For Peer Review

Clinical Journal of the American Society of NEPHROLOGY

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome

Journal:

Clinical Journal of the American Society of Nephrology

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Manuscript ID

Manuscript Type: Date Submitted by the Author:

Original Articles 06-Jul-2017 Warejko, Jillian; Boston Children's Hospital, Medicine Tan, Weizhen; Boston Children's Hospital, Medicine Daga, Ankana; Boston Children's Hospital, Medicine Schapiro, David; Boston Children's Hospital, Medicine Lawson, Jennifer; Boston Children's Hospital, Medicine Shril, Shirlee; Boston Children's Hospital, Medicine Lovric, Svjetlana; Boston Children’s Hospital, of Medicine, Division of Nephrology Ashraf, Shazia; Boston Children’s Hospital, Department of Medicine, Division of Nephrology Rao, Jia; Boston Children's Hospital, Medicine Hermle, Tobias; Boston Children's Hospital, Medicine Jobst-Schwan, Tilman; Boston Children's Hospital, Medicine Widmeier, Eugen; Boston Children's Hospital, Medicine Majmundar, Amar; Boston Children's Hospital, Medicine Schneider, Ronen; Boston Children's Hospital, Medicine Gee, Heon Yung; Boston Children's Hospital, Medicine; Yonsei University College of Medicine, Pharmacology Schmidt, J. Magdalena; Boston Children's Hospital, Medicine Vivante, Asaf; Boston Children's Hospital, Medicine; Sheba Medical Center at Tel Hashomer, Talpiot Medical Leadership Program van der Ven, Amelie; Boston Children's Hospital, Medicine Ityel, Hadas; Boston Children's Hospital, Medicine Chen, Jing; Boston Children's Hospital, Medicine Sadowski, Carolin; Boston Children's Hospital, Medicine Kohl, Stefan; Boston Children's Hospital, Medicine Pabst, Werner; Boston Children's Hospital, Medicine Somers, Michael; Boston Children's Hospital, Medicine Rodig, Nancy; Boston Children's Hospital, Medicine Daouk, Ghaleb; Children's Hospital Boston, Medicine Baum, Michelle; Boston Children's Hospital, Medicine Stein, Deborah; Boston Children's Hospital, Medicine Ferguson, Michael; Boston Children's Hospital, Medicine Traum, Avram; Boston Children's Hospital, Medicine Soliman, Neveen; Kasr Al Ainy School of Medicine, Department of Pediatrics Kari, Jameela; King Abdulaziz University Hospital, Department of Pediatrics El Desoky, Sherif; King Abdulaziz University Hospital, Department of

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Complete List of Authors:

CJASN-0412-04-17.R1

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Pediatrics Fathy, Hanan; University of Alexandria, Pediatric Nephrology Unit Zenker, Martin; Otto von Guericke Universitat Magdeburg, Institute of Human Genetics Bakkaloglu, Sevcan; Gazi University Hospital, Müller, Dominik; Charité, Pediatric Nephrology Noyan, Aytül; Baskent Universitesi, Department of Pediatric Nephrology Ozaltin, Fatih; Hacettepe University, Pediatrics Cadnapaphornchai, Melissa; Children's Hospital Colorado, Department of Pediatrics Hashmi, Seema; Sindh Institute of Urology and Transplantation, Pediatrict Nephrology and Histopathology Hopcian, Jeffrey; University of Michigan, Pediatrics Kopp, Jeffrey; National Institute of Diabetes and Digestive and Kidney Diseases, Kidney Disease Section Benador, Nadine; University of California San Diego, Pediatric Nephrology Bockenhauer, Detlef; Great Ormond Street Hospital For Children NHS Trust, Nephrology Bogdanovic, Radovan; University of Belgrade, Nephrology Stajic, Natasa; University of Belgrade, Nephrology Chernin, Gil; Kaplan Medical Center, Departments of Nephrology and Hypertension Ettenger, Robert; Mattel Children's Hospital at UCLA, Pediatrics Fehrenbach, Henry; Klinikum Memmingen, Pediatric Nephrology Kemper, Markus; Asklepios Kliniken Hamburg GmbH, Loza Munarriz, Reyner; Universidad Peruana Cayetano Heredia, Pediatrics Podracka, Ludmila; Univerzita Komenskeho v Bratislave, Pediatrics Büscher, Rainer; Universitäts-Kinderklinik Essen, Pediatrics Serdaroglu, Erkin; Dr. Behcet Uz Children's Hospital , Pediatric Nephrology Tasic, Velibor; University Children's Hospital, Medical School Skopje, Peditaric Nephrology Mane, Shrikant; Yale University School of Medicine, Genetics Lifton, Richard; Yale University, Genetics Braun, Daniela; Boston Children's Hospital, Medicine Hildebrandt, Friedhelm; Boston Children's Hospital , of Medicine, Division of Nephrology

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genetic renal disease, nephrotic syndrome, pediatric, molecular genetics

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Keywords:

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Introduction: Steroid-resistant nephrotic syndrome overwhelmingly progresses to end-stage renal disease. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 29.5% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared to whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in a large international cohort of 300 families.

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Abstract:

Methods: 335 individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from 4/1998 to 6/2016. Age of onset was restricted to under 25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. Results: In 78/300 families (26%), we identified a causative mutation in one of 23 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 40% of consanguineous families and in 13% of non-



consanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 74 different mutations were detected in 23 of 33 steroid-resistant nephrotic syndrome genes. Twenty of these mutations were novel. NPHS1, PLCE1, NPHS2 and SMARCAL1 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 towards 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.

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Friedhelm Hildebrandt, MD William E. Harmon Professor of Pediatrics Harvard Medical School Chief, Division of Nephrology

To Rajnish Mehrotra, MD, MS, FASN Editor-in-Chief Clinical Journal of the American Society of Nephrology

Boston Children’s Hospital 300 Longwood Avenue, HU319 Boston MA 02115 617-355-6129 | fax 617-730-0569

Re: “Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome”, Jillian K. Warejko, et al (CJASN-0412-04-17)

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Dear Dr. Mehrotra,

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Thank you for your kind editorial letter of 05/21/2017. Please find attached a ‘Response to Reviewer’ file in which we respond point-by-point to all the reviewers’ comments, addressing them all. As requested, we now provide the attached document address the reviewers’ concerns.

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Following your suggestion, we have now changed the title to “Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome”

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We trust that we therewith have completely fulfilled all the Editor’s and Reviewers’ requests. Thank you very much for your consideration.

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Sincerely, Friedhelm

Friedhelm Hildebrandt, MD William E. Harmon Professor of Pediatrics Harvard Medical School Chief, Division of Nephrology



Response to Editor and Reviewer’s Comments The manuscript must be revised and submitted within 60 days of receipt (5/21/17) of this letter. In addition to new or revised figures, tables and text, a detailed, point-by-point response to all issues raised by the Editors and Referees should be included. This response should present each comment followed by your response and any changes to the manuscript (noting the location of the change). The revised text should have changes from the original submission highlighted to facilitate rapid review of the revisions by editors and reviewers. Response: As requested, we have provided the below responses. The manuscript text has had changes highlighted in yellow, and underlined in figures.

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Editors' notes for Author:

1. Please note that the corresponding author has listed the author disclosures as included below within the manuscript submission. Please also remember to add the correct statement to your word document before the reference section. Disclosures: Friedhelm Hildebrandt has intellectual property licensed to Claritas and is a cofounder of Goldfinch.

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Response: As requested, we have now adjusted the COI statement in the appropriate section of the manuscript, saying (page17, paragraph 3). “Disclosures: Friedhelm Hildebrandt has intellectual property licensed to Claritas and is a cofounder of Goldfinch.”

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2. The word limit for original manuscripts is 3,000 words, excluding the abstract word limit which is now 300 words. There can be some flexibility to accommodate reviewer recommendations. However, the word count should remain as close to 3,000 words as possible.

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Response: As requested, we have now shortened the manuscript to 2979 words. The abstract has been shortened to 295 words. As such, we have shorted the text in all sections of the manuscript. Editor’s Comments 1. Please change the title of the manuscript to "Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome" Response: As requested, we have now changed the title accordingly. In regards to reviewer 3’s title suggestion, we changed the title to the one suggested by the editor.

1 ScholarOne support: 888-503-1050

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2. Please do not use any abbreviations or acronyms in the Abstract of the manuscript, other than the names of the genes with mutations. Please ensure that the word count for the Abstract remains 300 words or less. The Introduction section of the manuscript should be abbreviated, including removing the reference of the prior work (name of author and publication year). Response: Changes were introduced as requested. 3. Please present all proportions > 10% as whole numbers. Please review and update the Abstract, body of manuscript, Tables, Figures and Figure legends to ensure consistency of presentation. Response: We have adapted the abstract and manuscript, including all figures, figure legends, and tables as requested. We have highlighted changes in yellow, or in figures, underlined.

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4. Please limit the abbreviations/acronyms in the manuscript to CKD, ESRD, FSGS, PCR, SNP, LOD, DNA, and the name of the genes with mutations. Please expand other acronyms; some acronyms are used for other phrases in other settings - NS, not significant or nephrotic syndrome. Please try to keep the word count close to 3000 words nevertheless. Please review the Tables, figures, and figure legends to ensure consistency of data presentation.

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Response: As requested we have expanded abbreviations and acronyms as noted above.

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5. Please limit the use of the word "renal" to "end-stage renal disease" and "extrarenal" Please change renal to kidney - some sentences may need to restructured with the change.

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Response: We have changed the wording as requested.

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6. Please review the attached STROBE checklist and please include information on all items marked as NO in the body of the manuscript. Response: We have reviewed the STROBE checklist and noted that no changes are necessary to be made.



Associate Editor Comments to Author: 1. The Reviewers suggest an emphasis on a comparison between WES panel sequencing and the potential clinical benefit of the WES approach. Response: As requested, we have provided additional emphasis on the comparison between Fluidigm panel sequencing and WES in our manuscript. 94 of the 300 families studied by WES in this manuscript have been previously studied using Fluidigm panel sequencing (Sadowski, JASN 6:1279, 2015). We have now detailed this overlap in Supplementary Table 4. Interestingly, we found that, whereas in 20/78 families the causative mutation was previously detected using panel sequencing, 9 families had a diagnosis made by WES and not by panel sequencing. We have added this comparison of panel sequencing versus WES to the Results, saying (p. 10, paragraph 2):

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“94 of the 300 families studied by whole exome sequencing have been previously studied using Fluidigm panel sequencing (Sadowski, JASN 6:1279, 2015). The overlap between cohorts is given in Supplementary Table 4. We found that whereas in 20/78 (25%) families the causative mutation was previously detected using panel sequencing, 9/78 (12%) had a diagnosis made by whole exome sequencing and not by panel sequencing.”

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2. The authors should provide information as to whether patients in their previous study in 2015 were included in the present study, or whether they were a new population.

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Response: In response to this query, please see the above paragraphs. 3. They should also comment on the potential pathogenicity of the novel mutations identified.

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Response: In order to decide if a novel mutation in a gene previously described to cause steroid resistant nephrotic syndrome is disease causing, we used very stringent criteria as previously outlined in Methods. To portray these criteria in a more transparent way we have now added a supplementary table with sets of decision criteria, for recessive and dominant criteria (Supplementary Table 3). We refer to these tables in Methods, saying (p. 7, paragraph 2): “Mutations were designated as likely pathogenic based on criteria given by Supplementary Table 3.” Accordingly, we have now made the following addition to the discussion on page 13, paragraph 1: “To determine the pathogenicity of novel mutations in genes previously described to cause steroid-resistant nephrotic syndrome, we used a strict set of criteria separately for 3 ScholarOne support: 888-503-1050

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recessive or dominant. Criteria were based on evolutionary conservation, bioinformatic prediction programs of pathogenicity, and allele frequency in healthy control populations (Supplementary Table 3 and 9).” 4. Examples of the clinical benefit of the WES approach should be given, if possible. Response: As requested, we have added additional clinical consequences to our Discussion, saying (p.14, paragraph 2.) “In the case of the individual with COQ2 mutation, this individual was placed on COQ10 therapy and experienced a sustained remission of nephrosis.” Additionally, in our discussion of transplant consequences, we have added, saying, (p.15, paragraph 2): “Additionally, in families with INF2 mutations, the parents and family members should be screened for INF2 mutations, as this dominant disease has variable expressivity within families. Should a family member be positive for mutation, this would disqualify them from donation of a kidney for transplantation as they are at risk for developing proteinuria and kidney disease in the future.”

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Response to Referee 1: Comments to Author This is an important work with important clinical implications, such as the relatively high proportion of families with mutations in non-SRNS genes, and the high rate of mutations in candidate genes. These observations emphasize the clinical advantage of exome sequencing as compared to the panel sequencing. There are a few concerns, two of which affect the reported mutation rate. [Major comments] 1. NPHS2 and WT1, two of the three most commonly mutated genes in early-onset SRNS are underrepresented in the presented work, being together responsible for only 3.3% (Table 1) of the 300 cases, while they were previously reported to be responsible for 14.7% of the 1783 cases by the same group (J Am Soc Nephrol 26: 1279–1289, 2015). As the authors clearly state, this is due to the fact that they have not included families with previously identified NPHS2 and WT1 mutations in this analysis. However, this significantly decreases the overall mutation rate of SRNS genes in the patient cohort. I would suggest to include these data, even if they were published before, to provide clinically relevant mutation rates.

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Response: We agree that the overlap between the previous Sadowski publication (Sadowski, JASN 6:1279, 2015) and our study is somewhat unclear in the manuscript. We have therefore added the following paragraph to the discussion, saying (p. 13, paragraph 2):

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“NPHS2 and WT1, two of the three most commonly mutated genes in early-onset steroid-resistant nephrotic syndrome are underrepresented in the presented work, being together responsible for only 3.3% (Table 1) of 300 cases, while they were previously reported to be responsible for 14.7% of cases in 1783 cases ( Sadowski, JASN 6:1279, 2015). When all 1989 families studied in Sadowski, et al and in this study are combined together, mutation rates for NPHS2 and WT1 become more representative of what has been previously published. NPHS2 has a detection rate of 9.3% (185/1989) and WT1 has a detection rate of 4.4% (87/1989). Mutation rates for NPHS2 were previously 9.9% and for WT1 were 4.8%.”

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2. The pathogenicity of several mutations listed in Supplementary Table 6 is highly questionable on an epidemiological basis, like that of p. V801M of ACTN4, the p. V236M and c.1036-3C>G variants of ADCK4, the p.G1840S and p.N2157D variants of CUBN and the p.D571E of MYO1E. These variants have been reported in ExAc with a relatively high frequency, making very unlikely their pathogenicity: if they were pathogenic, they should have been frequently reported. Furthermore, variants reported in ExAc in the homozygous state, especially in genes responsible for dominant FSGS, are clearly unexpected to be pathogenic. The authors should have used different allele frequency criteria in dominant and recessive disorders. The reevaluation of the pathogenicity of these variants will also affect the number of genes mutated. Response: 5 ScholarOne support: 888-503-1050

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As requested, we have now made the criteria used to determine deleteriousness more transparent by adding Supplementary Table 3 for recessive and dominant alleles, respectively. In the case of p. V801M of ACTN4, this mutation meets those criteria in that 2/3 prediction programs predict deleteriousness of the variant to protein function. We agree that the heterozygous allele frequency is higher than we typically accept for dominant alleles (0.3% in this case versus our stated standard of accepting alleles if the heterozygous frequency is 10% are rounded to the nearest whole number.



A 42, 9, 112 (26%, 5.5%, 69%) 38, 5, 95 (28%, 3.6%, 69%)

Female Unknown

5, 1, 28 (15%, 2.9%, 82%)

Infants < or = 3 mo

15, 2, 14 (48%, 6.5%, 45%)

Children > 6 - 12 yr

12, 1, 41 (22%, 1.9%. 76%) 5, 2, 27 (15%, 5.9%, 79%)

9, 4, 39 (17%, 7.7%, 75%)

Not reported

0

Total individuals enrolled

No causative mutation detected

3, 0, 5 (38%, 0%, 62%)

85

15

(25%, 4.5%, 70%)

235

10 0

Young adults 18-25 yr

Causative mutation in phenocopy gene detected

19, 4, 71 (20%, 4.3%, 76%)

20 0

Age of onset

Children > 1- 6 yr

Children > 12 - 18 yr

Causative mutation in SRNS gene detected

22, 2, 38 (35%, 3.2%, 61%)

Infants >3 mo - 12 mo

30 0

Sex

Male

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Number of individuals

Pe p10% are rounded to the nearest whole number. (B) Median age of onset in patients with a causative mutation detected in a steroid-resistant nephrotic syndrome gene was 1.7 years versus 4 years in those without a mutation detected (range 0-24 years). For those with a causative mutation detected in a steroid-resistant nephrotic syndrome gene, the range was 0-21 years. Mann-Whitney U test p 2 individuals in ExAC) - Non-segregation in the case of compound heterozygotes

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Consider excluding allele as disease causing if:

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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)).

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Consider excluding allele as disease causing if:

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



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. Total families evaluated by panel sequencing and WES

94/300 (31%)

Total families evaluated by WES only

206/300 (69%)

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Total families with a causative mutation detected by panel sequencing and WES

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Total families with a causative mutation detected by WES and not by panel sequencing

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Total families evaluated by WES only with a causative mutation detected

WES (whole exome sequencing).

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20/78 (26% of solved cases) 9/78 (12%) 50/78 (64%)

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Supplementary Figure 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. Family ID

Gene

Zygosity

Accession #

c. position

p. position

Continuously conserved to

MT/SFT/PPi

ExAC

A1756 A4684

DDX53 MXRA5

Hemi Hemi

NM_182699.3 NM_015419.3

c.24G>A c.204_205insT

p.Trp8* p.Ala69Cysfs*22

Truncating Frameshift

-

NR NR

B51

DHTKD1

Hom

NM_018706.6

c.886G>A

p.Val296Met

Dm

DC/Del/1

0/3/121366

B52

DHTKD1

Hom

NM_018706.6

c.886G>A

p.Val296Met

Dm

DC/Del/1

0/3/121366

A5013 B50

CDK20 OSGEP

Hom Hom

NM_001039803.2 NM_017807.3

c.610T>C c.40A>T

p.Phe204Leu p.Ile14Phe

Dm Dr

DC/Del/1 DC/Del/0.023

NR NR

B57

OSGEP

Hom

NM_017807.3

c.40A>T

p.Ile14Phe

Dr

DC/Del/0.023

NR

B123 B377 B787 B1356

TPRKB OSGEP SLC35F1 COG1

Hom Hom Hom Hom

NM_016058.2 NM_017807.3 NM_001029858.3 NM_018714.2

c.407T>C c. 740G>A c.878T>G c.1070+5G>A

p.Leu136Pro p.Arg247Gln p.Met293Arg Splice

Xt Dm Ce Splice

DC/Del/1 DC/Tol/0.998 DC/Del/0.999 -

NR 0/8/121400 NR NR

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Clinical diagnosis. ) SRNS SRNS Deidentified Deidentified SRNS SRNS Deidentified SRNS CNS SRNS 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.

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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 an steroid-resistant nephrotic syndrome gene or a phenocopy gene. Age and sex demographics are given for a subset of 85 individuals from 78 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 78 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 Characteristics of Total Cohort

Clinical Characteristics of Individuals with Causative Mutation Detected Number of individuals with SRNS mutation detected (%)

Number of individuals with mutation detected - phenocopy gene (%)

42/85 (49%) 38/85 (45%) 5/85 (5.9%) 85/85 (100%)

9/15 (60%) 5/15 (33%) 1/15 (6.7%) 15/15 (100%)

4 (0-24)

1.7 (0-21)

4 (0-16)

31/335 (9.3%) 62/335 (19%) 94/335 (28%) 54/335 (16%) 34/335 (10%) 8/335 (2.4%) 52/335 (16%) 335/335 (100%)

15/85 (18%) 22/85 (26%) 19/85 (22%) 12/85 (14%) 5/85 (5.9%) 3/85 (3.5%) 9/85 (11%) 85/85 (100%)

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2/15 (13%) 2/15 (13%) 4/15 (27%) 1/15 (6.7%) 2/15 (13%) 0/15 (0%) 4/15 (27%) 15/15 (100%)

Number of families (%)



77/300 (26%) 59/300 (20%) 29/300 (10%) 22/300 (7.3%) 21/300 (7%) 15/300 (5%) 8/300 (2.7%) 9/300 (3%)

30/78 (39%) 10/78 (13%) 12/78 (15%) 2/78 (2.6%) 7/78 (9%) 4/78 (5.1%) 3/78 (3.8%) 0/78 (0%)

3/11 (27%) 3/11 (27%) 2/11 (18%) 1/11 (9.1%) 0/11 (0%) 0/11 (0%) 0/11 (0%) 0/11 (0%)

6/300 (2%)

1/78 (1.3%)

1/11 (9.1%)

1/300 (0.3%) 1/300 (0.3%) 52/300 (17%) 300/300 (100%)

0/78 (0%) 1/78 (1.3%) 8/78 (10%) 78/78 (100%)

0/11 (0%) 0/11 (0%) 1/11 (9.1%) 11/11 (100%)

Number of individuals (%) Gender Male Female Unknown Total

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Children > 12 yr or 1 yr and ≤ 6 yr Children > 6 and ≤ 12yr

163/335 (49%) 138/335 (41%) 34/335 (10%) 335/335 (100%)

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SRNS, steroid-resistant nephrotic syndrome.


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Supplementary Table 7: Presence of consanguinity, homozygosity, or multiple affected statuses in 300 families with steroid-resistant nephrotic syndrome. A subset of 78 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 steroidresistant nephrotic syndrome compared to a subset of 85 individuals from 78 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 Characteristics of Total Cohort

Clinical Characteristics of Individuals/Families with Causative Mutation Detected


Number of families with SRNS mutation detected (%)

Number of families with mutation detected phenocopy gene (%)

146/300 (49%)

59/78 (76%)

6/11 (55%)

135/300 (45%)

18/78 (23%)

4/11 (36%)

19/300 (6.3%)

1/78 (1.3%)

1/11 (9.1%)

147/300 (49%)

58/78 (74%)

5/11 (45%)

153/300 (51%)

20/78 (26%)

6/11 (55%)

Pedigree Consanguineous Non-consanguineous Unknown consanguinity

2/11 (18%)

65/300 (22%)

21/78 (27%)

5/11 (46%)

7/78 (9%)

3/11 (27%)

28/300 (9.3%) 33/300 (11%)

5/78 (6.4%)

1/11 (9.1%)

300/300 (100%)

78/78 (100%)

11/11 (100%)

Number of individuals (%) 91/335 (27%)

Number of individuals (%) 22/85 (26%)

219/335 (65%)

62/85 (73%)

7/15 (47%)

25/335 (7.5%)

1/85 (1.2%)

2/15 (13%)

85/85 (100%)

15/15 (100%)

ew

No Unknown/de-identified sample

45/78 (58%)

vi

Yes

174/300 (58%)

Re

Total families Extra-renal manifestations

er

Families with one affected individual Families with 2 affected individuals Families with 3 or greater affected individuals Unknown/de-identified sample

Pe

Homozygosity on mapping >100Mbp Homozygosity on mapping 10% are rounded to the nearest whole number.

Clinical Characteristics of Total Cohort

Clinical Characteristics of Individuals/Families with Causative Mutation Detected


Number of families with SRNS mutation detected (%)

Number of families with mutation detected phenocopy gene (%)

SRNS

205/300 (68%)

51/78 (65%)

9/11 (82%)

CNS

32/300 (11%)

17/78 (22%)

0/11 (0%)

9/300 (3%)

1/78 (1.3%)

0/11 (0%)

1/300 (0.3%)

1/78 (1.3%)

0/11 (0%)

4/300 (1.3%)

1/78 (1.3%)

0/11 (0%)

6/300 (2%)

1/78 (1.3%)

0/11 (0%)

7/300 (2.3%)

1/78 (1.3%)

0/11 (0%)

36/300 (12%)

5/78 (6.4%)

2/11 (18%)

Clinical diagnosis

r Fo

Infantile nephrotic syndrome Nephrotic syndrome with ESRD on presentation

ESRD on presentation, FSGS or DMS on biopsy Nephrotic syndrome with FSGS or DMS on biopsy

De-identified sample

78/78 (100%) Number of individuals (%) Diagnosis on biopsy (n=54)

153/223 (69%)

DMS

14/223 (6.3%)


39/54 (72%)

3/9 (33%)

3/54 (5.6%)

1/9 (11%)

MCNS

20/223 (9%)

5/54 (9.3%)

0/9 (0%)

MPGN

10/223 (4.5%)

2/54 (3.7%)

1/9 (11%)

CNS/Finnish type

5/223 (2.2%)

ew

FSGS

vi

Diagnosis on biopsy


Re

Total families enrolled

er

Nephrotic range proteinuria with FSGS or DMS of biopsy

Pe

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 116

1/54 (1.9%)

0/9 (0%)

Membranous GN

1/223 (0.4%)

0/54 (0%)

0/9 (0%)

Other

20/223 (9%)

4/54 (7.4%)

4/9 (44%)

112/335 (33%)

31/85 (37%)

6/15 (40%)

No bx data available

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.


Page 55 of 116

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Supplementary Table 9: Summary of causative mutations detected in one of 23 steroid-resistant nephrotic syndrome causing genes and 8 phenocopy genes in 90 of 300 families with steroid-resistant nephrotic syndrome by family and clinical phenotype. Gene

Family ID and indiv. #

c. change

p. change

Zygosity

Cons.

SFT

MT

PPi

ExAC (home/het/ total alleles)

Age (years)

Sex (M/F)

Race/ Ethnicity

Consang. (Y/N)

# affected per family

Synd . (Y/N)

Clin. Dx

Kidney biopsy results

ACTN4

B881_2 1

c. 2401G>A

p. V801M

Het

Dr

Del

DC

0.018

1/430/108228

20

F

C/E

N

1

N

SRNS

FSGS

c.706G>A

p. V236M

Mm

-

-

0.094

1/29/120426

ADCK4

A630_2 1

12

F

A/AA

Y

1

N

SRNS

FSGS

2

M

C/E

N

1

N

SRNS

FSGS

1

N

SRNS

MCNS

N

SRNS

FSGS

N

SRNS

MPGN

COQ2

CUBN

Fo Comp het Comp het

Splice

-

-

-

1/62/121000

FS

-

-

-

NR

Dm

Tol

rP

c.1036-3C>G

Splice

c.176_177in sT

p.F59fs

Comp het

c.683A>G

p.N228S

Comp het

B1498_ 21

c.6469A>G

p.N2157D

Hom

Dm

Del

A4431_ 21

c.610del

p.T204Qfs*6

Hom

FS

-

B1425_ 21

ee DC

0.918

DC

0.99

2/680/120822

1

M

Arabic

Y

-

-

NR

17

F

C/E

Y

DGKE

INF2

0/20/120566

rR

ev

A4431_ 22

c.610del

p.T204Qfs*6

Hom

FS

-

-

-

B788_2 1

c.532T>G

p.F178V

Het

Dr

Tol

DC

0.996

NR

A1605_ 21

c.2593del

p.D865Tfs*38

Hom

FS

-

-

-

NR

C

p. R628P

Hom

Dm

Del

DC

0.3

0/3/118974

B324_2 1

c.2401T>C

p.Y801H

Hom

Dm

Del

DC

1

A1757_ 21

c.143A>C

p.Y48S

Hom

Dr

Del

DC

A1757_ 22

c.143A>C

p.Y48S

Hom

Dr

Del

B819_2 1

c.395C>T

p.A132V

Hom

Xt

A2356_ 23

c.736C>T

p. R246W

Hom

Dm

NR

8

21

F

C/E

iew

Y

M

C/E

N

7

N

SRNS

FSGS

M

Turkish

Y

1

N

SRNS

FSGS

4 mo

F

Asian

Y

1

N

CNS

No bx

0/121/120924

2 mo

F

Roma

N

4

Y

CNS

MCNS

1

0/70/117226

13

M

Hispanic

N

N

SRNS

FSGS

DC

1

0/70/117226

13

F

Hispanic

N

N

SRNS

FSGS

T

P

0.002

0/2/121366

0

F

Turkish

Y

2

N

CNS

No bx

Del

DC

1

0/1/119680

3mo

M

Arabic

Y

2

Y

CNS

CNS

ITGA3

KANK4

3

2

LAMB2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 56 of 116

A5284_ 12

c.1731+1G> A

Splice

Hom

Splice

-

-

-

0/1/118980

1 mo

F

Asian

Y

1

Y

CNS

No bx

A2263_ 23

c.4537C>T

p.Q1513*

Hom

Trunc.

-

-

-

NR

2 mo

M

Arabic

Y

1

Y

SRNS

No bx

B1219_ 21

c.4573C>T

p.Q1525*

Hom

Trunc.

-

-

-

0/1/121384

0.2

F

Arabic

Y

1

Y

CNS

No bx

A200_2 1

c.737G>A

p.R246Q

Het

Dm

Del

DC

0.998

NR

8

F

Turkish

Y

2

N

SRNS

FSGS

A4642

c.737G>A

p.R246Q

Hom

Dm

Del

DC

0.998

NR

unk

unk

unk

unk

unk

unk

Deidentified

unk

B112_2 1

c.701A>G

p.Y234C

Ce

Del

DC

0.998

0/5/121374 2 mo

F

C/E

N

1

N

NS, DMS on bx

DMS

18

M

unk

Y

1

N

ESRD at presentatio n

Other chronic renal failure

NR

45 do

M

Asian

Y

1

N

CNS

MCD

0/2/114206

4 mo

M

Arabic

Y

1

N

SRNS

No bx

F

Arabic

Y

2

N

SRNS

No bx

LMX1B

Fo Comp het Comp het

c.1713C>G

p.D571E

Dr

Tol

DC

0.02

0/94/121412

A146_2 1

c.1228G>A

p.E410K

Hom

Sc

Del

DC

0.98

NR

A3656_ 21

c.1978C>T

p.Q660*

Hom

Trunc.

-

-

-

A5151_ 21

c.139del

p.A47Pfs81*

Hom

FS

-

-

-

A4472_ 22

c.515_517de l

p.T172del

Hom

Inframe del

-

-

-

c.1048T>C

p.S350P

Comp het

Dm

Del

P

0.76

c.2506+5G> T

Splice

Comp het

Splice

-

-

-

0/1/120468

B1238

c.1379G>A

p.R460Q

Hom

Ce

Tol

Pol

0.48

B55

c.1760T>G

p.L587R

Hom

Dr

Del

DC

c.2014G>A

p.A672T

Comp het

Dr

Del

c. 3250dupG

p.V1084Gfs*

Comp het

FS

A3432_ 24

c.2020C>A

p.P674T

Hom

A1500_ 21

c.2728T>C

p.S910P

Hom

MYO1E

B1122_ 21

rP

ee

rR NR

ev

60 do

iew

0/2/121174

unk

F

C/E

Y

1

N

CNS

unk

0/1/119280

0.25

M

Arabic

Y

4

N

CNS

No bx

0.99

NR

unk

unk

unk

Y

unk

Y

Deidentified

No bx

DC

0.99

0/1/82174 5 mo

M

Turkish

N

1

Y

Infantile NS

FSGS

-

-

-

0/1/82174

Dr

Del

DC

0.3

0/3/118974

1 mo

M

Arabic

Y

4

N

CNS

No bx

Dr

Del

DC

0.959

NR

1

M

A/AA

N

1

N

SRNS

MCNS

NPHS1

A5275_ 21


Page 57 of 116

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Clinical Journal of the American Society of NEPHROLOGY A3509_ 21

c.3478C>T

p.R1160*

Hom

Trunc.

-

-

-

0/8/121256

0

F

Asian

Y

1

N

CNS

No bx

A3594_ 21

c.3478C>T

p.R1160*

Hom

Trunc.

-

-

-

0/8/121256

0

F

Arabic

Y

1

N

CNS

No bx

A3708_ 21

c.3478C>T

p.R1160*

Hom

Trunc.

-

-

-

0/8/121256

2 mo

M

A/AA

Y

1

N

CNS

FSGS

A4427_ 23

c.3478C>T

p.R1160*

Hom

Trunc.

-

-

-

0/8/121256

0

F

Other/mul tiple races

N

1

N

CNS

No bx

B1357_ 21

c.3478C>T

p.R1160*

Hom

Trunc.

-

-

-

0/8/121256

0.1

F

African/ Arabic

Y

1

N

CNS

No bx

A4681_ 21

c.1A>T

p.M1?

-

-

-

NR

7

F

Arabic

Y

1

N

SRNS

FSGS

A679 _21

c.397del

p.R133Efs*2

unk

M

C/E

N

N

SRNS

No bx

c.413G>A

p.R138Q

Comp het

c. 397del

p.R133Efs*2

Comp het

N

SRNS

No bx

c.413G>A

c.419del

N

SRNS

FSGS

N

SRNS

FSGS

N

SRNS

No bx

N

SRNS

FSGS

N

SRNS

No bx

CNS

Other diffuse mesangi al hypercel lularity

A679 _22

A3133_ 21

NPHS2

Fo Hom

Comp het

Start loss

rP FS

-

-

-

NR

Dm

Del

DC

0.999

0/82/121298

FS

-

-

-

NR

p.R138Q

Comp het

Dm

Del

DC

0.999

0/82/121298

p.G140Dfs*41

Hom

FS

-

-

-

0/1/121308

ee

2

rR

unk

M

C/E

N

5.8

F

Arabic

Y

ev

2

A3133_ 43

c.419del

p.G140Dfs*41

Hom

FS

-

-

-

0/1/121308

2

B963_2 1

c.538G>A

p.V180M

Hom

Dr

Del

DC

0.58

0/3/120452

9 mo

F

Arabic

Y

c.686G>A

p.R229Q

Comp het

Xt

Tol

P

0.313

69/3526/11910 8 14

F

C/E

N

c.916A>T

p.R306W

Comp het

Dr

Del

DC

0.98

NR

c.686G>A

p.R229Q

Comp het

Xt

Tol

P

0.313

69/3526/11910 8

c.916A>T

p.R306W

Comp het

Dr

Del

DC

0.98

NR

A667_2 1

F

Arabic

iew

Y

1

2 A667_2 2

A4309_ 21

9

c.705_713de l9

p.L236del

Hom

Inframe del

-

-

-

NR

3 mo


M

M

C/E

Asian

N

Y

1

Y


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

NUP205

Page 58 of 116

B1090

c.800A>T

p.D267V

Hom

Ce

Del

DC

1

NR

8

M

African/ Arabic

Y

1

N

SRNS

FSGS

B188

c.855_856de l

p.R286Tfs*17

Hom

FS

-

-

-

0/8/115938

3

F

Hispanic

Y

2

N

SRNS

MCNS

B140_2 1

c.3095G>A

p.C1032Y

Hom

Dm

Tol

DC

1

NR

3

M

Arabic

Y

1

Y

SRNS

FSGS

A1733_ 21

c.5984T>C

p.F1995S

Hom

Dm

Tol

DC

0.99

NR

3.5

F

Turkish

N

N

SRNS

FSGS

N

SRNS

No bx

2 A1733_ 22

c.5984T>C

p.F1995S

A1626_ 21

c.1772G>T

p.G591V

A1671_ 21

c.1886A>G

p.Y629C

A2241_ 22

c.1886A>G

p.Y629C

B1311_ 21

c.2017C>T

p.R673W

Hom

A3853_ 22

c.1145C>T

p.Ser382Leu

B913_2 1

c.1709del

A1678_ 21

c.2576_2577 insT

A3617_ 25 A3921_ 22

c.3379_3380 del c.4506+2T> C

A59_21 B354_2 2

Dm

Tol

DC

0.99

NR

3

M

Turkish

N

Hom

Sc

Del

DC

1

0/14/121252

2.5

M

Turkish

Y

1

N

SRNS

FSGS

Hom

Sc

Del

rP

DC

0.997

0/1/120978

1.3

M

Turkish

N

1

N

SRNS

IgA

Sc

Del

DC

0.997

0/1/120978

11 mo

M

Turkish

Y

2

N

SRNS

No bx

Dr

Tol

DC

1

NR

1

F

Arabic

Y

2

N

CNS

FSGS

Hom

Dr

Del

DC

1

0/4/121372

1

M

Arabic

Y

2

Y

SRNS

No bx

p.S570Tfs*29

Hom

FS

-

-

-

NR

9 mo

M

Turkish

Y

1

Y

SRNS

FSGS

p.Q859Hfs*31

Hom

FS

-

-

-

M

Turkish

Y

1

N

SRNS

DMS

p.N1127*

Hom

Trunc.

-

-

-

NR

Splice

Hom

Splice

-

-

-

NR

c.4887del

p.A1630Qfs*4 0

Hom

FS

-

-

-

c.4978_4981 CAGA

p.Q1660Lfs*9

Hom

FS

-

-

c.5521A>G

p.K1841E

Hom

Sc

Del

DC

NUP93

PDSS2

PLCE1

A4654_ 21 A4654_ 22 A3869_ 24

Hom

Fo Hom

ee

rR NR

ev 7.9

iew

9 mo

F

Arabic

Y

3

N

SRNS

FSGS

6 mo

F

Arabic

Y

2

N

SRNS

FSGS

NR

7 mo

F

Turkish

N

1

N

SRNS

FSGS

-

NR

1

M

Arabic

Y

2

N

SRNS

DMS

1

NR

4

F

Arabic

Y

N

SRNS

FSGS

N

SRNS

FSGS

2 c.5521A>G

p.K1841E

Hom

Sc

Del

DC

1

NR

2.4

F

Arabic

Y

c.5521A>G

p.K1841E

Hom

Dm

Del

DC

1

NR

7 mo

M

Arabic

Y

1

N

SRNS

FSGS

-

-

-

0.5

M

Arabic

Y

1

N

SRNS

FSGS

-

-

-

6 mo

M

Arabic

Y

1

N

SRNS

FSGS

B1432_ 24

c.5950_5952 delAAC

p.N1984del

Hom

A4043_ 21

c.59515953delACA

p.N1984del

Hom

Inframe del. Inframe del.

NR


Page 59 of 116

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

SGPL1

SMARCAL1

TRPC6

Clinical Journal of the American Society of NEPHROLOGY A5171_ 21

c.5951_5953 del

p.N1984del

Hom

Inframe del.

-

-

-

NR

5

Male

Arabic

Y

2

N

SRNS

FSGS

A280_2 1

c.665G>A

p.R222Q

Hom

Dm

Del

DC

1

0/2/120744

2.5

M

Asian

Y

3

Y

SRNS

FSGS

B46

c.1037G>T

p.S346I

Hom

Sc

Del

DC

1

NR

unk

unk

unk

Y

unk

Y

Deidentified

No bx

B56

c.1037G>T

p.S346I

Hom

Sc

Del

DC

1

NR

unk

unk

unk

Y

unk

Y

Deidentified

No bx

A925_2 1

c.1736C>A

p.S579*

Hom

Trunc.

-

-

-

NR

2.9

M

Arabic

Y

1

Y

SRNS

FSGS

A1683_ 21

c.1756C>T

p.R586W

Hom

Dm

Del

DC

1

0/1/121386

7.6

M

Turkish

Y

1

Y

SRNS

FSGS

F1367_ 21

c.1756C>T

p.R586W

Hom

Dm

Del

DC

1

0/1/121386

4

F

unk

Y

1

N

ESRD, FSGS on bx

FSGS

B1067

c.1822T>C

p.F608L

DC

1

NR

14

M

Arabic

Y

1

Y

SRNS

FSGS

B672_2 1

c.1940A>C

p.K647T

Hom

NR

6

F

Arabic

Y

2

N

SRNS

No bx

B142_2 2

c.2290C>T

p.R764W

NR

8

M

Arabic

Y

N

SRNS

FSGS

B1319_ 21

c.2290C>T

p.R764W

NR

unk

F

African/ Arabic

Y

Y

SRNS

MPGN

FSGS

Fo Hom

rP Dm

Del

Dm

Del

Hom

Dm

Del

Hom

Dm

Del

ee DC

1

DC

1

DC

1

rR

ev

1

M

C/E

N

1

Y

Nephrotic range proteinuria, FSGS on bx

F

Arabic

N

1

N

SRNS

FSGS

F

African/ Arabic

Y

1

N

SRNS

FSGS

5 mo

M

Asian

Y

1

N

SRNS

No bx

NR

T

p.E848*

Hom

Trunc.

-

-

-

0/14/121298

11

A4685_ 21

c.523C>T

p.R175W

Het

Dr

Del

DC

1

NR

7

A5262_ 21

c.626C>T

p.P209L

Hom

Dr

Tol

DC

1

0/8/121264

8

A5002_ 21

c.2569G>A

p.Ala857Thr

Hom

Ce

Del

DC

0.983

NR

B49

c.287G>A

p.R96K

Hom

Dr

Tol

DC

1

B129_2 1

c.703C>T

p.Q235*

Hom

Trunc.

-

-

B41

c.940C>T

p.Q315*

Hom

Trunc.

-

-

iew

TTC21B

WDR73



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 60 of 116

B1018_ 21

c.1432+5G> A

Splice

Het

Splice

-

-

-

NR

3

F

Arabic

N

1

N

SRNS

Otherfocal mesangi al prolifera tion

B1244_ 21

c.1432+5G> A

Splice

Het

Splice

-

-

-

NR

5

F

C/E

N

1

N

SRNS

No bx

A63_21

c.33dup

p.K12Qfs*156

Hom

FS

-

-

-

NR

4 mo

M

Turkish

Y

1

Y

SRNS

No bx

B465_2 3

c.863G>A

p. W288*

Hom

Trunc.

-

-

-

NR

4 mo

M

Arabic

Y

3

unk

Deidentified

No bx

A3094_ 22

c.933G>C

p.E311D

Del

DC

1

NR

12

M

C/E

Y

2

Y

SRNS

FSGS

A1221_ 21

c.4825C>T

p.Arg1609*

-

-

0/3/121000

5

F

C/E

N

Y

SRNS

FSGS

Y

SRNS

Other Alport's

WT1

AGXT

CLCN5

Fo Hemi

Sc

Het

Trunc.

-

-

0/3/121000

5

M

C/E

N

Hemi

rP Dm

Del

DC

0.999

NR

unk

unk

unk

unk

unk

unk

Deidentified

No bx

p.G1241D

Hemi

Dr

Del

DC

1

NR

16

M

Hispanic

N

>3

N

SRNS

FSGS

c.3722G>A

p.G1241D

Hemi

Dr

Del

DC

1

NR

11 mo

M

Turkish

Y

N

SRNS

MPGN

A169_2 2

c.3722G>A

p.G1241D

Hemi

Dr

Del

DC

1

NR

unkn

M

Turkish

Y

N

SRNS

Other Cresentr ic GN

B249_2 1

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

4

F

Arabic

Y

N

SRNS

No bx

B249_2 2

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

4

F

Arabic

Y

N

SRNS

No bx

B249_3 1

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

unk

F

Arabic

Y

N

SRNS

No bx

FN1

A4936_ 21

c.6836T>C

p.V2279A

Het

Dr

Del

DC

0.696

NR

1

F

C/E

N

2

N

SRNS

Other IgM nephrop athy

GLA

B912_2 1

c.504A>C

p. K168N

Hemi

Dr

Del

DC

1

NR

14

M

Arabic

Y

1

Y

SRNS

Other Fabry's disease

WDR19

B1119_ 21

c.3533G>A

p.R1178Q

Hom

Ce

T

DC

0.948

0/9/69008

1

M


N

2

Y

SRNS

DMS

COL4A3

COL4A5

CTNS

-

A1221_ 22

c.4825C>T

p.Arg1609*

Het

Trunc.

-

A4644_ 21

c.3088G>A

p.G1030S

A2058_ 21

c.3722G>A

A169_2 1

Inframe del. Inframe del. Inframe del.

2

ee

rR

ev

2

iew


4

Page 61 of 116

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


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.

Fo

rP

ee

rR

ev

iew



8, 1, 43 (15%, 1.9%, 83%)

Unknown/not indicated Roma

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

Ashkenazi Jewish

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

Race or ethnicity

Other/multiple races indicated

1, 1, 4 (17%, 17%, 67%)

Arabic and Asian

0, 0, 9 (0%, 0%, 100%)

African/African American

3, 0, 5 (37%, 0%, 63%)

Asian

7, 0, 14 (33%, 0%, 67%)

Hispanic/Latino

2, 1, 19 (9%, 4.5%, 86%)

Turkish

Arabic Total families enrolled

Causative mutation in a phenocopy gene detected

4, 0, 11 (27%, 0%, 73%)

African and Arabic

European/Caucasian



12, 2, 15 (41%, 6.9%, 52%) 10, 3, 46 (17%, 5.1%, 78%)

r Fo

30, 3, 44 (39%, 3.9%, 57%)

78

0

11

211

0 10

0 20 Number of families

er

Pe

(26%, 3.7%, 70%)

0 30

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 indivuals 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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Patients without extra-renal manifestions

62, 7, 150 (28%, 3.2%, 69%)

Patients with extra-renal manifestions

p= 0.67

22, 6, 63 (24%, 6.6%, 69%)

85

Total patients enrolled

15

235

(25%, 4.5%, 70%)

1, 2, 22 (4%, 8%, 88%)

Unknown/de-identified sample

0

0 10

0 20

r Fo

Causative mutation in SRNS gene detected Causative mutation in phenocopy gene detected No causative mutation detected

0 30

Number of patients

Supplementary Figure 2: Distribution of affected individuals regarding gene identification status (steroidresistant nephrotic syndrome (SRNS) gene, phenocopy gene, or no mutation detected) for extrarenal 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 indivuals 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=0.67).

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Nephrotic range proteinuria, FSGS or DMS of biopsy

1, 0, 6 (14%, 0%, 86%)

Nephrotic syndrome, FSGS or DMS on biopsy

1, 0, 5 (17%, 0%, 83%)

ESRD on presentation, FSGS or DMS on biopsy

De-identified sample


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

Infantile NS

SRNS


1, 0, 3 (25%, 0%, 75%)

Nephrotic syndrome, ESRD on presentation

CNS


1, 0, 8 (11%, 0%, 89%) 17, 0, 15 (53%, 0%, 47%)

r Fo

51, 9, 145 (25%, 4.4%, 71%)

5, 2 , 29 (14%, 5.6%, 81%)

78

Total families enrolled

11

10

211

0

er

0

Pe

(26%, 3.7%, 70%)

20

0

Number of families

Re

30

0

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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Page 64 of 116


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4, 4, 12 (20%, 20%, 60%)

Other

Histologic diagnosis


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

Membranous GN

1, 0, 4 (20%, 0%, 80%)

CNS/Finnish type


2, 1, 7 (20%, 10%, 70%)

MPGN


5, 0, 15 (25%, 0%, 75%)

MCNS

3, 1, 10 (21%, 7.1%, 71%)

DMS

39, 3, 111 (25%, 2%, 73%)

FSGS

31


0

6

75

r Fo 50

(28%, 5.4%, 67%)

0 10 Number of patients

0 15

Pe

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 steroidresistant 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 indivuals 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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Hildebrandt F: Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet, 38: 1397-1405, 2006 22. Barua M, Shieh E, Schlondorff J, Genovese G, Kaplan BS, Pollak MR: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis. Kidney Int, 85: 124-133, 2014 23. Lovric S, Goncalves S, Gee HY, Oskouian B, Srinivas H, Choi WI, Shril S, Ashraf S, Tan W, Rao J, Airik M, Schapiro D, Braun DA, Sadowski CE, Widmeier E, Jobst-Schwan T, Schmidt JM, Girik V, Capitani G, Suh JH, Lachaussee N, Arrondel C, Patat J, Gribouval O, Furlano M, Boyer O, Schmitt A, Vuiblet V, Hashmi S, Wilcken R, Bernier FP, Innes AM, Parboosingh JS, Lamont RE, Midgley JP, Wright N, Majewski J, Zenker M, Schaefer F, Kuss N, Greil J, Giese T, Schwarz K, Catheline V, Schanze D, Franke I, Sznajer Y, Truant AS, Adams B, Desir J, Biemann R, Pei Y, Ars E, Lloberas N, Madrid A, Dharnidharka VR, Connolly AM, Willing MC, Cooper MA, Lifton RP, Simons M, Riezman H, Antignac C, Saba JD, Hildebrandt F: Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency. J Clin Invest, 127: 912-928, 2017 24. Boerkoel CF, Takashima H, John J, Yan J, Stankiewicz P, Rosenbarker L, Andre JL, Bogdanovic R, Burguet A, Cockfield S, Cordeiro I, Frund S, Illies F, Joseph M, Kaitila I, Lama G, Loirat C, McLeod DR, Milford DV, Petty EM, Rodrigo F, Saraiva JM, Schmidt B, Smith GC, Spranger J, Stein A, Thiele H, Tizard J, Weksberg R, Lupski JR, Stockton DW: Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia. Nat Genet, 30: 215-220, 2002 25. Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis N, Kwan SY, Ebersviller S, Burchette JL, Pericak-Vance MA, Howell DN, Vance JM, Rosenberg PB: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science, 308: 1801-1804, 2005 26. Huynh Cong E, Bizet AA, Boyer O, Woerner S, Gribouval O, Filhol E, Arrondel C, Thomas S, Silbermann F, Canaud G, Hachicha J, Ben Dhia N, Peraldi MN, Harzallah K, Iftene D, Daniel L, Willems M, Noel LH, Bole-Feysot C, Nitschke P, Gubler MC, Mollet G, Saunier S, Antignac C: A homozygous missense mutation in the ciliary gene TTC21B causes familial FSGS. J Am Soc Nephrol, 25: 2435-2443, 2014 27. Colin E, Huynh Cong E, Mollet G, Guichet A, Gribouval O, Arrondel C, Boyer O, Daniel L, Gubler MC, Ekinci Z, Tsimaratos M, Chabrol B, Boddaert N, Verloes A, Chevrollier A, Gueguen N, Desquiret-Dumas V, Ferre M, Procaccio V, Richard L, Funalot B, Moncla A, Bonneau D, Antignac C: Loss-of-function mutations in WDR73 are responsible for microcephaly and steroid-resistant nephrotic syndrome: Galloway-Mowat syndrome. Am J Hum Genet, 95: 637648, 2014 28. Mendelsohn HB, Krauss M, Berant M, Lichtig C: Familial early-onset nephrotic syndrome: diffuse mesangial sclerosis. Clinico-pathological study of a kindred. Acta Paediatr Scand, 71: 753758, 1982 29. Nishiyama K, Funai T, Katafuchi R, Hattori F, Onoyama K, Ichiyama A: Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene. Biochem Biophys Res Commun, 176: 1093-1099, 1991 30. Lemmink HH, Mochizuki T, van den Heuvel LP, Schroder CH, Barrientos A, Monnens LA, van Oost BA, Brunner HG, Reeders ST, Smeets HJ: Mutations in the type IV collagen alpha 3 (COL4A3) gene in autosomal recessive Alport syndrome. Hum Mol Genet, 3: 1269-1273, 1994 31. Mochizuki T, Lemmink HH, Mariyama M, Antignac C, Gubler MC, Pirson Y, Verellen-Dumoulin C, Chan B, Schroder CH, Smeets HJ, et al.: Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat Genet, 8: 77-81, 1994

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32. Barker DF, Hostikka SL, Zhou J, Chow LT, Oliphant AR, Gerken SC, Gregory MC, Skolnick MH, Atkin CL, Tryggvason K: Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science, 248: 1224-1227, 1990 33. Lloyd SE, Pearce SH, Fisher SE, Steinmeyer K, Schwappach B, Scheinman SJ, Harding B, Bolino A, Devoto M, Goodyer P, Rigden SP, Wrong O, Jentsch TJ, Craig IW, Thakker RV: A common molecular basis for three inherited kidney stone diseases. Nature, 379: 445-449, 1996 34. Town M, Jean G, Cherqui S, Attard M, Forestier L, Whitmore SA, Callen DF, Gribouval O, Broyer M, Bates GP, van't Hoff W, Antignac C: A novel gene encoding an integral membrane protein is mutated in nephropathic cystinosis. Nat Genet, 18: 319-324, 1998 35. Castelletti F, Donadelli R, Banterla F, Hildebrandt F, Zipfel PF, Bresin E, Otto E, Skerka C, Renieri A, Todeschini M, Caprioli J, Caruso RM, Artuso R, Remuzzi G, Noris M: Mutations in FN1 cause glomerulopathy with fibronectin deposits. Proc Natl Acad Sci U S A, 105: 25382543, 2008 36. Bernstein HS, Bishop DF, Astrin KH, Kornreich R, Eng CM, Sakuraba H, Desnick RJ: Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene. J Clin Invest, 83: 1390-1399, 1989 37. Kantarci S, Al-Gazali L, Hill RS, Donnai D, Black GC, Bieth E, Chassaing N, Lacombe D, Devriendt K, Teebi A, Loscertales M, Robson C, Liu T, MacLaughlin DT, Noonan KM, Russell MK, Walsh CA, Donahoe PK, Pober BR: Mutations in LRP2, which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes. Nat Genet, 39: 957-959, 2007 38. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium. Cell, 90: 797-807, 1997 39. Attree O, Olivos IM, Okabe I, Bailey LC, Nelson DL, Lewis RA, McInnes RR, Nussbaum RL: The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase. Nature, 358: 239-242, 1992

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Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome Jillian K. Warejko1*, Weizhen Tan1*, Ankana Daga1, David Schapiro1, Jennifer A. Lawson1, Shirlee Shril1, Svjetlana Lovric1, Shazia Ashraf1, Jia Rao1, Tobias Hermle1,Tilman JobstSchwan1, 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

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authors contributed equally to this work. Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 2Department of Pharmacology, Brain Korea 21 PLUS 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, NHS 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 1

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19Departments

of Nephrology and Hypertension, Kaplan Medical Center, Hebrew University School of Medicine, Rehovot, Israel. 20Department of Pediatrics, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California 21Department of Pediatric Nephrology, Hospital Memmingen, Memmingen, Germany 22Department of Pediatrics, Asklepios Medical School, AK 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 26 Department 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

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Correspondence should be addressed to: Friedhelm Hildebrandt, M.D. Division of Nephrology Boston Children's Hospital 300 Longwood Avenue Boston, Massachusetts 02115 Phone: +1 617-355-6129 Fax: +1 617-730-0365 Email: [email protected] 2 ScholarOne support: 888-503-1050


ABSTRACT (295/300 words) Introduction: Steroid-resistant nephrotic syndrome overwhelmingly progresses to end-stage renal disease. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 29.5% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared to whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in a large international cohort of 300 families.

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Methods: 335 individuals with steroid-resistant nephrotic syndrome from 300 families were

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recruited from 4/1998 to 6/2016. Age of onset was restricted to under 25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes.

er Results: In 78/300 families (26%), we identified a causative mutation in one of 23 genes known

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to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in

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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

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causative mutation in a known steroid-resistant nephrotic syndrome gene in 40% of consanguineous families and in 13% of non-consanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 74 different mutations were detected in 23 of 33 steroid-resistant nephrotic syndrome genes. Twenty of these mutations were novel. NPHS1, PLCE1, NPHS2 and SMARCAL1 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.


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Conclusions: Whole exome sequencing is a sensitive approach towards 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.

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2979/3000 words INTRODUCTION Nephrotic syndrome in childhood is characterized by proteinuria (>40 mg/m2/hour), hypoalbuminemia, edema and hyperlipidemia. It can cause hypertension, severe infections and thrombotic events. Patients are classified by their response to steroid therapy. In children and young adults, about 80% of patients respond to standard steroid therapy and are termed steroid sensitive (1). In contrast, individuals with steroid-resistant nephrotic syndrome overwhelmingly progress to chronic kidney disease (CKD) and end-stage renal disease (ESRD). At this time,

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there is no effective therapy to curtail the relentless progression to ESRD.

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The most frequent kidney histologic feature of SRNS is focal segmental glomerulosclerosis (FSGS). In patients with FSGS, the risk of recurrence after kidney transplantation is estimated to

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be ~33% (2-4). FSGS constitutes the third most prevalent cause of ESRD in the first 2 decades of life (5). To date, more than 30 monogenic causes of steroid-resistant nephrotic syndrome

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have been identified (6), many of which implicate the glomerular podocyte and slit membrane as

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the primary sites where the pathogenesis of steroid-resistant nephrotic syndrome unfolds (7). The majority of genes known to cause steroid-resistant nephrotic syndrome are recessively

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inherited. Patients with mutations in these genes manifest with steroid-resistant nephrotic syndrome in childhood and adolescence, whereas dominant steroid-resistant nephrotic syndrome genes often manifest later in life.

We showed previously by targeted panel sequencing of 27 known steroid-resistant nephrotic syndrome genes that in 29.5% of steroid-resistant nephrotic syndrome cases with onset before 25 years, a causative mutation can be detected (8). However, panel sequencing by multiplex PCR is limited by requiring large numbers of Sanger sequencing to confirm individual genetic 5 ScholarOne support: 888-503-1050

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variants (8). Additionally, evaluation of genes by panel sequencing is limited to approximately 30 genes. With the growing number of genes available, we sought a mechanism by which we could evaluate a patient for a high number of steroid-resistant nephrotic syndrome genes, as well as detect novel causes of nephrosis should no known gene be identified.

In a cohort of patients with CKD and the phenotype of increased kidney echogenicity, we identified a causative mutation in 63% using whole exome sequencing (9). We evaluated here the utility of whole exome sequencing in an international cohort with steroid-resistant nephrotic

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syndrome. To date, this cohort is the largest to undergo whole exome sequencing (10). Given the very high rate of establishing an etiologic diagnosis and the significant implications for

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clinical management, pre-transplant and post-transplant care, whole exome sequencing should be considered in all individuals with steroid-resistant nephrotic syndrome diagnosed before age 25 years.

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MATERIALS AND METHODS

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Human subjects. The study was approved by the institutional review board of the University of

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Michigan and Boston Children’s Hospital. From April 1998 to June 2016, patients were enrolled after obtaining informed consent. Inclusion criteria were: onset of symptoms before 25 years AND a clinical diagnosis of steroid-resistant nephrotic syndrome (e.g. proteinuria, hypoalbuminemia, edema) OR nephrotic range proteinuria with kidney histology of FSGS or diffuse mesangial sclerosis (Supplementary Table 1). 335 individuals from 300 families were enrolled. Prior to December 2013, enrolled individuals were screened for mutations in WT1 and NPHS2. Those that screened positive were not included in this study.



Whole exome sequencing and variant calling. Whole exome sequencing and variant burden analysis were performed as previously described (11-13). Genomic DNA was isolated from blood lymphocyte or saliva samples and subjected to exome capture using Agilent SureSelect™ human exome capture arrays (Life technologies™) followed by next generation sequencing on the Illumina HighSeq™ sequencing platform. Sequence reads were mapped to the human reference genome assembly (NCBI build 37/hg19) using CLC Genomics Workbench™ (version 6.5.2) software (CLC bio, Aarhus, Denmark). Following alignment to the human reference genome (GRCh37/hg19), variants were filtered for most likely non-deleterious variants as

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previously described (8, 11). Variants with minor allele frequencies >1% in the dbSNP (version 142) or in the 1,000 Genomes Project (1,094 subjects of various ethnicities; May, 2011 data

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release) databases were excluded as they were unlikely to be deleterious. We used manual inspection for the p.Arg229Gln mutation in the NPHS2 gene which is reported to be deleterious

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with other variants, which would be filtered out using this method (14). Synonymous variants and intronic variants that were not located within splice site regions were excluded. Remaining

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variants included non-synonymous variants and splice site variants.

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Mutation calling in known SRNS genes. We evaluated whole exome sequencing data for

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causative mutations in 33 steroid-resistant nephrotic syndrome genes known at the time (Supplementary Table 2). Mutation calling was applied as stated above, followed by filtering remaining variants for changes in the regions of the 33 genes. Remaining variants were ranked based on their probable impact on the function of the encoded protein considering evolutionary conservation among orthologues using ENSEMBL Genome Browser and assembled using Clustal Omega, as well as PolyPhen-2 (15), SIFT (16), and MutationTaster (17). Mutations were designated as likely pathogenic based on criteria given by Supplementary Table 3. Mutation calling was performed by clinician scientists/geneticists, with knowledge of the clinical 7 ScholarOne support: 888-503-1050

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phenotypes and pedigree structure, as well as experience with homozygosity mapping and whole exome sequencing evaluation. Remaining variants were confirmed in patient DNA by Sanger sequencing as previously described (8). Whenever parental DNA was available, segregation analysis was performed. If no causative mutation was identified, we evaluated for mutations in genes that may represent phenocopies of steroid-resistant nephrotic syndrome (Supplementary Table 2). Variants were evaluated as above. Correlation of genotype and phenotype was examined and, if matching, the genetic variant was deemed a causative mutation.

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Mutation calling to identify novel causes of steroid-resistant nephrotic syndrome. If no causative mutation was found in a known steroid-resistant nephrotic syndrome gene and a

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family had homozygosity (>100Mbp) detected following homozygosity mapping, we then

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evaluated whole exome sequencing data for homozygous variants. Single heterozygous variants were excluded. We applied homozygosity mapping in consanguineous families or

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linkage analysis in sibling cases to filter variants (18). Remaining variants were ranked as described above. Variants were confirmed as described above.

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Homozygosity mapping and linkage analysis. Prior to 2014, for genome-wide homozygosity mapping, the GeneChip® Human Mapping 250k d Array (Affymetrix) was used. Alternatively, homozygosity mapping data was generated from whole exome sequencing data. Nonparametric LOD scores were calculated using a modified version of the program GENEHUNTER2.1 (19, 20) through stepwise use of a sliding window with sets of 110 SNPs and the program ALLEGRO (21) in order to identify regions of homozygosity as described (18, 22) using a disease allele frequency of 0.0001 and Caucasian marker allele frequencies. To generate homozygosity mapping after 2014, downstream processing of aligned BAM files was 8 ScholarOne support: 888-503-1050


done using Picard and SAMtools4 (23). Single nucleotide variants calling was performed using Genome Analysis Tool Kit (GATK) (24) and the generated VCF file was subsequently used in Homozygosity Mapper (25). Web Resources UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway; 1000 Genomes Browser http://browser.1000genomes.org; Clustal Omega, http://www.ebi.ac.uk/Tools/msa/clustal;

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Ensembl Genome Browser, http://www.ensembl.org; Exome Variant Server, http://evs.gs.washington.edu/EVS; Exome Aggregation Consortium, exac.broadinstitute.org;

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HGMD Professional 2016.3, https://portal.biobase-international.com/hgmd; Online Mendelian Inheritance in Man (OMIM), http://www.omim.org;

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Polyphen2, http://genetics.bwh.harvard.edu/pph2;

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Sorting Intolerant From Tolerant (SIFT), http://sift.jcvi.org; MutationTaster http://www.mutationtaster.org

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141

RESULTS Identification of causative mutations in one of 23 steroid-resistant nephrotic syndrome genes in 26% of steroid-resistant nephrotic syndrome cases Whole exome sequencing was performed in 335 individuals from 300 families and evaluated for mutations in the 33 steroid-resistant nephrotic syndrome genes known at the time (Table 1). In 78 families (26 %), a causative mutation in one of 23 known steroid-resistant nephrotic syndrome genes was detected (Figure 1, Table 1). NPHS1 (13 families), PLCE1 (11 families), NPHS2 (8 families), and SMARCAL1 (8 families) were the most common genes in which mutations were identified,

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comprising 51% of all mutations identified (Figure 1, Table 1).

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94 of the 300 families studied by whole exome sequencing have been previously studied using Fluidigm panel sequencing. The overlap between cohorts is given in Supplementary Table 4. We

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found that whereas in 20/78 (25%) families the causative mutation was previously detected using panel sequencing, 9/78 (12%) had a diagnosis made by whole exome sequencing and not by panel

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sequencing. In an additional 28% of families, we detected a likely causative mutation in one or more potential novel SRNS genes (Figure 1), given in Supplementary Table 5.

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Novel mutations detected in known steroid-resistant nephrotic syndrome genes We detected 74 different mutations in 23 of 33 known steroid-resistant nephrotic syndrome genes, 54 of which had previously been reported in the literature (Table 1). 20 novel mutations that have not been reported previously (Table 1). Individual families in whom a causative mutation was detected are described in Supplementary Table 9.

Whole exome sequencing identifies phenocopies in 11 of 90 families with a causative mutation detected 10 ScholarOne support: 888-503-1050

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We detected a causative mutation in 11 of 300 families with steroid-resistant nephrotic syndrome (3.7%) (Figure 1, Table 1). Mutations were found in 8 phenocopy genes, specifically COL4A5, COL4A3, CLCN5, GLA, AGXT, CTNS, FN1 and WDR19. A total of 10 different mutations were detected, 5 of which are novel (Table 1).

Novel candidate genes are identified using whole exome sequencing In 61/146 (42%) consanguineous families with no causative mutation found in a known steroidresistant nephrotic syndrome gene, one or more candidate genes were detected using homozygosity

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mapping (Figure 2, Supplementary Table 5). In non-consanguineous families >1 individual affected, linkage analysis was used to identify a potentially causative mutation in 18 of 135 families (13%).

Description of cohort

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Onset of illness ranged from birth to 24 years of age (Figure 3A and Supplementary Table 6). The

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median age in individuals in whom a causative mutation was detected in a steroid-resistant nephrotic

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syndrome gene was 1.7 years versus 4 years in those without a causative mutation identified, which was statistically significant (Figure 3B).

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146/300 (49%) of families were consanguineous, 59 (40%) of whom we detected a causative mutation in a steroid-resistant nephrotic syndrome gene (Figure 4, Supplementary Table 7). In 58/147 of families with >100 Mbp of homozygosity on mapping (40%), a causative mutation was detected in a steroid-resistant nephrotic syndrome gene. In contrast, in 18/135 (13%) of nonconsanguineous families and 20/153 (13%) of families with 3

N

SRNS

FSGS

c.3722G>A

p.G1241D

Hemi

Dr

Del

DC

1

NR

11 mo

M

Turkish

Y

N

SRNS

MPGN

A169_2 2

c.3722G>A

p.G1241D

Hemi

Dr

Del

DC

1

NR

unkn

M

Turkish

Y

N

SRNS

Other Cresentr ic GN

B249_2 1

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

4

F

Arabic

Y

N

SRNS

No bx

B249_2 2

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

4

F

Arabic

Y

N

SRNS

No bx

B249_3 1

c.809_811de l

p.S270del

Hom

-

-

-

0/1/120874

unk

F

Arabic

Y

N

SRNS

No bx

FN1

A4936_ 21

c.6836T>C

p.V2279A

Het

Dr

Del

DC

0.696

NR

1

F

C/E

N

2

N

SRNS

Other IgM nephrop athy

GLA

B912_2 1

c.504A>C

p. K168N

Hemi

Dr

Del

DC

1

NR

14

M

Arabic

Y

1

Y

SRNS

Other Fabry's disease

WDR19

B1119_ 21

c.3533G>A

p.R1178Q

Hom

Ce

T

DC

0.948

0/9/69008

1

M


N

2

Y

SRNS

DMS

COL4A3

COL4A5

CTNS

-

A1221_ 22

c.4825C>T

p.Arg1609*

Het

Trunc.

-

A4644_ 21

c.3088G>A

p.G1030S

A2058_ 21

c.3722G>A

A169_2 1

Inframe del. Inframe del. Inframe del.

2

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2

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4


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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.

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8, 1, 43 (15%, 1.9%, 83%)

Unknown/not indicated Roma

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

Ashkenazi Jewish

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

Race or ethnicity

Arabic and Asian

0, 0, 9 (0%, 0%, 100%)

African/African American

3, 0, 5 (37%, 0%, 63%)

Asian

7, 0, 14 (33%, 0%, 67%)

Hispanic/Latino

2, 1, 19 (9%, 4.5%, 86%)

Turkish

Arabic Total families enrolled

Causative mutation in a phenocopy gene detected

4, 0, 11 (27%, 0%, 73%)

African and Arabic

European/Caucasian


1, 1, 4 (17%, 17%, 67%)

Other/multiple races indicated


12, 2, 15 (41%, 6.9%, 52%) 10, 3, 46 (17%, 5.1%, 78%)

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30, 3, 44 (39%, 3.9%, 57%)

78

0

11

211

0 10

(26%, 3.7%, 70%)

0 20 Number of families

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0 30

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 indivuals 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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Patients without extra-renal manifestions

62, 7, 150 (28%, 3.2%, 69%)

Patients with extra-renal manifestions

22, 6, 63 (24%, 6.6%, 69%)

85

Total patients enrolled

15

p= 0.67

235

(25%, 4.5%, 70%)

Causative mutation in SRNS gene detected Causative mutation in phenocopy gene detected No causative mutation detected

1, 2, 22 (4%, 8%, 88%)

Unknown/de-identified sample

0

10

0

20

0

Number of patients

30

0

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Supplementary Figure 2: Distribution of affected individuals regarding gene identification status (steroidresistant nephrotic syndrome (SRNS) gene, phenocopy gene, or no mutation detected) for extrarenal 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 indivuals 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=0.67).

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Nephrotic range proteinuria, FSGS or DMS of biopsy

1, 0, 6 (14%, 0%, 86%)

Nephrotic syndrome, FSGS or DMS on biopsy

1, 0, 5 (17%, 0%, 83%)

ESRD on presentation, FSGS or DMS on biopsy

1, 0, 3 (25%, 0%, 75%)

Nephrotic syndrome, ESRD on presentation

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

Infantile NS

1, 0, 8 (11%, 0%, 89%)

De-identified sample Total families enrolled

Causative mutation in phenocopy gene detected No causative mutation detected

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

CNS SRNS


r Fo

51, 9, 145 (25%, 4.4%, 71%)

5, 2 , 29 (14%, 5.6%, 81%)

78

0

11

211

Pe 10

0

(26%, 3.7%, 70%)

0 20

Number of families

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0 30

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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4, 4, 12 (20%, 20%, 60%)

Other

Histologic diagnosis

Causative mutation in SRNS gene detected Causative mutation in phenocopy gene detected

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

Membranous GN

1, 0, 4 (20%, 0%, 80%)

CNS/Finnish type

2, 1, 7 (20%, 10%, 70%)

MPGN


5, 0, 15 (25%, 0%, 75%)

MCNS

3, 1, 10 (21%, 7.1%, 71%)

DMS

39, 3, 111 (25%, 2%, 73%)

FSGS

31


0

6

r Fo 50

75

(28%, 5.4%, 67%)

0 10 Number of patients

15

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0

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 steroidresistant 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 indivuals 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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