MAGI2 Mutations Cause Congenital Nephrotic Syndrome

CLINICAL RESEARCH www.jasn.org

† ‡ Agnieszka Bierzynska,* Katrina Soderquest, Philip Dean, Elizabeth Colby,* Ruth Rollason,* | Caroline Jones,§ Carol D. Inward,* Hugh J. McCarthy,* Michael A. Simpson, † ‡ † Graham M. Lord, Maggie Williams, Gavin I. Welsh,* Ania B. Koziell, and Moin A. Saleem* on behalf of NephroS, the UK study of Nephrotic Syndrome

*Bristol Renal and Children’s Renal Unit, School of Clinical Sciences, University of Bristol, Bristol, United Kingdom; †Division of Transplantation Immunology and Mucosal Biology, Department of Experimental Immunobiology, and |Division of Genetics and Molecular Medicine, Faculty of Life Sciences and Medicine, King’s College London, London, United Kingdom; ‡Bristol Genetics Laboratory, North Bristol National Health Service Trust, Bristol, United Kingdom; and §Alder Hey Children’s Hospital, Liverpool, United Kingdom

ABSTRACT Steroid–resistant nephrotic syndrome (SRNS), a heterogeneous disorder of the renal glomerular filtration barrier, results in impairment of glomerular permselectivity. Inheritance of genetic SRNS may be autosomal dominant or recessive, with a subset of autosomal recessive SRNS presenting as congenital nephrotic syndrome (CNS). Mutations in 53 genes are associated with human SRNS, but these mutations explain #30% of patients with hereditary cases and only 20% of patients with sporadic cases. The proteins encoded by these genes are expressed in podocytes, and malfunction of these proteins leads to a universal end point of podocyte injury, glomerular filtration barrier disruption, and SRNS. Here, we identified novel disease–causing mutations in membrane–associated guanylate kinase, WW, and PDZ domain–containing 2 (MAGI2)through whole-exome sequencing of a deeply phenotyped cohort of patients with congenital, childhood–onset SRNS. Although MAGI2 has been shown to interact with nephrin and regulate podocyte cytoskeleton and slit diaphragm dynamics, MAGI2 mutations have not been described in human SRNS. We detected two unique frameshift mutations and one duplication in three patients (two families); two siblings shared the same homozygous frameshift mutation, whereas one individual with sporadic SRNS exhibited compound heterozygosity. Two mutations were predicted to introduce premature stop codons, and one was predicted to result in read through of the normal translational termination codon. Immunohistochemistry in kidney sections from these patients revealed that mutations resulted in lack of or diminished podocyte MAGI2 expression. Our data support the finding that mutations in the MAGI2 gene are causal for congenital SRNS.

J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016040387

Steroid–resistant nephrotic syndrome (SRNS), a Correct podocyte morphology is essential for disorder of glomerular ﬁltration, results in severe maintaining glomerular ﬁltration barrier (GFB) proteinuria, hypoalbuminaemia, and edema. Currently, 53 genes are implicated (Supplemental Received April 1, 2016. Accepted October 18, 2016. Table 1), but these account for only 20%–30% of A.B.K. and M.A.S. contributed equally to this work. patients with hereditary cases and only 10%–20% Published online ahead of print. Publication date available at of patients with sporadic cases, supporting signif- www.jasn.org. icant genetic heterogeneity.1–8 To date, all known – Correspondence: Dr.MoinA.Saleem,UniversityofBristol, SRNS associated genes encode proteins ex- Children’s Renal Unit and Bristol Renal, Dorothy Hodgkin Build- pressed in podocytes (or associated basement ing, Whitson Street, Bristol BS1 3NY, United Kingdom. Email: membrane), polarized cells connected by highly [email protected] specialized junctions called slit diaphragms. Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 28: ccc–ccc, 2016 ISSN : 1046-6673/2805-ccc 1 CLINICAL RESEARCH www.jasn.org integrity, and the podocyte actin cytoskeleton is tightly reg- Whole-exome sequencing was performed on patients 175 ulated by cell surface receptors, including the multiprotein and 180. Subsequent segregation analysis confirmed that two complex at the slit diaphragm. Known SRNS gene mutations siblings (175 and 175S) shared the same homozygous frame- disrupt key cellular functions, resulting in podocyte injury shift deletion MAGI2:NM_012301:exon22:c.3998delG:p. and disruption of glomerular permselectivity. Typically, pa- (Gly1333Alafs*141). The patient with the sporadic case tientswithamutationinanSRNS–associated gene are less (180) exhibited compound heterozygosity: a deletion (pater- likely to respond to immunosuppressive treatment but nal) resulting in a premature stop codon MAGI2:NM_012301: have a reduced risk of disease recurrence after kidney trans- exon1:c.64_71delAGGAACCC:p.(Arg22Glyfs*7) together plant.9 SRNS may present at birth (congenital nephrotic with a duplication (maternal) MAGI2:NM_012301:exon20: syndrome [CNS]), usually with an early and severe pheno- c.3526_3533dupCTGGCAGA:p.(Glu1178Aspfs*9). All three type, with about 80%10 of patients with cases ascribed to variants were absent in the ExAC database and had high only six causal genes: NPHS1, NPHS2, LAMB2, WT1, CADD scores (34 [175 and 175S] and 35 [180]), supporting PLCE1,andLMX1B,11 all key players in podocyte biology; pathogenicity of these mutations. Translation of the mutated the genetic cause of the remainder is unknown. The majority protein (ExPASy; http://web.expasy.org/translate) indicated of patients with SRNS in childhood have an autosomal re- that the two frameshift mutations located in exons 1 and 20 cessive mode of inheritance. resulted in stop codons and premature termination of protein translation, and they were located in PDZ domains. The exon 22 frameshift deletion resulted in predicted read RESULTS through of the normal termination (stop) codon, resulting in likely continued translation of the mRNA farther down- Screening the Known Nephrotic Genes stream into the 39-untranslated region. We expanded the study originally described in the work by Patients 175, 175S, and 180 did not show any other signif- McCarthy et al.1 and performed whole-exome sequencing on a icant rare or novel variants in other genes expressed in podo- deeply phenotyped cohort of 187 patients with childhood cytes during exome screening, supporting MAGI2 as the most SRNS (onset ,18 years old; 11.8% familial and 7% consan- likely causative gene. Furthermore, there were no rare frame- guineous; 48.7% girls and 51.3% boys; 69.5% white and 30.5% shift mutations at the same site as any of the MAGI2 mutations South Asian, mixed race, African, and East Asian) collected within 100 bp in Ensembl or other public databases, although via a United Kingdom–wide registry. one in-house control carried a heterozygous frameshift inser- The cohort was first screened for the presence of disease- tion in exon 20:c.3512_3513insTGTA:p.(Leu1171Phefs*27). causing mutations in the 53 published genes known associate Other MAGI2 variants were not detected in these or other with SRNS (Supplemental Table 1, mapping statistics are pre- control samples. sented in Supplemental Table 2). Our findings correlated with The MAGI2 homozygous frameshift deletion previous published studies,2,12 in that mutations in known p.(Gly1333Alafs*141) was verified by Sanger sequencing, SRNS genes were only detected in approximately 25% of pa- and this was the only homozygous variant present in both tients. Assuming that mutations in the exome were present 175 and 175S. It was also present in the mother (175M) as a in a proportion of the remaining 75% of patients, variants heterozygote but could not be verified in the father, because detected in the whole exome were filtered to identify potential DNA was not available. mutations in genes not previously directly associated with Of the eight rare/novel variants found in 175, two (RAD51D SRNS. and NUP155) were absent in 175S, and two (GABRD and POLR2M) were only present as heterozygous variants. Fur- Membrane–Associated Guanylate Kinase, WW, and thermore, one variant (AKR1C1) was present as homozy- PDZ Domain–Containing 2 Mutations Identified by gous in the ExAC database, and one (IGLL5)waspredicted Whole-Exome Sequencing to be tolerated by in silico tools, excluding both as likely After filtering and additional analysis of potentially pathogenic candidates. Both heterozygous variants in SKOR1 found in mutations in genes not previously directly associated with 175 were inherited from the mother and thus, present on the SRNS, we identified three novel, likely disease–causing frame- same allele. Only one rare heterozygous potentially known shift mutations (Figure 1) in membrane–associated guanylate SRNS gene variant was shared by the siblings: COL4A3 kinase, WW, and PDZ domain–containing 2 (MAGI2; MIM: c.4421T.C:p.(Leu1474Pro); it was detected before enroll- 606382) in one patient with a sporadic case (180) (Supple- ment into the study by the Bristol Genetics Laboratory mental Figure 1) and two patients with familial cases (175 and (SRNS 37 gene panel; www.nbt.nhs.uk/severn-pathology/ 175S) (Supplemental Figure 2) presenting with nonsyndromic pathology-services/bristol-genetics-laboratory-bgl). This congenital SRNS. Because the parents of patients 175 and 175S variant was seen over 300 times as a heterozygote in the were consanguineous, autosomal recessive inheritance was ExAC database and is, therefore, of unlikely significance. considered the most likely mechanism of inheritance (Supple- Because DNA from both parents was available for 180, we mental Figure 3). were able to verify that the novel MAGI2 mutations were in

2 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 www.jasn.org CLINICAL RESEARCH

Figure 1. MAGI2 mutations causing CNS. (A) The exon structure of MAGI2 cDNA (NM_012301.3); 22 coding exons with start and stop codons are indicated. (B) Domain structure of the MAGI2 protein. Six PDZ domains (PDZ0–PDZ5) are shown in blue, one guanylate kinase (GK) domain is shown in yellow, and two WW domains are shown in green. (C) Frameshift variants found in three patients with CNS. Individual 180 is a compound heterozygote: the variant in exon 1 was inherited from the father (180F), and the variant in exon 20 was inherited from the mother (180M). Individual 175 and her sister, 175S, are homozygous for a single-nucleotide deletion in exon 22. The mother of the siblings is heterozygous for the variant. trans; p.(Arg22Glyfs*7) was inherited on a paternal allele, and the sole candidate gene for causing nephrotic syndrome in our p.(Glu1178Aspfs*9) was inherited on a maternal allele. The patients. (Data are presented in Supplemental Table 3.) frameshift variations in MAGI2 were not present in the ExAC There was no associated extrarenal phenotype in patient database. Six other nonsense variants in MAGI2 were present; 175, whereas patient 175S had some minor cardiac (possibly however, each was seen only once and as a heterozygote. Sim- unrelated) abnormalities. Patient 180 had associated polydac- ilarly, one splice acceptor and two splice donor variants are tyly and a previous pyloric stenosis, although the patient did also present in the ExAC database but again, only present as not show any other features of any characterized syndrome. heterozygotes. If we include non-PASS variants, an additional The inheritance pattern in all three patients was compatible nonsense and three frameshift variants are also present; how- with autosomal recessive disease. All had presented with ever, again, all were seen only as heterozygotes. significant proteinuria and hypoalbuminaemia within the first Five other genes were also left after the filtering steps; how- 4 months of life compatible with a CNS. Patient 175 was ever, variants in three genes were maternally inherited in cis, diagnosed after renal biopsy with Finnish–type nephrotic and insertions in MICALCL and ZIC5 were considered of un- syndrome, which is usually caused by NPHS1 mutations; how- likely significance due to the presence of similar insertions ever, the renal histology in early life may be relatively nonspe- around this region in the ExAC database. MAGI2 remained cific in appearance, and no NPHS1 mutations were detected

J Am Soc Nephrol 28: ccc–ccc, 2016 MAGI2 in Congenital Nephrotic Syndrome 3 CLINICAL RESEARCH www.jasn.org

on screening the entire gene. Patient 175 had rapid disease 2.33 9.08 progression and required a kidney transplant at age 3.5 years Up, yr Follow- Length of old; in contrast, the sibling, 175S, has followed a more benign course. Similarly, patient 180 has had persistent proteinuria

3.2 9.58 for 9 years with only mild renal impairment to date. Pheno- 17 g/L) 17 g/L) albumin albumin mg/mmol

Albumin-to- typic variability between different family members is not Most Recent Creatinine Ratio, unusual in SRNS and has been previously described. Inter- estingly, the most severely affected patient (175) also 24 2160 (Serum 73 32 83 (Serum carried a single heterozygous variant in LAMB2, resulting mol/L Serum m . ﬁ

Creatinine, in c.4274G C:p.(Gly1425Ala), the signi cance of which is Most Recent unknown,butitwasnotpresentasahomozygoteonthe ExAC database and had an MAF of 0.000075. Disease mod- iﬁcation due to bigenic heterozygosity or triallelic hits may Used occur but is unusual in SRNS.13 Phenotypic details are pre- Treatment cyclosporin, tacrolimus, prednisolone, mycophenolate mofetil Captopril Enalapril, sented in Table 1.

Immunohistochemistry digit No None – Kidney biopsy sections (from individual 180 and an individual Extrarenal Phenotype with minimal change disease), a nephrectomy section from branch pulmonary stenosis, patent foramen ovale postaxial polydactyly, thrombocytosis single individual 175, and an unsuitable for transplantnormal human kidney section used for immunohistochemistry were formalin ﬁxed and parafﬁn embedded (Figure 2). Disease

Recurrence? Glomeruli from patient 175 showed marked lobulation, fi fi Tx? with brosis or global sclerosis, interstitial brosis, and diffuse inflammation. From patient 180, changes were milder (con-

Time sistent with milder renal impairment), with two of 59 glomer- to ESRF ulishowingglobal sclerosisandthe rest looking normal on light

1 N/A N/A N/A Mild peripheral microscopy. Electron microscopy (Supplemental Figure 4) Tx 1 yr, 9 mo Yes No from this specimen shows a normal mesangial area, normal CKD Stage glomerular basement membrane, and diffuse podocyte foot process effacement, similar to the reported mouse model.14 N/A Native Biopsy Second MAGI2 staining was absent in patient 180, consistent with Finnish-type CNS (26 mo)

End stage of an early truncation resulting in nontranslation of the MAGI2

type protein through nonsense-mediated decay. MAGI2 staining – was, however, weakly positive in podocytes of patient 175. First Native Biopsy The terminal position of the frameshift mutation (exon 22) CNS (22 mo) and predicted read through into the 39-untranslated region supportweakexpressionofatranslatedbutdysfunctional protein. Steroids

Resistance to We observed MAGI2 staining in the tubules in control sections, which was absent in patient 180 with this antibody (Sigma-Aldrich). We, therefore, stained sections with a different antibody (Santa Cruz), which showed different degrees of

Consanguinity tubular staining in all sections, weak glomerular staining in the normal kidney, weak/negative staining in patient 175, and mo Age at Onset, negative staining in patient 180 (Supplemental Figure 5). The best fit explanation for the difference in tubular staining with the Santa Cruz antibody is that this is due to a compo- Familial/ Sporadic nent of nonspecific staining with this antibody, and therefore, we cannot be conclusive about the specificity of tubular staining. Clinical features of the affected individuals with MAGI2 mutations Nephrin expression seemed to be decreased and localization was altered in both patients 175 and 180 compared with control (Figure 3), similar to what was observed in MAGI2 mouse 15,16 175S F W Familial 1 Yes Presumed N/A 175 F W Familial 4 Yes Presumed Late Finnish Table 1. Patient No. Sex Ethnicity ESRF, end stage renal failure; Tx, transplanted; M, boy; W, white; MCD, minimal change disease; N/A, not applicable; F, girl. 180 M W Sporadic 3 No Presumed MCD (28 mo) N/A 2models. N/A No N/A Pyloric stenosis,

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family by having a guanylate kinase domain at the N terminus as well as WW and PDZ domains in reverse orientation to MA- GUK.20 MAGI2 is expressed in podocyte foot processes together with IQGAP1, CASK, spectrins, and a-actinin and is known to directly bind the cytosolic tail of nephrin, an essential component of the slit diaphragm, as part of the nephrin multiprotein complex.21 It also plays a role in RhoA-dependent regulation of the actin cytoskeleton, an important process in podocytes.22 Moreover, MAGI2 has recently been described as a WT1 target gene and required for podocyte development in both zebrafish23 and mice. Mice with MAGI2 deletion display disruption of the slit diaphragm formation, podocyte foot process effacement, and severe glomerular pathology compatible with human SRNS, with early Figure 2. MAGI2 expression is altered in patients with mutations. Immunohisto- death from nephrotic syndrome and kidney chemical staining with human anti–MAGI2 antibody (Sigma-Aldrich) is shown. (A) A failure.16 In humans, expression data from kidney that was not suitable for transplant was used as a control. (B) Biopsy specimen microdissected renal biopsies (Nephromine; obtained from an individual with minimal change disease; MAGI2 staining is seen in http://www.nephromine.org/) indicated that the glomeruli at the periphery of capillary loops, consistent with podocyte localization. MAGI2 was one of the top deregulated genes Staining in tubules is also seen. (C) Nephrectomy specimen obtained from patient 175 in glomerular disease, with 3.2- and 6.2-fold (homozygous mutation in MAGI2) shows weak but positive glomerular MAGI2 stain- decreases in FSGS and diabetic nephropathy, ing. (D) Biopsy specimen obtained from patient 180 (compound heterozygous mu- respectively, further underlining its potential tation in MAGI2) shows complete lack of MAGI2. Original magnification, 320. Scale 15 bar, 100 mm. importance in human SRNS. Aside from published evidence that MAGI2 is a component of the multiprotein complex at the slit diaphragm, MAGI2- DISCUSSION deleted mice die soon after birth from podocyte injury, severe proteinuria, and end stage renal failure, indicating a profound We performed whole-exome sequencing using an Illumina developmental slit diaphragm defect.17,19,20,22 Furthermore, platform on 187 patients with SRNS as an extension of a pre- MAGI2 is a WT1 target during podocyte development and vious analysis of a cohort of childhood SRNS.1 Patients lacking has been previously implicated in RhoA regulation/signaling mutations in the known 53 nephrotic genes were analyzed known to play a critical role in actin cytoskeletal regulation in further, particularly those with CNS, which is generally auto- podocytes.22–25 somal recessive and a developmental disorder. The exome data In conclusion, we present genetic data that support MAGI2 were, therefore, analyzed to look for novel candidate genes. After mutations as a cause of congenital SRNS in humans. Although filtering and additional analysis of potentially pathogenic muta- additional functional studies are required to establish exactly tions in genes expressed in podocytes but not previously directly how MAGI2 mutations lead to human CNS, we propose that associated with SRNS, we identified likely disease–causing frame- MAGI2 gene mutations should be added to gene panels when shift mutations in MAGI2 in one patient with a sporadic case investigating SRNS and considered in patients with congeni- (180) and two patients with familial cases (175 and 175S). tal-onset SRNS, particularly where mutation in other known Although a recently ascribed podocyte gene, MAGI2 genesisnotfound. mutations have not previously been directly associated with human SRNS. MAGI2 together with its paralogs MAGI1 and MAGI3 belong to the membrane–associated guanylate kinase CONCISE METHODS (MAGUK) family of scaffolding proteins highly expressed in – neurons and normally associated with neurologic function.17 19 Sequencing MAGI proteins function as molecular scaffolds, coordinating Patients 175 and 180 underwent exome sequencing. DNA from pe- signaling complexes by linking cell surface receptors to the ripheral blood was extracted with the Gentra Puregene Blood Kit cytoskeleton, but differ from other members of the MAGUK (Qiagen). DNA libraries were prepared from 3 mg dsDNA using the

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Figure 3. Nephrin immunostaining is altered in patients with MAGI2 mutations. Nephrin immunostaining on parafﬁn–embedded renal tissue sections. Confocal microscopy images show nephrin staining in green and DAPI (49,6-diamidino-2-phenylindole) nuclear staining in blue. (A) Positive control; normal human kidney. (B) Negative control (no nephrin antibody); normal human kidney. (C) Nephrectomy specimen obtained from patient 175 (homozygous mutation in MAGI2). (D) Biopsy specimen obtained from patient 180 (compound heterozygous mutation in MAGI2). Upper panels show the whole glomerulus (34063), and lower panels in A, C, and D show higher original magniﬁcation (310067). Lower panels show decreased/disrupted nephrin expression between normal and patient podocytes.

SureSelect Human All Exon 50 Mb Kit (Agilent). Samples were multi- (4) Analysis of potential synonymous and splice site variants was plexed (four samples on each lane), and 100-bp paired end sequenc- performed using Alamut Visual 2.7 (SpliceSiteFinder-like, MaxEntScan, ing was performed on the Illumina HiSeq System. Sequence data were NNSPLICE, GeneSplicer, and Human Splicing Finder) to check if aligned to the hg19 human reference genome using Novoalign there was a consistent predicted splice effect across the majority of (Novocraft Technologies SDN BHD), and variants were called with tools. MAGI2 frameshift mutations were examined for deleterious- SAMtools (Sequence Alignment/Map Tools)26 and annotated via ness using CADD (http://cadd.gs.washington.edu). (5)Variants multiple passes through Annovar.27 Initial exploration of datasets present within 50 bp and subsequently shown to be present in cis was performed using the Integrative Genome Browser (https:// were excluded and not considered compound heterozygotes. (6) www.broadinstitute.org/igv). Any variants that did not meet criteria above were discarded. (7) All mutations of interest were confirmed by Sanger sequencing in Variant Filtering probands and parents. (1) Only variants with a minor allele frequency (MAF),0.01 were To ascertain additional annotative information about potential considered (1000 Genomes Project; http://www.1000genomes.org/; genes of interest, we also examined genomic, proteomic, transcrip- NHLBI Exome Sequencing Project [ESP]; http://evs.gs.washington. tomic, genetic, and functional information on all new candidates edu/EVS/; http://exac.broadinstitute.org/; KCL in–house dataset using databases, such as GeneCards (http://www.genecards.org/), [King’s College London; in-house data from 5000 control individuals Ensembl (http://ensembl.org), and Uniprot (http://www.uniprot. without kidney disease]). (2) Variants seen as a homozygote in the org), as well as the Human Protein Atlas (http://www.proteinatlas. ExAC database (http://exac.broadinstitute.org/), ESP, or 1000 org/) and literature searches to help decisions regarding potential Genomes Project (http://www.internationalgenome.org/) were pathogenicity of findings. Mouse Genome Informatics (http:// excluded from additional analysis. (3) For the novel/not previously www.informatics.jax.org/) was used for murine data and establishing described missense variants, information from Alamut Visual (http:// whether SRNS disease phenotypes might occur in mice. Additional interactive-biosoftware.com/alamut-visual) and UCSC (http:// details are provided in Supplemental Tables 3 and 4. genome.ucsc.edu/) was used to check whether an amino acid is conserved. For filtering, the amino acid must be conserved, and the new Immunohistochemistry amino acid must not be present in another multicellular organism. Two MAGI2 antibodies were used.

6 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 www.jasn.org CLINICAL RESEARCH

MAGI2 (HPA013650; Sigma-Aldrich) was used 1:250. Heat– Yoshikawa N, Kitamura A, Cheong HI, Kagami S, Yamashita M, Fujita A, induced epitope retrieval was performed using a domestic stainless Miyatake S, Tsurusaki Y, Nakashima M, Saitsu H, Ohashi K, Imamoto N, steel pressure cooker; 5 minutes were counted as soon as the cooker Ryo A, Ogata K, Iijima K, Matsumoto N: Biallelic mutations in nuclear pore complex subunit NUP107 cause early-childhood-onset steroid- reached full pressure. (10 mM sodium citrate tribasic, pH 6), and resistant nephrotic syndrome. Am J Hum Genet 97: 555–566, 2015 10% goat serum (Sigma-Aldrich) was used for blocking. 5. Braun DA, Sadowski CE, Kohl S, Lovric S, Astrinidis SA, Pabst WL, Gee MAGI2 (sc-25664; Santa Cruz) was used 1:50. The heat–induced HY, Ashraf S, Lawson JA, Shril S, Airik M, Tan W, Schapiro D, Rao J, Choi antigen retrieval method was used (boiled for 5 minutes; 10 mM WI, Hermle T, Kemper MJ, Pohl M, Ozaltin F, Konrad M, Bogdanovic R, sodium citrate tribasic, pH 6); 3% BSA and 3% goat serum were Büscher R, Helmchen U, Serdaroglu E, Lifton RP, Antonin W, Hildebrandt F: Mutations in nuclear pore genes NUP93, NUP205 and used for blocking. XPO5 cause steroid-resistant nephrotic syndrome. Nat Genet 48: 457– 465, 2016 Fluorescent Immunohistochemistry Staining of 6. Gee HY, Zhang F, Ashraf S, Kohl S, Sadowski CE, Vega-Warner V, Zhou Paraffin–Embedded Tissue Sections W, Lovric S, Fang H, Nettleton M, Zhu JY, Hoefele J, Weber LT, Nephrin (AF4269; R&D) was used 1:200. Donkey anti–sheep IgG Podracka L, Boor A, Fehrenbach H, Innis JW, Washburn J, Levy S, Lifton RP, Otto EA, Han Z, Hildebrandt F: KANK deficiency leads to podocyte (H+L) secondary antibody and Alexa Fluor 488 conjugate (A11015; dysfunction and nephrotic syndrome. JClinInvest125: 2375–2384, Life Technologies) were used 1:300. Proteinase K was used as antigen 2015 retrieval (20 mg/ml); 1.5% BSA (A9647; Sigma-Aldrich) plus 5% 7. Ebarasi L, Ashraf S, Bierzynska A, Gee HY, McCarthy HJ, Lovric S, donkey serum (D9663; Sigma-Aldrich) was used for blocking. Sadowski CE, Pabst W, Vega-Warner V, Fang H, Koziell A, Simpson MA, Dursun I, Serdaroglu E, Levy S, Saleem MA, Hildebrandt F, Majumdar A: Defects of CRB2 cause steroid-resistant nephrotic syndrome. Am J Hum Genet 96: 153–161, 2015 8. De Mutiis C, Pasini A, La Scola C, Pugliese F, Montini G: Nephrotic- ACKNOWLEDGMENTS range albuminuria as the presenting symptom of Dent-2 disease. Ital J Pediatr 41: 46, 2015 We thank Lauren Flanagan, Liz Bailey, and Hannah Leyland for their 9. Jungraithmayr TC, Hofer K, Cochat P, Chernin G, Cortina G, Fargue S, help with data and sample collection. Grimm P, Knueppel T, Kowarsch A, Neuhaus T, Pagel P, Pfeiffer KP, Schäfer F, Schönermarck U, Seeman T, Toenshoff B, Weber S, Winn MP, The research was supported by the National Institute for Health Zschocke J, Zimmerhackl LB: Screening for NPHS2 mutations may help ’ Research (NIHR) Biomedical Research Centre based at Guy sand predict FSGS recurrence after transplantation. JAmSocNephrol22: St. Thomas’ National Health Service (NHS) Foundation Trust and King’s 579–585, 2011 College London. Funding sources were Kids Kidney Research, Nephrotic 10. Machuca E, Benoit G, Nevo F, Tête MJ, Gribouval O, Pawtowski A, Syndrome Trust, NephCure, NIHR, and Guy’sandSt.Thomas’ Hospital Brandström P, Loirat C, Niaudet P, Gubler MC, Antignac C: Genotype- phenotype correlations in non-Finnish congenital nephrotic syndrome. Charity. The United Kingdom Renal Rare Disease Registry is funded by JAmSocNephrol21: 1209–1217, 2010 ’ Kidney Research UK and the British Kidney Patients Association. 11. Sweeney E, Fryer A, Mountford R, Green A, McIntosh I: Nail patella The views expressed are those of the author(s) and are not nec- syndrome: A review of the phenotype aided by developmental bi- essarily those of the NHS, the NIHR, or the Department of Health. ology. JMedGenet40: 153–162, 2003 12. 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J Am Soc Nephrol 28: ccc–ccc, 2016 MAGI2 in Congenital Nephrotic Syndrome 7 CLINICAL RESEARCH www.jasn.org

18. Shoji H, Tsuchida K, Kishi H, Yamakawa N, Matsuzaki T, Liu Z, Nakamura transcriptomic analyses identiﬁes Nphs2, Mafb, and Magi2 as Wilms’ T, Sugino H: Identiﬁcation and characterization of a PDZ protein that Tumor 1 target genes in podocyte differentiation and maintenance. interacts with activin type II receptors. J Biol Chem 275: 5485–5492, JAmSocNephrol26: 2118–2128, 2015 2000 24. Wang L, Ellis MJ, Gomez JA, Eisner W, Fennell W, Howell DN, Ruiz P, 19. Wood JD, Yuan J, Margolis RL, Colomer V, Duan K, Kushi J, Kaminsky Z, Fields TA, Spurney RF: Mechanisms of the proteinuria induced by Rho Kleiderlein JJ, Sharp AH, Ross CA: Atrophin-1, the DRPLA gene GTPases. Kidney Int 81: 1075–1085, 2012 product, interacts with two families of WW domain-containing proteins. 25. Babayeva S, Zilber Y, Torban E: Planar cell polarity pathway regulates Mol Cell Neurosci 11: 149–160, 1998 actin rearrangement, cell shape, motility, and nephrin distribution in 20. Nagashima S, Kodaka M, Iwasa H, Hata Y: MAGI2/S-SCAM outside podocytes. Am J Physiol Renal Physiol 300: F549–F560, 2011 brain. J Biochem 157: 177–184, 2015 26. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, 21. Lehtonen S, Ryan JJ, Kudlicka K, Iino N, Zhou H, Farquhar MG: Cell Abecasis G, Durbin R; 1000 Genome Project Data Processing junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and Subgroup: The sequence alignment/map format and SAMtools. Bio- alpha-actinin are components of the nephrin multiprotein complex. informatics 25: 2078–2079, 2009 Proc Natl Acad Sci USA 102: 9814–9819, 2005 27. Wang K, Li M, Hakonarson H: ANNOVAR: Functional annotation of 22. Iida J, Ishizaki H, Okamoto-Tanaka M, Kawata A, Sumita K, Ohgake S, genetic variants from high-throughput sequencing data. Nucleic Acids Sato Y, Yorifuji H, Nukina N, Ohashi K, Mizuno K, Tsutsumi T, Mizoguchi Res 38: e164, 2010 A, Miyoshi J, Takai Y, Hata Y: Synaptic scaffolding molecule alpha is a scaffold to mediate N-methyl-D-aspartate receptor-dependent RhoA activation in dendrites. Mol Cell Biol 27: 4388–4405, 2007 23. Dong L, Pietsch S, Tan Z, Perner B, Sierig R, Kruspe D, Groth M, Witzgall This article contains supplemental material online at http://jasn.asnjournals. R, Grone HJ, Platzer M, Englert C: Integration of cistromic and org/lookup/suppl/doi:10.1681/ASN.2016040387/-/DCSupplemental.

8 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2016 SUPPLEMENTAL METHODS

List of the known genes associated with nephrotic syndrome:

Gene Inheritance Disease ACTN4 AD Familial and sporadic SRNS (usualy adult) ADCK4 AR SRNS ALG1 AR Congenital disorder of glycosylation ANLN AD FSGS (mainly adult) ARHGAP24 AD FSGS ARHGDIA AR Congenital nephrotic syndrome

NS,pretibial bullous skinlesions, neurosensory deafness, bilateral lacrimal CD151 AR duct stenosis, nail dystrophy, and -thalassemia minor

CD2AP AD/AR FSGS/SRNS CFH AR MPGN type II + NS COL4A3 AR Alport’s disease/FSGS COL4A4 AR Alport’s disease/FSGS COL4A5 X-linked AR Alport’s disease/FSGS COQ2 AR Mitochondrial disease/isolated nephropathy COQ6 AR NS +/- sensorineural deafness; DMS CRB2 AR SRNS

CUBN AR Intermittent nephrotic range proteinuria +/- with epilepsia

DGKE AR Hemolytic-Uremic Syndrome + SRNS E2F3 AD FSGS+mental retardation (whole gene deletion) EMP2 AR Childhood-onset SRNS and SSNS Familial and sporadic SRNS, FSGS-associated Charcot-Marie-Tooth INF2 AD neuropathy Congenital interstitial lung disease, nephrotic syndrome, and mild ITGA3 AR epidermolysis bullosa ITGB4 AR epidermolysis bullosa and pyloric atresia + FSGS KANK1 AR SSNS KANK2 AR SSNS/SDNS +/- hematuria KANK4 AR SRNS + haematuria LAMB2 AR Pierson syndrome LMNA AD Familial partial lipodystrophy + FSGS

LMX1B AD Nail patella syndrome; also FSGS without extrarenal involvement

MYO1E AR Familial SRNS NUP93 AR Childhood SRNS NUP107 AR Childhood SRNS NUP205 AR Childhood SRNS NPHS1 AR Congenital nephrotic syndrome/SRNS NPHS2 AR CNS, SRNS

NXF5 X-linked recessive FSGS with co-segregating heart block disorder

OCRL X-linked recessive Dent disease2, Lowe syndrome, +/- FSGS, +/- nephrotic range proteinuria

PAX2 AD adult onset FSGS without extrarenal manifestations

PDSS2 AR Leigh syndrome PLCe1 AR Congenital nephrotic syndrome/SRNS PMM2 AR Congenital disorder of glycosylation PODXL AD FSGS PTPRO AR NS

SCARB2 AR Action myoclonus renal failure syndrome +/-hearing loss

SMARCAL1 AR Schimke immuno-osseous dysplasia SYNPO AD sporadic FSGS (promoter mutations) TRPC6 AD Familial and sporadic SRNS (mainly adult) TTC21B AR FSGS with tubulointerstitial involvment

WDR73 AR Galloway-Mowat syndrome (microcephaly and SRNS)

Sporadic SRNS (children—may be associated with abnormal genitalia); WT1 AD Denys-Drash and Frasier syndrome

XPO5 AR Childhood SRNS ZMPSTE24 AR Mandibuloacral dysplasia with FSGS

MYH9 AD/assoc. MYH9-related disease; Epstein and Fechtner syndromes

Increased susceptibility to FSGS and ESRD in African Americans, Hispanic APOL1 G1, G2 risk alleles Americans and in individuals of African descent

Table S1. 53 genes associated with steroid-resistant nephrotic syndrome (SNRS) of congenital, childhood, or adult onset, familial and sporadic origin, different syndrome association/risk factors are shown. 1-8

Mapping statistics - Coverage – whole exome

Mean Std. Error Reads mapped to target (%) 73.16 0.25 Reads mapped to target plus 150bp (%) 82.25 0.35 Mean coverage 115.90 1.48 Accessible target bases 1x (%) 98.50 0.04 Accessible target bases 5x (%) 97.35 0.07 Accessible target bases 10x (%) 96.06 0.11 Target bases 20x (%) 92.60 0.21

Table S2. Coverage – whole exome

SUPPLEMENTAL RESULTS

Figure S1. Family pedigree of patient 180.

Black filled square represents patient 180 diagnosed with SRNS at the age of 9 years who is a compound heterozygote forMAGI2; variant in exon 1 was inherited from the Father and in exon 20 from the Mother. Crossed triangles represent miscarriages and the crossed circle represents stillborn female. DNA samples from other members of the family were not available.

Figure S2. Family pedigree of sisters 175 and 175S.

Black filled circles represent sisters 175 and 175S whose parents are first cousins. Both siblings are homozygous for a single nucleotide deletion in exon 22 of MAGI2 gene. Mother of the siblings is heterozygous for the variant. DNA samples from other members of the family was not available.CH - Celiac and heart disease.

Figure S3. Homozygosity plot.

Homozygosity mapping (http://www.homozygositymapper.org/) was performed for individual 175 since the parents are first cousins. Homozygosity plot is shown with chromosome numbers presented on x-axis and score on y-axis. Red bars represent regions of homozygosity, which reach maximum significance (height of red bar) on chromosomes: 1, 3, 5, 9, 10, 13, 15, 17and 22. Only 6 homozygous variants with MAF <0.01 were found within the homozygosity regions, none of these variants seemed however like a good SRNS gene candidate. One (AKR1C1, RS139588200) was found 9 times as a homozygote in ExAC and therefore not considered causative. Four other (GABRD, POLR2M, RAD51D and NUP155) were either found to be heterozygous or not present in the other affected sibling. One variant (IGLL5 RS201956362 - not checked with Sanger sequencing here) affects not conserved amino acid and the substituted threonine is present in many other species at this position and therefore was not considered as likely causative in this patient.

Figure S4. Electron microscopy images of a renal biopsy sample from patient 180.

One glomerulus was examined by electron microscope method. The mesangial area is normal and normocellular with no evidence of mesangial electron dense deposits. There are no peripheral electron dense deposits. The capillary loops are open and the endothelial fenestration is intact. The majority of the capillary loops have normal GBM (glomerular basement membrane). Diffuse podocyte foot process fusion is seen on the outer surface of the GBM.

Immunohistochemistry with Santa Cruz MAGI2 antibody

Figure S5. MAGI2 (Santa Cruz) staining

Immunohistochemical staining with human anti-MAGI2 antibody (Santa Cruz) is shown (20x). A - A kidney which was not suitable for transplant was used as a control. B – A nephrectomy specimen obtained from individual 175 (homozygous mutation in MAGI2). C – Biopsy specimen obtained from individual 180 (compound heterozygous mutation in MAGI2) shows relative absence of MAGI2.

ExAC frequency

Phred scaled Allele Number of Allele gene and variant dbSNP144 variant Allele count Additional significant information* Number homozygotes freq quality

KTN1:NM_001079521:exon40: Both variants are inherited from Mother (in rs372815686 170 . . . . c.3697C>A:p.Leu1233Met cis conformation) and therefore not considered as likely causative here. Kinectin 1 (Kinesin Receptor). Mice homozygous for a targeted null mutation or a floxed allele exhibit no discernable phenotype; mice are KTN1:NM_001079521:exon6:c. rs569480873 192 6 120590 0 4.98E-05 viable and fertile up to one year of age. 964-7A>G Expression: cells in glomeruli – medium/high; cells in tubules – medium/high

MAGI2:NM_012301:exon1:c.64 This compound heterozygote is in trans. No 217 . . . . _71del:p.Arg22Glyfs*7 Deletion inherited from the Father and insertion from the Mother.Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 2. Mice homozygous for a knock-out allele die within 24 hours of birth and possess hippocampal neurons with MAGI2:NM_012301:exon20:c.3 altered dendritic spine morphology. 526_3533dup:p.Glu1178Aspfs* No 217 . . . . Homozygotes for different knock-out allele 9 die shortly after birth, exhibiting anuria, increased plasma creatinine levels, and abnormal podocyte morphology. Expression: cells in glomeruli – medium; cells in tubules – low

MCC:NM_001085377:exon1:c. Both variants are inherited from Mother (in No 217 . . . . 58_63dup:p.Gly20_Gly21dup cis conformation) and therefore not considered as likely causative here. Mutated In Colorectal Cancers (MCC); Mice homozygous for hypomorphic or null MCC:NM_001085377:exon6:c. mutations are viable and fertile with no RS185322500 156 5 121250 0 4.12E-05 994G>A:p.Glu332Lys, gross abnormalities. Expression: cells in glomeruli – not detected/medium; cells in tubules – not detected/high

Similar inframe insertion around this region are present in ExAC and therefore this MICALCL:NM_032867:exon3:c. variant is less likely to be significant here. 1412_1413insTCC:p.Thr471_Al No 80.4 . . . . MICAL C-Terminal Like (MICALCL). Variant a472insPro (H) not checked with Sanger sequencing. Expression: cells in glomeruli – low; cells in tubules –high/medium

ZIC5:NM_033132:exon1:c.1248 Deletion inherited from Father. Similar rs778996938 217 . . . . _1262del:p.Pro420_Pro424del inframe insertion/deletions around this region are present in ExAC and therefore this variant is less likely to be significant here. p.Ser610Phe inherited from the Mother. Zic Family Member 5; Homozygous null mice display postnatal lethality and reduced life ZIC5:NM_033132:exon2:c.1829 rs201876139 184 63 121024 0 0.00052 spans with exencephaly, abnormal cerebral C>T:p.Ser610Phe cortex and diencephalon morphology, abnormal gait and posture, and impaired growth. Expression: cells in glomeruli – not available; cells in tubules – not available

Table S3. Rare variants found in patient 180.

H – homozygous, * - Additional information obtained from GeneCards (http://www.genecards.org/) and Mouse GenomeInformatics (http://www.informatics.jax.org/), ExAC - The Exome Aggregation Consortium (http://exac.broadinstitute.org/), The Human Protein Atlas (http://www.proteinatlas.org/) ExAC frequency Phred scaled Allele Allele Number of gene and variant dbSNP144 variant Allele freq Additional significant information* count Number homozygotes quality Present 9 times as a homozygote in individuals without a kidney phenotype. Variant not considered AKR1C1:NM_001353 significant and thus not checked for :exon5:c.509G>A:p.A RS139588200 222 912 121406 9 0.007512 presence in 175S. Aldo-Keto rg170His (H) HM Reductase Family 1, Member C1. Expression: cells in glomeruli – not detected; cells in tubules – high Variant present within a homozygosity run. This variant is present as a GABRD:NM_000815: heterozygote in the 175S. Gamma- exon3:c.C184C>T:p.P No 222 . . . . Aminobutyric Acid (GABA) A Receptor, ro62Ser (H) HM, EA Delta. Expression: cells in glomeruli – not detected; cells in tubules – not detected Substitution affects weakly conserved amino acid, Thr present in several species in this position. Variant not IGLL5:NM_00117812 considered significant and thus not 6:exon3:c.523G>A:p. RS201956362 222 177 121084 0 0.001462 checked for presence in 175S. Ala175Thr (H) HM Immunoglobulin Lambda-Like Polypeptide 5. Expression: cells in glomeruli – not detected; cells in tubules – high Patient 175S is also homozygous for this deletion. Membrane Associated Guanylate Kinase, WW And PDZ Domain Containing 2. Mice homozygous for a knock-out allele die within 24 hours of birth and possess MAGI2:NM_012301: hippocampal neurons with altered exon22:c.3998delG: No 150 . . . . dendritic spine morphology. p.Gly1333Alafs*141 Homozygotes for different knock-out (H) EA allele die shortly after birth, exhibiting anuria, increased plasma creatinine levels, and abnormal podocyte morphology. Expression: cells in glomeruli – medium; cells in tubules – low Variant present within a homozygosity run. This variant is not present in NUP155:NM_15348 patient 175S. Nucleoporin 155kDa. 5:exon6:c.723G>A:p. No 222 . . . . Expression: cells in glomeruli – Gln241Gln (H) HM, EA medium/low; cells in tubules – medium/low Variant present within a homozygosity run. This variant is present only as a POLR2M:NM_01553 heterozygous in the sister - 175S. 2:exon2:c.606G>A:p. RS139384982 222 4 121362 0 0.00003296 Polymerase (RNA) II (DNA Directed) Ala202Ala (H) HM, EA Polypeptide M. Expression: cells in glomeruli – not available; cells in tubules – not available Variant present within a homozygosity run. This variant is not present in the RAD51D:NM_00287 sister-175S. RAD51 Paralog D. 8:exon1:c.26G>C:p.C RS140825795 222 42 119626 0 0.0003511 Expression: cells in glomeruli – not ys9Ser (H) HM, EA available; cells in tubules – not available SKOR1:NM_0012580 Both variants are present also in the 24:exon5:c.775A>C: RS776369126 215 1 110882 0 0.000009019 sister 175S and their mother thus p.Lys259QGln EA both variants are on the same allele. SKI Family Transcriptional Corepressor SKOR1:NM_0012580 1. Expression: cells in glomeruli – not 24:exon2:c.80G>C:p. No 174 . . . . detected; cells in tubules – not Arg27Thr EA detected

Table S4. Rare variants found in patient 175

Supplementary References:

1. McCarthy, HJ, Bierzynska, A, Wherlock, M, Ognjanovic, M, Kerecuk, L, Hegde, S, Feather, S, Gilbert, RD, Krischock, L, Jones, C, Sinha, MD, Webb, NJ, Christian, M, Williams, MM, Marks, S, Koziell, A, Welsh, GI, Saleem, MA, Group, RtUSS: Simultaneous sequencing of 24 genes associated with steroid-resistant nephrotic syndrome. Clinical journal of the American Society of Nephrology : CJASN, 8: 637-648, 2013. 2. Sadowski, CE, Lovric, S, Ashraf, S, Pabst, WL, Gee, HY, Kohl, S, Engelmann, S, Vega-Warner, V, Fang, H, Halbritter, J, Somers, MJ, Tan, W, Shril, S, Fessi, I, Lifton, RP, Bockenhauer, D, El- Desoky, S, Kari, JA, Zenker, M, Kemper, MJ, Mueller, D, Fathy, HM, Soliman, NA, Group, SS, Hildebrandt, F: A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome. Journal of the American Society of Nephrology : JASN, 26: 1279-1289, 2015. 3. Gee, HY, Zhang, F, Ashraf, S, Kohl, S, Sadowski, CE, Vega-Warner, V, Zhou, W, Lovric, S, Fang, H, Nettleton, M, Zhu, JY, Hoefele, J, Weber, LT, Podracka, L, Boor, A, Fehrenbach, H, Innis, JW, Washburn, J, Levy, S, Lifton, RP, Otto, EA, Han, Z, Hildebrandt, F: KANK deficiency leads to podocyte dysfunction and nephrotic syndrome. The Journal of clinical investigation, 125: 2375-2384, 2015. 4. Miyake, N, Tsukaguchi, H, Koshimizu, E, Shono, A, Matsunaga, S, Shiina, M, Mimura, Y, Imamura, S, Hirose, T, Okudela, K, Nozu, K, Akioka, Y, Hattori, M, Yoshikawa, N, Kitamura, A, Cheong, HI, Kagami, S, Yamashita, M, Fujita, A, Miyatake, S, Tsurusaki, Y, Nakashima, M, Saitsu, H, Ohashi, K, Imamoto, N, Ryo, A, Ogata, K, Iijima, K, Matsumoto, N: Biallelic Mutations in Nuclear Pore Complex Subunit NUP107 Cause Early-Childhood-Onset Steroid-Resistant Nephrotic Syndrome. American journal of human genetics, 97: 555-566, 2015. 5. Braun, DA, Sadowski, CE, Kohl, S, Lovric, S, Astrinidis, SA, Pabst, WL, Gee, HY, Ashraf, S, Lawson, JA, Shril, S, Airik, M, Tan, W, Schapiro, D, Rao, J, Choi, WI, Hermle, T, Kemper, MJ, Pohl, M, Ozaltin, F, Konrad, M, Bogdanovic, R, Buscher, R, Helmchen, U, Serdaroglu, E, Lifton, RP, Antonin, W, Hildebrandt, F: Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome. Nature genetics, 2016. 6. 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. American journal of human genetics, 95: 637-648, 2014. 7. Bierzynska, A, Soderquest, K, Koziell, A: Genes and podocytes - new insights into mechanisms of podocytopathy. Frontiers in endocrinology, 5: 226, 2014. 8. De Mutiis, C, Pasini, A, La Scola, C, Pugliese, F, Montini, G: Nephrotic-range Albuminuria as the presenting symptom of Dent-2 disease. Italian journal of pediatrics, 41: 46, 2015.