA, AGXT, CTNS, difference in mutation detection between consanguineous FN1,andWDR19. A total of ten different mutations were fi and nonconsanguineous families and between homozy- detected, ve of which are novel (Table 1). gous and nonhomozygous families was statistically signif- icant using a chi-squared test (P,0.001) (Figure 4). There Novel Candidate Genes Are Identified Using Whole Exome was no significant difference in the rate of mutation Sequencing detectionwhencomparingfamilies with one affected In 61 of 146 (42%) consanguineous families with no individual versus two affected individuals or $3affected causative mutation found in a known steroid-resistant individuals (Figure 4). nephrotic syndrome gene, one or more candidate genes In 24% of those with additional systemic manifestations were detected using homozygosity mapping (Figure 2, in addition to kidney disease, a causative mutation was Supplemental Table 5). In nonconsanguineous families detected in a steroid-resistant nephrotic syndrome gene, . with 1 individual affected, linkage analysis was used compared with 27% of those with no additional systemic to identify a potentially causative mutation in 18 of 135 manifestations with a causative mutation detected in a families (13%). steroid-resistant nephrotic syndrome gene (Supplemental Figure 2, Supplemental Table 8). This difference was not Description of Cohort statistically significant. Onset of illness ranged from birth to 24 years of age The most common clinical diagnosis was steroid-resis- (Figure 3A, Supplemental Table 6). The median age in tant nephrotic syndrome in 205 of 300 (68%) (Supplemental individuals in whom a causative mutation was detected Figure 3, Supplemental Table 8). It was the most common in a steroid-resistant nephrotic syndrome gene was 1.7 clinical diagnosis in those families with a causative muta- years versus 4 years in those without a causative mutation tion identified (48 of 74 families, 65%) (Supplemental identified, which was statistically significant (Figure 3B). Figure 3, Supplemental Table 8). One hundred forty-six of 300 (49%) families were In 24% of individuals with FSGS on biopsy and in 14% of consanguineous, in 56 (38%) of whom we detected a individuals with diffuse mesangial sclerosis on biopsy, a causative mutation in a steroid-resistant nephrotic syn- causative mutation was detected in a steroid-resistant Clin J Am Soc Nephrol 13: ccc–ccc, January, 2018 Exome Sequencing for Steroid-Resistant Nephrotic Syndrome, Warejko et al. 5

Figure 2. | A causative mutation in a steroid-resistant nephrotic syndrome gene, phenocopy gene, or novel candidate gene is detected in a greater proportion of consanguineous families compared to non-consanguineous families. We detected a causative mutation in 38% of consanguineous families and 13% of nonconsanguineous families. Through homozygosity mapping and a recessive hypothesis, we were able to identify potentially causative mutations in 42% of consanguineous families. Potential causative mutations in novel candidate genes were detected in nonconsanguineous families by evaluating for overlapping genes in siblings. Percentages.10% are rounded to the nearest whole number. SRNS, steroid-resistant nephrotic syndrome. nephrotic syndrome gene (Supplemental Figure 4, Supple- separately for recessive or dominant. Criteria were on the mental Table 8). Two hundred twenty-three of 335 (66.6%) basis of evolutionary conservation, bioinformatic prediction individuals had kidney histology data available. programs of pathogenicity, and allele frequency in healthy control populations (Supplemental Tables 3 and 9). Before 2014, our lab screened for mutations in NPHS2 and Discussion WT1, which may account for lower prevalence in our cohort. Summary and Effect of this Work NPHS2 and WT1, two of the three most commonly mutated To date, this is the largest international cohort in which genes in early-onset steroid-resistant nephrotic syndrome, are molecular causes of steroid-resistant nephrotic syndrome underrepresented in the presented work, being together were evaluated using whole exome sequencing. Our rate of responsible for only 3.3% (Table 1) of 300 cases, whereas they mutation detection is 25%, consistent with our previous work were previously reported to be responsible for 15% of cases in (8). Recently, in 187 individuals, a causative mutation was 1783 cases (8). When all 1989 families studied in Sadowski detected in one of 53 steroid-resistant nephrotic syndrome et al. (8) and in this study are combined together, mutation genes in 26% of individuals (10). rates for NPHS2 and WT1 become more representative of We detected causative mutations in 20 of 33 known causes what has been previously published. NPHS2 has a detection of steroid-resistant nephrotic syndrome, with a total of 68 rate of 9.3% (185 of 1989) and WT1 has a detection rate of different mutations, 15 of which have not been reported in 4.4% (87 of 1989). Mutation rates for NPHS2 were previously the literature. To determine the pathogenicity of novel 9.9% and for WT1 were 4.8%. mutations in genes previously described to cause steroid- We detected mutations in eight genes that are pheno- resistant nephrotic syndrome, we used a strict set of criteria copies for steroid-resistant nephrotic syndrome, with five 6 Clinical Journal of the American Society of Nephrology

Figure 3. | After dividing the 335 individuals from 300 families with steroid-resistant nephrotic syndrome by gene identification status (steroid-resistant nephrotic syndrome gene, phenocopy gene, no mutation detected) and sorting by age and sex, the median age of individuals with a mutation detected in a steroid resistant nephrotic syndrome gene is significantly lower than the median age of individuals with a mutation detectedin aphenocopygene or individuals with no mutation detected. (A) Families in whom a causativemutation in a knownsteroid- resistantnephrotic syndromegene(blue)orphenocopygene(orange)wasdetectedas comparedwiththosefamilieswherenocausativemutation wasdetected(gray). Bars andnumbers atendofbars representnumberof affected individuals in eachcategory,divided intothosewith acausative mutation detected in a steroid-resistant nephrotic syndrome gene (blue), those with a causative mutation detected in a phenocopy gene (orange), and those without a causative mutation detected (gray). Percentages at end of each bar reflect the same three categories. Percentages.10% are Clin J Am Soc Nephrol 13: ccc–ccc, January, 2018 Exome Sequencing for Steroid-Resistant Nephrotic Syndrome, Warejko et al. 7

Figure 4. | Gene identification in a steroid-resistant nephrotic syndrome gene occurs in a statistically significant greater proportion of homozygous families (homozygosity >100 Mb) when compared to non-homozygous families (homozygosity <100 Mb) and in a statistically significant greater proportion of consanguineous families when compared to non-consanguineous families. Families in whom causative mutations in a known steroid-resistant nephrotic syndrome gene (blue) or a phenocopy gene (orange) were detected, compared with those families in whom no causative mutation was detected (gray). Bars and numbers at end of bars represent total number of families in each category, divided into those families with a causative mutation detected (blue), those families with a causative mutation detected in a phenocopy gene (orange), and those families without a causative mutation detected (gray). Percentages at end of each bar reflect the same three categories. Percentages.10% are rounded to the nearest whole number. Rate of detection of a causative mutation in a steroid-resistant nephrotic syndrome gene did not vary with number of affected individuals per family. Number of affected individuals per family did not have a statistically significant difference between one affected individual per family versus two affected individuals, or between one affected individual and $3 individuals. Mutation detection rate in a steroid-resistant nephrotic syndrome gene was significantly higher in those families that were reported clinically as consanguineous or had homozygosity on mapping .100 Mbp than those that were nonconsanguineous or had homozygosity,100 Mbp on mapping (two-sided chi-squared test P,0.001 for each condition). Data of the characteristics of the steroid-resistant nephrotic syndrome cohort compared with the subcohort of those families with a causative mutation detected in a steroid-resistant nephrotic syndrome gene or phenocopy gene are given in Supplemental Table 4. *Statistically significant. SRNS, steroid-resistant nephrotic syndrome. novel mutations and five mutations previously reported in may phenocopy steroid-resistant nephrotic syndrome and the literature. Because these genes may be excluded from provide the opportunity for novel gene discovery. panels that target steroid-resistant nephrotic syndrome specifically, these families may have been left without a Therapeutic Implications molecular diagnosis. Identification of a monogenic cause of steroid-resistant Whole exome sequencing allows for the identification of nephrotic syndrome has significant therapeutic implications. novel genes using homozygosity mapping in consanguine- (1) In children, treatment often requires prolonged steroid ous families and linkage analysis in related individuals. exposure and potentially exposure to multiple immunosup- Panels are limited as to how many genes could be evaluated pressant medications. All of these medications carry signif- in a given experiment. However, whole exome sequencing icant side effect profiles, including growth failure (steroids), allows for the evaluation of all genes, including those that bone marrow suppression (mycophenolate mofetil,

rounded to the nearest whole number. (B) Median age of onset in patients with a causative mutation detected in a steroid-resistant nephrotic syndromegenewas1.7yearsversus4yearsinthosewithout amutation detected(range,0–24 years).For thosewith acausativemutationdetected in a steroid-resistant nephrotic syndrome gene, the range was 0–21 years. Mann–Whitney U test P,0.01. Median age of individuals with a phenocopy mutation detected was 4 years (range, 0.3–16), which was not statistically significant. Data of the characteristics of the steroid- resistant nephrotic syndrome cohort compared with the subcohort of those individuals with a causative mutation detected in a steroid-resistant nephrotic syndrome gene or phenocopy gene are given in Supplemental Table 3. SRNS, steroid-resistant nephrotic syndrome. 8 Clinical Journal of the American Society of Nephrology

tacrolimus, azathioprine), kidney dysfunction (tacrolimus), Limitations and unacceptable cosmetic effects (cyclosporine), among One limitation to our study is that approximately 70% of other side effects. This generates an indication for fast, families remain undiagnosed. Our lab is currently perform- efficient exome sequencing in order to avoid unfavorable ing trio analysis, in which both parents and the proband side effects which may be experienced while taking medi- have sequencing performed, which allows for evaluation of cations that may not provide clinical benefit. (2) Identification nonconsanguineous individuals. We anticipate that this of a causative mutation may reveal that a potential therapy is may increase the number of candidate genes identified and available for some rare single-gene causes of nephrosis. For lead to future molecular genetic diagnoses. example, if a mutation in a gene of coenzyme Q10 bio- synthesis (COQ2, COQ6, ADCK4,orPDSS2) is detected, Cost of Whole Exome Sequencing – treatment with coenzyme Q10 may be indicated (26 28). In Whole exome sequencing has several advantages over the case of the individual with COQ2 mutation, this indi- panel sequencing. It has the theoretic likelihood of 86% of vidual was placed on COQ10 therapy and experienced a detecting recessive disease mutations. Whole exome se- fi sustained remission of nephrosis. (3)Identi cation of caus- quencing examines all exons in a genome, whereas panel ative mutations in WT1 can also lead to surgical evaluation sequencing typically examines only approximately 30. and intervention to remove streak gonads with potential for With falling costs of sequencing, a research exome is malignant transformation (29). (4) Genotype and phenotype approximately $650, which ultimately is more cost effective fi correlations in the future may lead to strati cation in clinical than panel sequencing because hundreds of panels would fi trials for novel therapeutics. In our study, we identify ve be required to cover the whole exome and would not families with the p.R1160* mutation in NPHS1. This mutation provide the opportunity to identify for novel causes of has been shown to cause congenital nephrotic syndrome; disease. Once an exome is performed, the data can be however, in some patients with this mutation, a milder revisited as more genes are discovered. With the introduc- phenotype has been reported (30). (5) Furthermore, identi- tion of trio analysis, nonconsanguineous families can be fi cation of mutations in genes that may represent pheno- evaluated for novel genes, potentially allowing for a copies of steroid-resistant nephrotic syndrome, such as conclusive monogenic diagnosis in the future. cystinosis, hyperoxaluria, or Fabry’s disease, can direct therapy. Mutations in these genes would be missed in panel Acknowledgments sequencing, because they are not canonic nephrosis genes, We thank the physicians and the participating families for their but may present with proteinuria, edema, and CKD. The contribution. F. Alkandary, Hawally (Kuwait), M. Al Shebadi, and ability to detect mutations in genes that represent pheno- B. Alhermi, Manama (Bahrain); A. Al Swaid, Riyadh (Saudi Arabia), copies of nephrosis is a benefit of whole exome sequencing D. Aviles, New Orleans, Louisiana; A. Bakkaloglu, Ankara (Turkey); over panel sequencing. A. Bagga, New Delhi (India); M. Banks, Denver, Colorado; B. Beck, Cologne (Germany); J. Behunova, Kosice (Slovakia); E. Ben Shalom, Jerusalem (Israel); A. Berdeli, Izmir (Turkey); C. Brix, Hamburg Implications for Kidney Transplant (Germany); J. Camacho, Irvine, California; B. Cavan, Cebu City Many patients progress to ESRD, requiring transplanta- (Philippines); R. Cohn, Chicago, Illinois; G.S. Ch’ng, Kuala Lumpur tion and dialysis. Given that approximately 30% of all cases (Malaysia); R. Cohen, Jerusalem (Israel); E. Comak, Antalya (Tur- of idiopathic FSGS can recur post-transplant, many centers key); I. Dursun, Kayseri (Turkey); E. Epstein, Oakland, California; employ increased immunosuppression before and after E. Evani and D. Milford, Birmingham (United Kingdom); R. Fine, transplant to prevent recurrence (31,32). Because mono- Los Angeles, California; J. Flynn, Seattle, Washington; G. Giannico, genic forms of FSGS are unlikely to recur, young children Nashville, Tennessee; K. Gunther, St. Gallen (Switzerland); could be spared exposure to aggressive immunosuppres- K. Haffner, Freiburg (Germany); M. Halty, Montevideo (Uruguay); sion and avoid many of the infectious complications seen in C. Hanna, Minneapolis, Minnesota; M. Hashim, Chennai (India); transplantation (10,33). Patients with a monogenic cause of J. Hernandez, Spokane, Washington; J. Hogan, Philadelphia, Penn- nephrosis identified are younger that those that do not sylvania; R. Jenkins, Portland, Oregon. R. Johnson, Denver, Colo- have a causative mutation identified (Figure 3, A and B, rado; C. Kempf, Berlin (Germany); W.T. Keng, Kuala Lumpus Supplemental Table 3), which puts them at greater risk for (Malaysia); S. Kalman, Ankara (Turkey); E. Kamil, Los Angeles, infections post-transplant, including Epstein–Barr virus California; D. Katalin, Leipzig (Germany); S. Kerr, Cord Springs, and cytomegalovirus. A monogenic diagnosis provides Florida; M. Khemchand, Karachi (Pakistan); K. Kranmiller, Salzburg the opportunity to reduce immunosuppression and reduce (Austria); J. Kwinta-Rybicka, Krakow (Poland); E. Lahav, Tel risk of infectious complication. Hashomer (Israel); D. Lewis, Sydney (Australia); K. Lhotta, Feldul- Furthermore, many pediatric patients receive a living rich (Austria); C. Licht and J. Dotsch, Koln (Germany); V. Logan and donor kidney transplant from a close relative, such as a P. Conlon, Dublin (Ireland); C. Mache,Graz (Austria); M. Mayr, Basel parent, and having a monogenic cause identified, such as (Switzerland); J. Misselwitz, Jena (Germany); T. Mueller, Vienna COL4A5, may have implications on donor selection. Ad- (Austria); J. Muhleder and M. Prenninger, Wels (Austria); ditionally, in families with INF2 mutations, the parents and C. Nailescu, Indianapolis, Indiana; S. Nampoothiri, Kerala (India); family members should be screened for INF2 mutations, S. Narayan, Orange, California; A. Nayir, Istanbul (Turkey); S. Nef, because this dominant disease has variable expressivity Zurich (Switzerland); T. Neuhaus, Zurich (Switzerland); W. Pietee, within families. Should a family member be positive for Kuala Lumpur (Malaysia); M. Pradhan, Philadelphia, Pennsylvania; mutation, this would disqualify them from donation of a A. Ramanathan,Chennai (India); C. Rinat and Y. FrishbergJerusalem kidney for transplantation because they are at risk for (Israel); I. Roberti, West Orange, New Jersey; R. Roszkowska, Bia- developing proteinuria and kidney disease in the future. łystok (Poland); J. Saland, New York City, New York; M. Schaeffer, Clin J Am Soc Nephrol 13: ccc–ccc, January, 2018 Exome Sequencing for Steroid-Resistant Nephrotic Syndrome, Warejko et al. 9

Hannover (Germany); R. Schild, Hamburg (Germany); A. Schulze Collaborative Studies (NAPRTCS) Registry. Pediatr Transplant 12: Everding, Muenster (Germany); W. Seeherunvong, Miami, Florida; 878–882, 2008 6. Lovric S, Ashraf S, Tan W, Hildebrandt F: Genetic testing in ste- P. Senguttvuan and A. Kumar, Chennai (India); J. Siegel-Bartel, roid-resistant nephrotic syndrome: When and how? Nephrol Dial Phoenix, Arizona; M. Soran, Adana (Turkey); A. Soylu, Izmir (Tur- Transplant 31: 1802–1813, 2016 key); M. Stephan, Munchen (Germany); C. Strehlau, Hannover 7. Wiggins RC: The spectrum of podocytopathies: A unifying view of (Germany); T. Sulakova, Ostrava (Czech Republic); K. Supe- glomerular diseases. Kidney Int 71: 1205–1214, 2007 Markovina, Stony Brook, New York; A. Szuminska, Białystok (Po- 8. Sadowski CE, Lovric S, Ashraf S, Pabst WL, Gee HY, Kohl S, Engelmann S, Vega-Warner V,Fang H, Halbritter J, Somers MJ, Tan land); R. Tapia, Willmington, Delaware; K. Taranta-Janusz, Białystok W, Shril S, Fessi I, Lifton RP, Bockenhauer D, El-Desoky S, Kari JA, (Poland); M. Theault, Minneapolis, Mennesota; Y. Tse, Newcastle Zenker M, Kemper MJ, Mueller D, Fathy HM, Soliman NA, upon Tyne (United Kingdom); L. Tranebjaerg, Copenhagen (Den- Hildebrandt F; SRNS Study Group: A single-gene cause in 29.5% mark); S. Twichell, Burlington, Vermont; M. Waszkiewicz-Stojda, of cases of steroid-resistant nephrotic syndrome. JAmSoc Białystok (Poland); R. Weiss, Valhalla, New York; J. Zeig, Prague Nephrol 26: 1279–1289, 2015 9. Braun DA, Schueler M, Halbritter J, Gee HY,Porath JD, Lawson JA, (Czech Republic). Airik R, Shril S, Allen SJ, Stein D, Al Kindy A, Beck BB, Cengiz N, F.H. is the William E. Harmon Professor of Pediatrics. This re- Moorani KN, Ozaltin F, Hashmi S, Sayer JA, Bockenhauer D, search was supported by grants from the National Institutes of Soliman NA, Otto EA, Lifton RP, Hildebrandt F: Whole exome Health to F.H. (DK076683) and J.K.W. (DK007726-31A1). The Yale sequencing identifies causative mutations in the majority of Center for Mendelian Genomics is funded by U54 HG006504 consanguineous or familial cases with childhood-onset increased renal echogenicity. Kidney Int 89: 468–475, 2016 granted to R.P.L. and by the National Institute of Diabetes and 10. Bierzynska A, McCarthy HJ, Soderquest K, Sen ES, Colby E, Ding Digestive and Kidney Diseases Intramural Research Program to J.B. WY, Nabhan MM, Kerecuk L, Hegde S, Hughes D, Marks S, K. Funding to W.T. through the American Society of Nephrology Feather S, Jones C, Webb NJ, Ognjanovic M, Christian M, Gilbert Benjamin J. Lipps Research Fellowship Award (FP01014311) and RD, Sinha MD, Lord GM, Simpson M, Koziell AB, Welsh GI, Saleem MA: Genomic and clinical profiling of a national ne- DK007726-31A1. H.Y.G. was supported by the Basic Science Re- phrotic syndrome cohort advocates a precision medicine ap- search Program through the National Research Foundation of Korea proach to disease management. Kidney Int 91: 937–947, 2017 (2015R1D1A1A01056685). T.H. is supported by a Research Fel- 11. Gee HY, Otto EA, Hurd TW, Ashraf S, Chaki M, Cluckey A, Vega- lowship from the Deutsche Forschungsgemeinschaft (DFG) (HE WarnerV,SaisawatP,DiazKA,FangH,KohlS,AllenSJ,AirikR,Zhou 7456/1-1). T.J.-S. is supported by the DFG (Jo 1324/1-1). A.J.M. is W,Ramaswami G, Janssen S, FuC,InnisJL, Weber S, VesterU,Davis EE, Katsanis N, Fathy HM, Jeck N, Klaus G, Nayir A, Rahim KA, Al supported by National Institutes of Health T32-AR053461-04 Attrach I, Al Hassoun I, Ozturk S, Drozdz D, Helmchen U, O’Toole Postdoctoral Fellow Training Grant. M.N. is supported by the Ii- JF, Attanasio M, Lewis RA, Nu¨rnberg G, Nu¨rnberg P, Washburn J, numa-Tsuchiya Foundation for Overseas Research. F.O. is sup- MacDonald J, Innis JW, Levy S, Hildebrandt F: Whole-exome re- ported by the European Community’s Seventh Framework sequencing distinguishes cystic kidney diseases from phenocopies Programme (FP7/2007-2013) (European Consortium for High- in renal ciliopathies. Kidney Int 85: 880–887, 2014 12. Boyden LM, Choi M, Choate KA, Nelson-Williams CJ, Farhi A, Toka Throughput Research in Rare Kidney Diseases, grant 2012-305608). HR, Tikhonova IR, Bjornson R, Mane SM, Colussi G, Lebel M, The Nephrogenetics Laboratory at Hacettepe University was estab- Gordon RD, Semmekrot BA, Poujol A, Va¨lima¨ki MJ, De Ferrari ME, lished by the Hacettepe University Infrastructure Project (grant SanjadSA,GutkinM,KaretFE,TucciJR,StockigtJR,Keppler-Noreuil 06A101008). A.V. is supported by the Manton Center for Orphan KM, Porter CC, Anand SK, Whiteford ML, Davis ID, Dewar SB, Diseases Research grant. A.T.v.d.V. is supported by the Postdoctoral Bettinelli A, Fadrowski JJ, Belsha CW, Hunley TE, Nelson RD, Trachtman H, Cole TR, Pinsk M, Bockenhauer D, Shenoy M, Research Fellowship (VE 916/1-1) from the DFG. E.W. is supported Vaidyanathan P, Foreman JW, Rasoulpour M, Thameem F, Al- by the German National Academy of Sciences Leopoldina (LPDS- Shahrouri HZ, Radhakrishnan J, Gharavi AG, Goilav B, Lifton RP: 2015-07). Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482: 98–102, 2012 13. Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang Disclosures H, Hurd TW, Zhou W, Cluckey A, Gee HY, Ramaswami G, Hong Friedhelm Hildebrandt has intellectual property licensed to C-J, Hamilton BA, Cervenkaˇ I, Ganji RS, Bryja V, Arts HH, van Claritas and is a cofounder of Goldfinch. 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Salviati L, Hurd TW, Vega-Warner V,Killen PD, Raphael Y,Ashraf asnjournals.org/lookup/suppl/doi:10.2215/CJN.04120417/-/ S, Ovunc B, Schoeb DS, McLaughlin HM, Airik R, Vlangos CN, DCSupplemental. Author List Supplement

Jillian K. Warejko1*, Weizhen Tan1*, Ankana Daga1, David Schapiro1, Jennifer A. Lawson1, Shirlee Shril1, Svjetlana Lovric1, Shazia Ashraf1, Jia Rao1, Tobias Hermle1,Tilman Jobst-Schwan1, Eugen Widmeier1, Amar J. Majmundar1, Ronen Schneider1, Heon Yung Gee1,2, J. Magdalena Schmidt1, Asaf Vivante1,3, Amelie T. van der Ven1, Hadas Ityel1, Jing Chen1, Carolin E. Sadowski1, Stefan Kohl1, Werner L. Pabst1, Makiko Nakayama1, Michael J.G. Somers1, Nancy M. Rodig1, Ghaleb Daouk1, Michelle Baum1, Deborah R. Stein1, Michael A. Ferguson1, Avram Z. Traum1, Neveen A. Soliman4, Jameela A. Kari5, Sherif El Desoky5, Hanan Fathy6, Martin Zenker7, Sevcan A. Bakkaloglu8, Dominik Müller9, Aytul Noyan10, Fatih Ozaltin11, Melissa A. Cadnapaphornchai12, Seema Hashmi13, Jeffrey Hopcian14, Jeffrey B. Kopp15, Nadine Benador16, Detlef Bockenhauer17, Radovan Bogdanovic18, Nataša Stajić18, Gil Chernin19, Robert Ettenger20, Henry Fehrenbach21, Markus Kemper22, Reyner Loza Munarriz23, Ludmila Podracka24, Rainer Büscher25, Erkin Serdaroglu 26, Velibor Tasic27, Shrikant Mane28, Richard P. Lifton29, Daniela A. Braun1, and Friedhelm Hildebrandt1

* These authors contributed equally to this work. 1 Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 2Department of Pharmacology, Brain Korea 21 Program for Leading Students & Universities, Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 3Talpiot Medical Leadership Program, Sheba Medical Center, Tel-Hashomer, Israel 4Department of Pediatics,Cairo University Center of Pediatric Nephrology &Transplantation, Kasr Al Ainy Medical School, Cairo University, Cairo, Egypt 5Department of Pediatrics, King AbdulAziz University, Jeddah, Saudi Arabia 6Pediatric Nephrology Unit, University of Alexandria, Alexandria, Egypt 7Institute of Human Genetics, University Hospital Magdeburg, Otto-von-Guericke University, Magdeburg, Germany 8Department of Pediatric Nephrology, Gazi University, Ankara, Turkey 9Department of Pediatric Nephrology, Charité, Berlin, Germany 10Department of Pediatric Nephrology, Adana Teaching and Research Center, Baskent University, Adana, Turkey 11Department of Pediatric Nephrology, Nephrogenetics Laboratory, Hacettepe University Faculty of Medicine, Ankara, Turkey 12Division of Renal Disease and Hypertension, Department of Pediatrics, Children's Hospital Colorado, University of Colorado, Aurora, Colorado 13Department of Pediatric Nephrology and Histopathology, Sindh Institute of Urology and Transplantation, Karachi, Pakistan 14Department of Pediatrics, C.S. Mott Children’s Hospital, University of Michigan, Ann Arbor, Michigan 15Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 16Department of Pediatrics, Rady Children's Hospital, University of California San Diego, San Diego, California 17Department of Pediatric Nephrology, Great Ormond Street Hospital, National Health Service Foundation Trust, Great Ormond Street, London, United Kingdom 18Department of Nephrology, Institute for Mother and Child Health Care of Serbia, "Dr Vukan Čupić" Faculty of Medicine, University of Belgrade, Belgrade, Serbia 19Departments of Nephrology and Hypertension, Kaplan Medical Center, Hebrew University School of Medicine, Rehovot, Israel. 20Department of Pediatrics, David Geffen School of Medicine at the University of California Los Angeles, University of California Los Angeles, Los Angeles, California 21Department of Pediatric Nephrology, Hospital Memmingen, Memmingen, Germany 22Department of Pediatrics, Asklepios Medical School, Asklepios Klinik Nord Heidberg, Hamburg, Germany 23Department of Pediatrics, Cayetano Heredia Hospital, Lima, Peru 24Department of Pediatrics, Faculty of Medicine and University Children’s Hospital, Comenius University, Bratislava, Slovakia 25Department of Pediatrics, Universitäts-Kinderklinik Essen, Essen, Germany 26Department of Pediatric Nephrology, Dr. Behçet Uz Children’s Hospital, Izmir, Turkey 27University Children's Hospital, Medical Faculty Skopje, Skopje, Macedonia 28Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 29 Department of Genetics, Howard Hughes Medical Institute, and Yale Center for Mendelian Genomics, Yale University, New Haven, Connecticut

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

Inclusion criteria Exclusion criteria Age <25 years at nephrotic syndrome onset Patients with non-nephrotic range proteinuria or -AND- hematuria only Clinical diagnosis of nephrotic syndrome (e.g. SSNS, SDNS, acute GN (e.g. hypocomplementemia, proteinuria, hypoalbuminemia, edema) gross hematuria), acute kidney injury. -AND/OR- Renal histology of FSGS or DMS Patient age >25 year at nephrotic syndrome onset Supplementary Table 1: Inclusion and exclusion criteria for enrollment in study.

DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis; GN, glomerulonephritis; SDNS, steroid dependent nephrotic syndrome; SSNS, steroid sensitive.

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. Supplementary Table 2: Thirty-three genes known to cause monogenic steroid-resistant nephrotic syndrome and mode of inheritance. Underlined genes were discovered and published by the laboratory of F.H. during performance of the whole exome sequencing study. References for original publications are given on the right. Blue background indicates known steroid-resistant nephrotic syndrome gene and orange background a phenocopy gene for steroid-resistant nephrotic syndrome. Citations for these references are provided on page 20-23 of the supplementary information.

Gene Mode of inheritance OMIM ID Reference ACTN4 AD #604638 Kaplan, JM (2000) (1) ADCK4 AR #615567 Ashraf, SA (2013) (2) ARHGDIA AR #601925 Gee, HY (2013) (3) CD2AP AR/AD #604241 Kim, JM (2003) (4) Diomedi-Camassei, F (2007) COQ2 AR #609825 (5) COQ6 AR #614647 Heeringa, SF (2011) (6) CRB2 AR #609720 Ebarasi, L (2015) (7) CUBN AR #602997 Sadowski, CA (2014) (8) DGKE AR #601440 Sadowski, CA (2014) (8) FAT1 AR #600976 Gee, HY (2016) (9) INF2 AD #610982 Brown, EJ (2010) (10) ITGA3 AR #605025 Has, C (2012) (11) KANK1 AR #607704 Gee, HY (2015) (12) KANK2 AR #614610 Gee, HY (2015) (12) KANK4 AR #614612 Gee, HY (2015) (12) LAMB2 AR #150325 Zenker, M (2004) (13) LMX1B AD #602575 Boyer, O (2013) (14) MYO1E AR #601479 Mele, C (2011) (15) NPHS1 AR #602716 Kestila, M (1998) (16) NPHS2 AR #604766 Boute, N (2000) (17) NUP205 AR #614352 Braun, DA (2016) (18) NUP93 AR #614351 Braun, DA (2016) (18) PAX2 AD #167409 Barua, M (2014) (19) PDSS2 AR #610564 Lopez, LC (2006) (20) PLCE1 AR #608414 Hinkes, B (2006) (21) PODXL AD #602632 Barua, M (2014) (22) SGPL1 AR #603729 Lovric, S (2017) (23) SMARCAL1 AR #606622 Boerkoel, CF (2002) (24) TRPC6 AD #603652 Winn, MP (2005) (25) TTC21B AR #612014 Huynh Cong, E (2014) (26) WDR73 AR #616144 Colin, E (2014) (27) WT1 AD #607102 Mendelsohn, HB (1982) (28) XPO5 AR #607845 Braun, DA (2016) (18) AGXT AR #604285 Nishiyama, K (1991) (29) COL4A3 AR #120070 Lemmink, HH (1994) (30) COL4A4 AR #120131 Mochizuki, T. (1994) (31) COL4A5 X-LINKED #303630 Barker, DF (1990) (32) CLCN5 X-LINKED #300008 Lloyd, SE (1996) (33) CTNS AR #606272 Town, M (1998) (34) FN1 AD #135600 Castelletti, F (2008) (35) GLA X-LINKED #300644 Bernstein, HS (1989) (36) LRP2 AR #600073 Kantarci, S (2007) (37) International FMF Consortium MEVF AD/AR #608107 (1997) (38) OCRL X-LINKED # 300535 Attree, O (1992) (39) AR, autosomal recessive; AD, autosomal dominant.

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. Supplementary Table 3: Variant calling as disease causing for autosomal recessive and dominant disease in genes known to cause steroid-resistant nephrotic syndrome.

Autosomal recessive variant calling in known genes Include allele as Truncating mutation (stop, abrogation of start or stop, obligatory disease splice, frameshift). causing if: Missense mutation: - Continuously conserved at least among vertebrates -or- -Previously reported as disease causing -or- - Loss of function in human allele is supported by functional data. -or- - Phenotype correlates with the published phenotype for the gene. - or- - Predicted deleterious for the protein function (at least in two among three prediction programs (Polyphen (>0.5), SIFT (Del), Mutation taster (DC)).

Consider Allele Frequency excluding allele - Heterozygous allele frequency >0.1% (in ExAC) as disease - Homozygous allele frequency (> 2 individuals in ExAC) causing if: - Non-segregation in the case of compound heterozygotes

Autosomal dominant variant calling in known genes Include allele as Truncating mutation (Stop, abrogation of start or stop, obligatory disease splice, frameshift). causing if: Missense mutation: - Continuously conserved at least among vertebrates -or- -Previously reported as disease causing -or- - Loss of function in human allele is supported by functional data. -or- - Phenotype correlates with the published phenotype for the gene. - or- - Predicted deleterious for the protein function (at least in two among three prediction programs (Polyphen (>0.5), SIFT (Del), Mutation taster (DC)).

Consider Allele Frequency excluding allele - Heterozygous allele frequency (>3 individuals in ExAC) as disease - If the variant is present homozygously in any individual in ExAC causing if: - Non-segregation (note that variable expressivity and incomplete penetrance must be taken into consideration when evaluating dominant genes).

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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.

Supplementary Table 4: Number of families evaluated by panel sequencing in Sadowski (8) and by whole exome sequencing in this study. 94 families were evaluated by whole exome sequencing and panel sequencing. In 20/94 families, a causative mutation was detected in one of 26 genes known to cause steroid resistant nephrotic syndrome gene by both whole exome sequencing and panel sequencing. In nine families of the 94 no causative mutation was detected by panel sequencing but a causative mutation was detected by whole exome sequencing. Phenotypes and genotypes of families with a causative mutation detected are given in Supplementary Table 9. 94/300 Total families evaluated by panel sequencing and WES (31%)

206/300 Total families evaluated by WES only (69%)

20/74 Total families with a causative mutation detected by panel (27% of solved sequencing and WES cases) Total families with a causative mutation detected by WES and not by 9/74 panel sequencing (12%)

Total families evaluated by WES only with a causative mutation 45/74 detected (61%) WES (whole exome sequencing).

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.

Supplementary Table 5: Selection of novel candidate genes for 11 families with steroid resistant nephrotic syndrome in whom a causative mutation in a known nephrosis or phenocopy gene was excluded. Each candidate gene represents the most deleterious mutation within a homozygous peak region of the respective families. Clinical Continuously Family ID Gene Zygosity Accession # c. position p. position MT/SFT/PPi ExAC diagnosis. conserved to ) A1756 DDX53 Hemi NM_182699.3 c.24G>A p.Trp8* Truncating - NR SRNS A4684 MXRA5 Hemi NM_015419.3 c.204_205insT p.Ala69Cysfs*22 Frameshift - NR SRNS De- B51 DHTKD1 Hom NM_018706.6 c.886G>A p.Val296Met Dm DC/Del/1 0/3/121366 identified De- B52 DHTKD1 Hom NM_018706.6 c.886G>A p.Val296Met Dm DC/Del/1 0/3/121366 identified A5013 CDK20 Hom NM_001039803.2 c.610T>C p.Phe204Leu Dm DC/Del/1 NR SRNS B50 OSGEP Hom NM_017807.3 c.40A>T p.Ile14Phe Dr DC/Del/0.023 NR SRNS De- B57 OSGEP Hom NM_017807.3 c.40A>T p.Ile14Phe Dr DC/Del/0.023 NR identified B123 TPRKB Hom NM_016058.2 c.407T>C p.Leu136Pro Xt DC/Del/1 NR SRNS B377 OSGEP Hom NM_017807.3 c. 740G>A p.Arg247Gln Dm DC/Tol/0.998 0/8/121400 CNS B787 SLC35F1 Hom NM_001029858.3 c.878T>G p.Met293Arg Ce DC/Del/0.999 NR SRNS B1356 COG1 Hom NM_018714.2 c.1070+5G>A Splice Splice - NR SRNS Bx, biopsy; Ce, Caenorhabditis elegans; CNS, congenital nephrotic syndrome; DC, disease causing; Del, deleterious; Dm, Drosophila melanogaster; Dr, Danio rerio; Hom, homozygous; Hemi, Hemizygous; Mm, Mus musculcus; MT, MutationTaster; NR, not reported; PPi, Polyphene score; Sc, Saccharomyces cerevisiae; SFT, SIFT; SRNS, steroid-resistant nephrotic syndrome; Tol, tolerated; Xt, Xenopus tropicalis.

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. Supplementary Table 6: Age, sex, and ethnic characteristics of the steroid-resistant nephrotic syndrome cohort and of the families in whom a causative monogenic mutation was detected in either a steroid-resistant nephrotic syndrome gene or a phenocopy gene. Age and sex demographics are given for a subset of 81 individuals from 74 families in whom a causative mutation was detected in a steroid-resistant nephrotic syndrome gene or 15 individuals from 11 families in whom a causative mutation was detected in a phenocopy gene are shown. Additionally, ethnic and racial data are given for all the families in the cohort, with a subset of 74 families in which a mutation was detected in a steroid- resistant nephrotic syndrome gene and in 11 families with a mutation detected in a phenocopy gene. Age and sex is represented graphically in Figure 3A, race and ethnicity are represented graphically in Supplementary Figure 1. Families from Egypt identified as being African and Arabic and families from Saudi Arabia identified as being Arabic and Asian. Percents >10% are rounded to the nearest whole number.

Clinical Clinical Characteristics of Individuals with Characteristics Causative Mutation Detected of Total Cohort Number of Number of individuals Number of individuals with with mutation individuals (%) SRNS mutation detected - phenocopy detected (%) gene (%) Gender Male 163/335 (49%) 41/81 (51%) 9/15 (60%) Female 138/335 (41%) 35/81 (43%) 5/15 (33%) Unknown 34/335 (10%) 5/81 (6%) 1/15 (6.7%) Total 335/335 (100%) 81/81 (100%) 15/15 (100%) Median age (range) at 4 (0-24) 1.7 (0-21) 4 (0.3-16) diagnosis (in years) Infants ≤ 90 days 31/335 (9.3%) 16/81 (20%) 0/15 (0%) Infants >3 mo and ≤ 12 mo 62/335 (19%) 19/81 (24%) 4/15 (27%) Children > 1 yr and ≤ 6 yr 94/335 (28%) 19/81 (24%) 4/15 (27%) Children > 6 and ≤ 12yr 54/335 (16%) 11/81 (14%) 1/15 (6.7%) Children > 12 yr or <18 yr 34/335 (10%) 5/81 (6%) 2/15 (13%) Young adults ≥ 18 yr or <25 yr 8/335 (2.4%) 2/81 (2.5%) 0/15 (0%) Not reported 52/335 (16%) 9/81 (11%) 4/15 (27%) Total 335/335 (100%) 81/81 (100%) 15/15 (100%) Race/Ethnicity Number of Number of families Number of families families (%) (%) (%) Arabic 77/300 (26%) 29/74 (39%) 3/11 (27%) European/Caucasian 59/300 (20%) 8/74 (11%) 3/11 (27%) Turkish 29/300 (10%) 12/74 (16%) 2/11 (18%) Hispanic/Latino 22/300 (7.3%) 2/74 (2.7%) 1/11 (9.1%) Asian 21/300 (7%) 7/74 (9.5%) 0/11 (0%) African and Arabic 15/300 (5%) 4/74 (5.4%) 0/11 (0%) African/African American 8/300 (2.7%) 2/74 (2.7%) 0/11 (0%) Arabic and Asian 9/300 (3%) 0/74 (0%) 0/11 (0%) Other/multiple races 6/300 (2%) 1/74 (1.4%) 1/11 (9.1%) indicated Ashkenazi Jewish 1/300 (0.3%) 0/74 (0%) 0/11 (0%) Roma 1/300 (0.3%) 1/74 (1.4%) 0/11 (0%) Unknown/not indicated 52/300 (17%) 8/74 (11%) 1/11 (9.1%)

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. Total 300/300 (100%) 74/74 (100%) 11/11 (100%) SRNS, steroid-resistant nephrotic syndrome.

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. Supplementary Table 7: Presence of consanguinity, homozygosity, or multiple affected statuses in 300 families with steroid-resistant nephrotic syndrome. A subset of 74 families in whom a causative mutation was detected in a steroid-resistant nephrotic syndrome gene or 11 families in whom a causative mutation was detected in a phenocopy gene are shown. In addition, the presence of extra-renal manifestations in 335 individuals from 300 families with steroid- resistant nephrotic syndrome compared to a subset of 81 individuals from 74 families in whom a causative mutation was detected in a steroid-resistant nephrotic syndrome gene or 15 individuals from 11 families in whom a causative mutation was detected in a phenocopy gene. Pedigree characteristics are represented graphically in Figure 4. Extra-renal manifestations are represented graphically in Supplementary Figure 2. Percents >10% are rounded to the nearest whole number.

Clinical Clinical Characteristics of Individuals/Families Characteristics of with Causative Mutation Detected Total Cohort Number of families Number of families with Number of families with SRNS mutation mutation detected - (%) detected (%) phenocopy gene (%) Pedigree Consanguineous 146/300 (49%) 56/74 (76%) 6/11 (55%) Non-consanguineous 135/300 (45%) 17/74 (23%) 4/11 (36%) Unknown 19/300 (6.3%) 1/74 (1.4%) 1/11 (9.1%) consanguinity

Homozygosity on 147/300 (49%) 56/74 (76%) 5/11 (45%) mapping >100Mbp Homozygosity on 153/300 (51%) 18/74 (24%) 6/11 (55%) mapping <100Mbp

Families with one 174/300 (58%) 41/74 (55%) 2/11 (18%) affected individual Families with 2 65/300 (22%) 21/74 (28%) 5/11 (46%) affected individuals Families with 3 or greater affected 28/300 (9.3%) 7/74 (9.5%) 3/11 (27%) individuals Unknown/de-identified 33/300 (11%) 5/74 (6.8%) 1/11 (9.1%) sample Total families 300/300 (100%) 74/74 (100%) 11/11 (100%) Extra-renal

manifestations Number of Number of Number of individuals (%) individuals (%) individuals (%) Yes 91/335 (27%) 22/81 (27%) 6/15 (40%) No 219/335 (65%) 58/81 (72%) 7/15 (47%) Unknown/de-identified 25/335 (7.5%) 1/81 (1.2%) 2/15 (13%) sample Total individuals 335/335 (100%) 81/81 (100%) 15/15 (100%) SRNS, steroid-resistant nephrotic syndrome.

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. Supplementary Table 8: Clinical and histologic diagnosis of 335 individuals from 300 families with steroid-resistant nephrotic syndrome. A subset of 85 individuals from 78 families in whom a causative mutation was detected in a steroid-resistant nephrotic syndrome gene and 15 individuals from 11 families in whom a phenocopy gene was detected are shown. Clinical diagnosis is represented graphically in Supplementary Figure 3; histologic diagnoses are represented graphically in Supplementary Figure 4. Percents >10% are rounded to the nearest whole number.

Clinical Characteristics Clinical Characteristics of Individuals/Families with

of Total Cohort Causative Mutation Detected Number of families with Number of families with Number of families (%) SRNS mutation detected mutation detected - (%) phenocopy gene (%) Clinical diagnosis SRNS 205/300 (68%) 48/74 (65%) 9/11 (82%) CNS 32/300 (11%) 17/74 (23%) 0/11 (0%) Infantile nephrotic syndrome 9/300 (3%) 1/74 (1.4%) 0/11 (0%) Nephrotic syndrome with 1/300 (0.3%) 1/74 (1.4%) 0/11 (0%) ESRD on presentation ESRD on presentation, FSGS 4/300 (1.3%) 1/74 (1.4%) 0/11 (0%) or DMS on biopsy Nephrotic syndrome with 6/300 (2%) 0/74 (0%) 0/11 (0%) FSGS or DMS on biopsy Nephrotic range proteinuria 7/300 (2.3%) 1/74 (1.4%) 0/11 (0%) with FSGS or DMS of biopsy De-identified sample 36/300 (12%) 5/74 (6.8%) 2/11 (18%) Total families enrolled 300/300 (100%) 74/74 (100%) 11/11 (100%) Number of individuals Number of individuals Number of individuals (%) (%) (%) Diagnosis on biopsy Diagnosis on biopsy Diagnosis on biopsy Diagnosis on biopsy (n=223) (n=50) (n=9) FSGS 153/223 (69%) 37/50 (74%) 3/9 (33%) DMS 14/223 (6.3%) 2/50 (4%) 1/9 (11%) MCNS 20/223 (9%) 4/50 (8%) 0/9 (0%) MPGN 10/223 (4.5%) 2/50 (4%) 1/9 (11%) CNS/Finnish type 5/223 (2.2%) 1/50 (2%) 0/9 (0%) Membranous GN 1/223 (0.4%) 0/50 (0%) 0/9 (0%) Other 20/223 (9%) 4/50 (8%) 4/9 (44%) No bx data available 112/335 (33%) 31/81 (38%) 6/15 (40%) CNS, congenital nephrotic syndrome; DMS, diffuse mesangial sclerosis; ESRD, end stage renal disease; FSGS, focal segmental glomerulosclerosis; GN, glomerulonephritis; MCNS, minimal change nephrotic syndrome; MPGN, membranoproliferative glomerulonephritis; SRNS, steroid resistant nephrotic syndrome.

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. Supplementary Table 9: Summary of causative mutations detected in one of 20steroid-resistant nephrotic syndrome causing genes and 8 phenocopy genes in 90 of 300 families with steroid-resistant nephrotic syndrome by family and clinical phenotype.

# Family ExAC Synd Kidney Zygo- Age Sex Race/ Consang. affected Clin. Gene ID and c. change p. change Cons. SFT MT PPi (home/het/ . biopsy sity (years) (M/F) Ethnicity (Y/N) per Dx indiv. # total alleles) (Y/N) results family

c.176_177in Comp p.F59fs FS - - - NR sT het B1425_ COQ2 2 M C/E N 1 N SRNS FSGS 21 Comp c.683A>G p.N228S Dm Tol DC 0.918 0/20/120566 het

A4431_ c.610del p.T204Qfs*6 Hom FS - - - NR 17 F C/E Y N SRNS FSGS 21 DGKE 3 A4431_ c.610del p.T204Qfs*6 Hom FS - - - NR 8 F C/E Y N SRNS MPGN 22

B788_2 INF2 c.532T>G p.F178V Het Dr Tol DC 0.996 NR 21 M C/E N 7 N SRNS FSGS 1

A1605_ c.2593del p.D865Tfs*38 Hom FS - - - NR <1 M Turkish Y 1 N SRNS FSGS 21 ITGA3 A3113_ c.1883G>C p. R628P Hom Dm Del DC 0.3 0/3/118974 4 mo F Asian Y 1 N CNS No bx 21

B324_2 KANK4 c.2401T>C p.Y801H Hom Dm Del DC 1 0/121/120924 2 mo F Roma N 4 Y CNS MCNS 1

A1757_ c.143A>C p.Y48S Hom Dr Del DC 1 0/70/117226 13 M Hispanic N N SRNS FSGS 21 2 A1757_ c.143A>C p.Y48S Hom Dr Del DC 1 0/70/117226 13 F Hispanic N N SRNS FSGS 22

B819_2 c.395C>T p.A132V Hom Xt T P 0.002 0/2/121366 0 F Turkish Y 2 N CNS No bx 1

A2356_ LAMB2 c.736C>T p. R246W Hom Dm Del DC 1 0/1/119680 3mo M Arabic Y 2 Y CNS CNS 23

A5284_ c.1731+1G> Splice Hom Splice - - - 0/1/118980 1 mo F Asian Y 1 Y CNS No bx 12 A

A2263_ c.4537C>T p.Q1513* Hom Trunc. - - - NR 2 mo M Arabic Y 1 Y SRNS No bx 23

B1219_ c.4573C>T p.Q1525* Hom Trunc. - - - 0/1/121384 0.2 F Arabic Y 1 Y CNS No bx 21

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. A200_2 c.737G>A p.R246Q Het Dm Del DC 0.998 NR 8 F Turkish Y 2 N SRNS FSGS 1 LMX1B De- A4642 c.737G>A p.R246Q Hom Dm Del DC 0.998 NR unk unk unk unk unk unk unk identified

Other - ESRD at A146_2 chronic c.1228G>A p.E410K Hom Sc Del DC 0.98 NR 18 M unk Y 1 N presentatio 1 renal n MYO1E failure

A3656_ c.1978C>T p.Q660* Hom Trunc. - - - NR 45 do M Asian Y 1 N CNS MCD 21

A5151_ c.139del p.A47Pfs81* Hom FS - - - 0/2/114206 4 mo M Arabic Y 1 N SRNS No bx 21 In- A4472_ c.515_517de p.T172del Hom frame - - - NR 60 do F Arabic Y 2 N SRNS No bx 22 l del Comp c.1048T>C p.S350P Dm Del P 0.76 0/2/121174 het B1122_ unk F C/E Y 1 N CNS unk 21 c.2506+5G> Comp Splice Splice - - - 0/1/120468 T het

B1238 c.1379G>A p.R460Q Hom Ce Tol Pol 0.48 0/1/119280 0.25 M Arabic Y 4 N CNS No bx

De- B55 c.1760T>G p.L587R Hom Dr Del DC 0.99 NR unk unk unk Y unk Y No bx identified

Comp c.2014G>A p.A672T Dr Del DC 0.99 0/1/82174 het A5275_ 5 mo M Turkish N 1 Y Infantile NS FSGS NPHS1 21 Comp c. 3250dupG p.V1084Gfs* FS - - - 0/1/82174 het

A3432_ c.2020C>A p.P674T Hom Dr Del DC 0.3 0/3/118974 1 mo M Arabic Y 4 N CNS No bx 24

A1500_ c.2728T>C p.S910P Hom Dr Del DC 0.959 NR 1 M A/AA N 1 N SRNS MCNS 21

A3509_ c.3478C>T p.R1160* Hom Trunc. - - - 0/8/121256 0 F Asian Y 1 N CNS No bx 21

A3594_ c.3478C>T p.R1160* Hom Trunc. - - - 0/8/121256 0 F Arabic Y 1 N CNS No bx 21

A3708_ c.3478C>T p.R1160* Hom Trunc. - - - 0/8/121256 2 mo M A/AA Y 1 N CNS FSGS 21

Other/mul A4427_ c.3478C>T p.R1160* Hom Trunc. - - - 0/8/121256 0 F tiple N 1 N CNS No bx 23 races

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. B1357_ African/ c.3478C>T p.R1160* Hom Trunc. - - - 0/8/121256 0.1 F Y 1 N CNS No bx 21 Arabic

A4681_ Start c.1A>T p.M1? Hom - - - NR 7 F Arabic Y 1 N SRNS FSGS 21 loss

A679 Comp c.397del p.R133Efs*2 FS - - - NR _21 het unk M C/E N N SRNS No bx Comp c.413G>A p.R138Q Dm Del DC 0.999 0/82/121298 het 2 A679 Comp c. 397del p.R133Efs*2 FS - - - NR _22 het unk M C/E N N SRNS No bx Comp c.413G>A p.R138Q Dm Del DC 0.999 0/82/121298 het

A3133_ c.419del p.G140Dfs*41 Hom FS - - - 0/1/121308 5.8 F Arabic Y N SRNS FSGS 21 2 A3133_ c.419del p.G140Dfs*41 Hom FS - - - 0/1/121308 2 F Arabic Y N SRNS FSGS 43

B963_2 c.538G>A p.V180M Hom Dr Del DC 0.58 0/3/120452 9 mo F Arabic Y 1 N SRNS No bx 1 NPHS2 Comp 69/3526/11910 c.686G>A p.R229Q Xt Tol P 0.313 het 8 A667_2 14 F C/E N N SRNS FSGS 1 Comp c.916A>T p.R306W Dr Del DC 0.98 NR het 2 Comp 69/3526/11910 c.686G>A p.R229Q Xt Tol P 0.313 het 8 A667_2 9 M C/E N N SRNS No bx 2 Comp c.916A>T p.R306W Dr Del DC 0.98 NR het

Other - diffuse In- A4309_ c.705_713de mesangi p.L236del Hom frame - - - NR 3 mo M Asian Y 1 Y CNS 21 l9 al del hypercel lularity

African/ B1090 c.800A>T p.D267V Hom Ce Del DC 1 NR 8 M Y 1 N SRNS FSGS Arabic

c.855_856de B188 p.R286Tfs*17 Hom FS - - - 0/8/115938 3 F Hispanic Y 2 N SRNS MCNS l

B140_2 c.3095G>A p.C1032Y Hom Dm Tol DC 1 NR 3 M Arabic Y 1 Y SRNS FSGS 1 NUP205 A1733_ c.5984T>C p.F1995S Hom Dm Tol DC 0.99 NR 3.5 F Turkish N 2 N SRNS FSGS 21

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. A1733_ c.5984T>C p.F1995S Hom Dm Tol DC 0.99 NR 3 M Turkish N N SRNS No bx 22

A1626_ c.1772G>T p.G591V Hom Sc Del DC 1 0/14/121252 2.5 M Turkish Y 1 N SRNS FSGS 21

A1671_ c.1886A>G p.Y629C Hom Sc Del DC 0.997 0/1/120978 1.3 M Turkish N 1 N SRNS IgA 21 NUP93 A2241_ c.1886A>G p.Y629C Hom Sc Del DC 0.997 0/1/120978 11 mo M Turkish Y 2 N SRNS No bx 22

B1311_ c.2017C>T p.R673W Hom Dr Tol DC 1 NR 1 F Arabic Y 2 N CNS FSGS 21

A3853_ PDSS2 c.1145C>T p.Ser382Leu Hom Dr Del DC 1 0/4/121372 1 M Arabic Y 2 Y SRNS No bx 22

B913_2 c.1709del p.S570Tfs*29 Hom FS - - - NR 9 mo M Turkish Y 1 Y SRNS FSGS 1

A1678_ c.2576_2577 p.Q859Hfs*31 Hom FS - - - NR 7.9 M Turkish Y 1 N SRNS DMS 21 insT A3617_ c.3379_3380 p.N1127* Hom Trunc. - - - NR 9 mo F Arabic Y 3 N SRNS FSGS 25 del A3921_ c.4506+2T> Splice Hom Splice - - - NR 6 mo F Arabic Y 2 N SRNS FSGS 22 C p.A1630Qfs*4 A59_21 c.4887del Hom FS - - - NR 7 mo F Turkish N 1 N SRNS FSGS 0

B354_2 c.4978_4981 p.Q1660Lfs*9 Hom FS - - - NR 1 M Arabic Y 2 N SRNS DMS 2 CAGA PLCE1 A4654_ c.5521A>G p.K1841E Hom Sc Del DC 1 NR 4 F Arabic Y N SRNS FSGS 21 2 A4654_ c.5521A>G p.K1841E Hom Sc Del DC 1 NR 2.4 F Arabic Y N SRNS FSGS 22 A3869_ c.5521A>G p.K1841E Hom Dm Del DC 1 NR 7 mo M Arabic Y 1 N SRNS FSGS 24 In- B1432_ c.5950_5952 p.N1984del Hom frame - - - 0.5 M Arabic Y 1 N SRNS FSGS 24 delAAC del. In- A4043_ c.5951- p.N1984del Hom frame - - - NR 6 mo M Arabic Y 1 N SRNS FSGS 21 5953delACA del. In- A5171_ c.5951_5953 p.N1984del Hom frame - - - NR 5 Male Arabic Y 2 N SRNS FSGS 21 del del. A280_2 c.665G>A p.R222Q Hom Dm Del DC 1 0/2/120744 2.5 M Asian Y 3 Y SRNS FSGS 1 De- B46 c.1037G>T p.S346I Hom Sc Del DC 1 NR unk unk unk Y unk Y No bx SGPL1 identified

De- B56 c.1037G>T p.S346I Hom Sc Del DC 1 NR unk unk unk Y unk Y No bx identified

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. A925_2 c.1736C>A p.S579* Hom Trunc. - - - NR 2.9 M Arabic Y 1 Y SRNS FSGS 1

A1683_ c.1756C>T p.R586W Hom Dm Del DC 1 0/1/121386 7.6 M Turkish Y 1 Y SRNS FSGS 21

ESRD, F1367_ c.1756C>T p.R586W Hom Dm Del DC 1 0/1/121386 4 F unk Y 1 N FSGS on FSGS 21 bx

B1067 c.1822T>C p.F608L Hom Dm Del DC 1 NR 14 M Arabic Y 1 Y SRNS FSGS

B672_2 c.1940A>C p.K647T Hom Dm Del DC 1 NR 6 F Arabic Y 2 N SRNS No bx SMARCAL1 1

B142_2 c.2290C>T p.R764W Hom Dm Del DC 1 NR 8 M Arabic Y N SRNS FSGS 2

B1319_ African/ c.2290C>T p.R764W Hom Dm Del DC 1 NR unk F Y 1 Y SRNS MPGN 21 Arabic

Nephrotic range B1134_ c.2542G>T p.E848* Hom Trunc. - - - 0/14/121298 11 M C/E N 1 Y proteinuria, FSGS 21 FSGS on bx

A4685_ TRPC6 c.523C>T p.R175W Het Dr Del DC 1 NR 7 F Arabic N 1 N SRNS FSGS 21

A5262_ African/ c.626C>T p.P209L Hom Dr Tol DC 1 0/8/121264 8 F Y 1 N SRNS FSGS 21 Arabic TTC21B A5002_ c.2569G>A p.Ala857Thr Hom Ce Del DC 0.983 NR 5 mo M Asian Y 1 N SRNS No bx 21

B49 c.287G>A p.R96K Hom Dr Tol DC 1 NR <1y M unk Y 2 Y SRNS No bx

B129_2 WDR73 c.703C>T p.Q235* Hom Trunc. - - - NR 3 M Arabic N 2 Y SRNS No bx 1

De- B41 c.940C>T p.Q315* Hom Trunc. - - - NR unk unk unk Y unk Y No bx identified

Other- focal B1018_ c.1432+5G> mesangi Splice Het Splice - - - NR 3 F Arabic N 1 N SRNS 21 A al WT1 prolifera tion

B1244_ c.1432+5G> Splice Het Splice - - - NR 5 F C/E N 1 N SRNS No bx 21 A

AGXT A63_21 c.33dup p.K12Qfs*156 Hom FS - - - NR 4 mo M Turkish Y 1 Y SRNS No bx

14

Supplemental material is neither peer-reviewed nor thoroughly edited by CJASN. The authors alone are responsible for the accuracy and presentation of the material. B465_2 De- c.863G>A p. W288* Hom Trunc. - - - NR 4 mo M Arabic Y 3 unk No bx 3 identified

A3094_ CLCN5 c.933G>C p.E311D Hemi Sc Del DC 1 NR 12 M C/E Y 2 Y SRNS FSGS 22

A1221_ c.4825C>T p.Arg1609* Het Trunc. - - - 0/3/121000 5 F C/E N Y SRNS FSGS 21 COL4A3 2 A1221_ Other - c.4825C>T p.Arg1609* Het Trunc. - - - 0/3/121000 5 M C/E N Y SRNS 22 Alport's

A4644_ De- c.3088G>A p.G1030S Hemi Dm Del DC 0.999 NR unk unk unk unk unk unk No bx 21 identified

A2058_ c.3722G>A p.G1241D Hemi Dr Del DC 1 NR 16 M Hispanic N >3 N SRNS FSGS 21

COL4A5 A169_2 c.3722G>A p.G1241D Hemi Dr Del DC 1 NR 11 mo M Turkish Y N SRNS MPGN 1 2 Other - A169_2 c.3722G>A p.G1241D Hemi Dr Del DC 1 NR unkn M Turkish Y N SRNS Cresentr 2 ic GN

In- B249_2 c.809_811de p.S270del Hom frame - - - 0/1/120874 4 F Arabic Y N SRNS No bx 1 l del. In- B249_2 c.809_811de CTNS p.S270del Hom frame - - - 0/1/120874 4 F Arabic Y 4 N SRNS No bx 2 l del. In- B249_3 c.809_811de p.S270del Hom frame - - - 0/1/120874 unk F Arabic Y N SRNS No bx 1 l del. Other - A4936_ IgM FN1 c.6836T>C p.V2279A Het Dr Del DC 0.696 NR 1 F C/E N 2 N SRNS 21 nephrop athy

Other - B912_2 GLA c.504A>C p. K168N Hemi Dr Del DC 1 NR 14 M Arabic Y 1 Y SRNS Fabry's 1 disease

Other/mul B1119_ WDR19 c.3533G>A p.R1178Q Hom Ce T DC 0.948 0/9/69008 1 M tiple N 2 Y SRNS DMS 21 races

Ce, Caenorhabditis elegans; Cs, Ciona savignyi; DC, disease causing; Del, deleterious; Dm, Drosophila melanogaster; DMS, diffuse mesangial sclerosis; do, days old; Dr, Danio rerio; F, female; FSGS, focal segmental glomerulosclerosis; GN, glomerulonephritis; Hom, homozygous; Het, heterozygous; Hemi, Hemizygous; indiv., individual; M, male; Mm, Mus musculcus; mo, months old; MPGN, membranoproliferative glomerulonephritis; MT, MutationTaster; NR, not reported; PPi, Polyphene score. Sc, Saccharomyces cerevisiae; SFT, SIFT; SRNS, steroid-resistant nephrotic syndrome; Tol, tolerated; Xt, Xenopus tropicalis. Orange shading indicates a gene that is a phenocopy for steroid-resistant nephrotic syndrome. 15

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

Unknown/not indicated 8, 1, 43 (15%, 1.9%, 83%)

Roma 1, 0, 0 (100%, 0%, 0%)

Ashkenazi Jewish 0, 0, 1 (0%, 0%, 100%) Causative mutation in SRNS gene Other/multiple races indicated 1, 1, 4 (17%, 17%, 67%) detected

Arabic and Asian 0, 0, 9 (0%, 0%, 100%)

y

t

i c

i African/African American 2, 0, 6 (25%, 0%, 75%) Causative mutation in a n

h phenocopy gene

t e

African and Arabic 4, 0, 11 (27%, 0%, 73%)

r detected

o

e

c Asian 7, 0, 14 (33%, 0%, 67%) a

R No causative Hispanic/Latino 2, 1, 19 (9%, 4.5%, 86%) mutation Turkish 12, 2, 15 (41%, 6.9%, 52%) detected

European/Caucasian 8, 3, 48 (14%, 5.1%, 81%)

Arabic 29, 3, 45 (38%, 3.9%, 58%)

Total families enrolled 74 11 215 (25%, 3.7%, 72%)

0 0 00 10 200 3 Number of families

Supplementary Figure 1: Distribution of families regarding gene identification status (steroid-resistant nephrotic syndrome (SRNS) gene, phenocopy gene, no mutation detected for race or ethnicity in 335 individuals with SRNS from 300 families. Families in whom a causative mutation in a known steroid-resistant nephrotic syndrome gene (blue) or a phenocopy gene (orange) was detected as compared to those families in whom no causative mutation was detected (gray). Bars and numbers represent number of affected individuals in each race or ethnic category, divided into those with a causative mutation detected in an steroid-resistant nephrotic syndrome gene (blue), those with a causative mutation detected in a phenocopy gene (orange) and those without a causative mutation detected (gray). Percent at end of each bar reflect the same three categories. Percents >10% are rounded to the nearest whole number. Percent of each race or ethnicity per total cohort population or per total population with a mutation detected in an steroid-resistant nephrotic syndrome or phenocopy gene is shown in Supplementary Table 6. Families from Saudi Arabia were identified as Arabic and Asian, and a portion of families from Egypt identified as Arabic and African.

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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.

Patients without extra-renal manifestions 58, 7, 154 (27%, 3.2%, 70%)

Patients with p=0.8 extra-renal manifestions 22, 6, 63 (24%, 6.6%, 69%)

Total patients enrolled 8115 239 (24%, 4.5%, 79%)

Unknown/de-identified sample 1, 2, 22 (4%, 8%, 88%)

0 0 0 1 200 300 Number of patients Causative mutation in SRNS gene detected

Causative mutation in phenocopy gene detected No causative mutation detected

Supplementary Figure 2: Distribution of affected individuals regarding gene identification status (steroid- resistant nephrotic syndrome (SRNS) gene, phenocopy gene, or no mutation detected) for extra-renal (additional systemic) manifestations in 335 individuals with steroid-resistant nephrotic syndrome from 300 families. Families in whom a causative mutation in a known steroid-resistant nephrotic syndrome gene (blue) or a phenocopy gene (orange) was detected are compared with those families in whom no causative mutation was detected (gray). Bars and numbers represent number of affected individuals in each category, divided into those with a causative mutation detected in an steroid-resistant nephrotic syndrome gene (blue), those with a causative mutation detected in a phenocopy gene (orange) and those without a causative mutation detected (gray). Percent at end of each bar reflect the same three categories. Percents >10% are rounded to the nearest whole number. Percent of each category per total cohort population or per total population with a mutation detected is shown in Supplementary Table 7. Rate of mutation identification in an steroid-resistant nephrotic syndrome gene in patients with extra-renal manifestations was not statistically different than those who did not have syndromic features by two-sided chi squared test (p=0.8).

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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.

Nephrotic range proteinuria, FSGS or DMS of biopsy 1, 0, 6 (14%, 0%, 86%) Causative mutation in SRNS gene detected Nephrotic syndrome, 0, 0, 6 (0%, 0%, 100%) FSGS or DMS on biopsy ESRD on presentation, Causative mutation in phenocopy gene detected FSGS or DMS on biopsy 1, 0, 3 (25%, 0%, 75%) Nephrotic syndrome, ESRD on presentation 1, 0, 0 (100%, 0%, 0%) No causative mutation detected Infantile NS 1, 0, 8 (11%, 0%, 89%)

CNS 17, 0, 15 (53%, 0%, 47%)

SRNS 48, 9, 148 (23%, 4.4%, 72%)

De-identified sample 5, 2 , 29 (14%, 5.6%, 81%)

Total families enrolled 7411 215 (25%, 3.7%, 72%)

0 0 0 0 0 1 2 300 Number of families

Supplementary Figure 3: Distribution of families regarding gene identification status (steroid-resistant nephrotic syndrome (SRNS) gene, phenocopy gene, or no mutation detected) for clinical diagnosis in 300 families with steroid-resistant nephrotic syndrome. Families in whom a causative mutation in a known steroid-resistant nephrotic syndrome gene (blue) or a phenocopy gene (orange) was detected are compared with those families where no causative mutation was detected (gray). Bars and numbers represent number of families in each category, divided into those families with a causative mutation detected (blue), those families with a causative mutation detected in a phenocopy gene (orange) and those families without a causative mutation detected (gray). Percent at end of each bar reflect the same three categories. Percents >10% are rounded to the nearest whole number. Percent of each category per total cohort population or per total population with a mutation detected in an steroid-resistant nephrotic syndrome gene or phenocopy gene is shown in Supplementary Table 8. CNS, congenital nephrotic syndrome; DMS, diffuse mesangial sclerosis: ESRD, end stage renal disease; FSGS, focal segmental glomerulosclerosis; NS, nephrotic syndrome.

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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.

Other 4, 4, 12 (20%, 20%, 60%) Causative mutation in SRNS gene detected Membranous GN 0, 0, 1 (0%, 0%, 100%) Causative mutation in phenocopy gene detected CNS/Finnish type 1, 0, 4 (20%, 0%, 80%)

MPGN 2, 1, 7 (20%, 10%, 70%) No causative mutation detected

MCNS 4, 0, 16 (20%, 0%, 80%)

DMS 2, 1, 11 (14%, 7.1%, 79%) Histologic diagnosis

FSGS 37, 3, 113 (24%, 2%, 74%)

No bx data available 31 6 75 (28%, 5.4%, 67%)

0 50 00 50 1 1 Number of patients

Supplementary Figure 4: Distribution regarding gene identification status (steroid-resistant nephrotic syndrome (SRNS) gene, phenocopy gene, or no mutation detected) for histologic diagnosis in 335 affected individuals with steroid-resistant nephrotic syndrome from 300 families. Individuals in whom a causative mutation in a known steroid- resistant nephrotic syndrome gene (blue) or a phenocopy gene (orange) was detected are compared with those families where no causative mutation was detected (gray). Bars and numbers at end of bars represent total number of affected individuals in each race or ethnic category, divided into those with a causative mutation detected in an steroid-resistant nephrotic syndrome gene (blue), those with a causative mutation detected in a phenocopy gene (orange) and those without a causative mutation detected (gray). Percent at end of each reflect the same three categories. Percents >10% are rounded to the nearest whole number. Percent of each category per total cohort population or per total population with a mutation detected is shown in Supplementary Table 8. CNS, congenital nephrotic syndrome; DMS, diffuse mesangial sclerosis; FSGS, focal segmental glomerulosclerosis; GN, glomerulonephritis; MCNS, minimal change nephrotic syndrome; MPGN, membranoproliferative glomerulonephritis.

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