Mutations in PAX2 Associate with Adult-Onset FSGS

BASIC RESEARCH www.jasn.org

† ‡ | Moumita Barua,* Emilia Stellacci, Lorenzo Stella, Astrid Weins,§ Giulio Genovese,* ¶** † †† ‡‡ Valentina Muto, Viviana Caputo, Hakan R. Toka,* Victoria T. Charoonratana,* † Marco Tartaglia, and Martin R. Pollak*

*Division of Nephrology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts; †Department of Hematology, Oncology and Molecular Medicine, National Institute of Health, Rome, Italy; ‡Department of Chemical Science and Technology, University of Rome Tor Vergata, Rome, Italy; §Department of Pathology, and ‡‡Division of Nephrology, Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts; |Stanley Center for Psychiatric Research, Cambridge, Massachusetts; ¶Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts; **Department of Genetics, Harvard Medical School, Boston, Massachusetts; and ††Department of Experimental Medicine, University “La Sapienza,” Rome, Italy

ABSTRACT FSGS is characterized by the presence of partial sclerosis of some but not all glomeruli. Studies of familial FSGS have been instrumental in identifying podocytes as critical elements in maintaining glomerular function, but underlying mutations have not been identiﬁed for all forms of this genetically heterogeneous condition. Here, exome sequencing in members of an index family with dominant FSGS revealed a nonconservative, disease-segregating variant in the PAX2 transcription factor gene. Sequencing in probands of a familial FSGS cohort revealed seven rare and private heterozygous single nucleotide substitutions (4% of individuals). Further sequencing revealed seven private missense variants (8%) in a cohort of individuals with congenital abnormalities of the kidney and urinary tract. As predicted by in silico structural modeling analyses, in vitro functional studies documented that several of the FSGS-associated PAX2 mutations perturb protein function by affecting proper binding to DNA and transactivation activity or by altering the interaction of PAX2 with repressor proteins, resulting in enhanced repressor activity. Thus, mutations in PAX2 may contribute to adult-onset FSGS in the absence of overt extrarenal manifestations. These results expand the phenotypic spectrum associated with PAX2 mutations, which have been shown to lead to congenital abnormalities of the kidney and urinary tract as part of papillorenal syndrome. Moreover, these results indicate PAX2 mutations can cause disease through haploinsufﬁciency and dominant negative effects, which could have implications for tailoring individualized drug therapy in the future.

J Am Soc Nephrol 25: 1942–1953, 2014. doi: 10.1681/ASN.2013070686

FSGS is a heterogeneous form of kidney injury yielded important insight into our current under- defined by partial sclerosis of some but not all standing of the glomerular filter.3–5 These studies glomeruli.1,2 FSGS can be idiopathic, a result of genetically determined changes in podocytes, or Received July 2, 2013. Accepted December 24, 2013. secondary to a variety of renal insults, including reduced nephron mass and vesicoureteral reflux. M.B. and E.S. contributed equally to this work. M.T. and M.R.P. contributed equally as the senior investigators for this work. It is a condition marked by significant proteinuria with or without features of nephrotic syndrome. All Published online ahead of print. Publication date available at forms of FSGS are challenging to treat and fre- www.jasn.org. quently lead to ESRD. Correspondence: Dr. Martin Pollak, Beth Israel Deaconess Only a minority of individuals with adult-onset Medical Center, 99 Brookline Avenue, RN 304, Boston, MA 02215, or Dr. Marco Tartaglia, Department of Hematology, On- FSGS have a family history of disease that suggests a cology and Molecular Medicine, National Institute of Health, monogenic origin. Nonetheless, the study of famil- Viale Regina Elena 299, 00161 Rome, Italy. Email: mpollak@ ial FSGSandthe discoveryof genesimplicated in this bidmc.harvard.edu or [email protected] disease, such as INF2, TRPC6, and ACTN4, have Copyright © 2014 by the American Society of Nephrology

1942 ISSN : 1046-6673/2509-1942 JAmSocNephrol25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH have provided evidence that dysfunction in the podocyte is manifestations also occur (Mendelian Inheritance in Man, central to disease, serving a critical role in glomerular ﬁltration. 120330). We report that heterozygous PAX2 mutations ac- Monogenic adult-onset FSGS is genetically heterogeneous, with count for 4% of adult FSGS and perturb PAX2 function by mutations in INF2, TRPC6, and ACTN4 accounting for 9%, 3%, affecting proper binding to DNA or enhancing its interaction and 2% of our own cohort of families, leaving a substantial with repressor proteins. number of unexplained pedigrees.6 Exome analysis is facilitating the discovery of disease- causing genetic alterations in small, previously uninformative RESULTS families. To identify additional FSGS genes, we exploited this technology, coupled with high-throughput Sanger sequencing, Genetics in a cohort of FSGS families with unexplained genetic etiology. Targeted enrichment was performed on genomic DNA ob- This sequencing effort identiﬁed a disease-segregating PAX2 tained from two affected individuals from family FG-EQ, who missense mutation in a family designated FG-EQ (Figure 1). were separated by three meioses (Figure 1A). Massively parallel The product of the PAX2 gene, one of the nine members of the sequencing resulted in 36,116,715 and 66,666,479 seventy- family of paired box (PAX) transcription factor genes, plays a four–base pair single-end reads. Following alignment, target critical role in kidney development.7–9 Mutations in this gene region coverage had an average sequencing depth of 303 and have been associated with congenital abnormalities of the 523 for the two samples. Collectively, the total number of kidney and urinary tract (CAKUT) as part of a syndrome variants called was 86,328 (81,338 single-nucleotide polymor- known as papillorenal syndrome (PRS) in which ocular phisms [SNPs] and 4990 small indels). Among them, 75,753

Figure 1. FG-EQ pedigree, sequencing and multisequence alignment demonstrating conservation of the affected residue, Gly189. (A) Pedigree for family FG-EQ. Affected individuals are indicated in gray. One indeterminate individual is indicated with a half-shaded icon. Individuals heterozygous for the PAX2 p.G189R mutation are denoted by a plus sign while individuals without the mutation are denoted by a minus sign. Individuals for whom no DNA was available have no notation. Exome sequencing was performed in individuals FG-EQ III(8) and FG-EQ IV(8). (B) Electropherograms obtained from Sanger sequencing of exon 5 of the PAX2 gene conﬁrming the c.565G.A missense change (p.G189R). (C) Alignment of PAX2 orthologs across 7 species demonstrating conservation of the affected residue, Gly189 (asterisk).

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1943 BASIC RESEARCH www.jasn.org

SNPs and 4394 indels had been annotated in dbSNP137 (ftp:// number of haplotypes was higher in the patient groups than ftp.ncbi.nih.gov/snp). Variants annotated in the 1000 Ge- in controls. nomes Project (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/) The PAX2 protein is a multidomain transcription factor and National Heart, Lung, and Blood Institute Exome Se- characterized by an N-terminal DNA-binding paired domain quencing Project (http://evs.gs.washington.edu/EVS/) were and a transactivation domain at the C terminus (Figure 2). In removed, leading to a total of 137 SNPs and 15 indels that the FSGS group, six of the seven heterozygous mutations were were previously unreported. Of these, 22 nonsynonymous missense and altered amino acid residues located outside of SNPs and 3 indels shared by the affected individuals were the transactivation domain (Figure 2A). One nonsense muta- retained in the analysis (Supplemental Table 1). An overall tion was identified that resulted in a premature stop codon prioritization score was obtained for each variant using rank- within the C terminus of the paired domain. All of the muta- ing parameters after functional annotation was performed, tions in the CAKUT cohort were missense, with the majority retrieving information from several data sources (Supple- involving residues clustering within the transactivation do- mental Tables 2 and 3). PAX2 was identified as the most prom- main (Figure 2A). We compared the location of these muta- ising candidate as a result of this analysis and given its known tions to those listed in the PAX2 variant database (www.lovd. association with CAKUT and PRS.10–12 The c.565G.A nl/PAX2), which catalogs published disease-causing muta- missense variant, predicting the p.Gly189Arg amino acid sub- tions (Figure 2B, Supplemental Figure 2). Most PRS-causing stitution, was validated by Sanger sequencing (Figure 1B). mutations were found to be truncating (28 nonsense, frame- Genotyping of members of the family for whom DNA was shift, or splice site changes, representing 79% of total cases). available documented cosegregation of the variant with dis- Of note, all the PRS-associated missense changes and small in- ease. Of note, Gly189 is a highly conserved residue within the frame indels (16 different changes, accounting for 21% of octapeptide motif, a functionally important and conserved cases) affected the paired domain, in striking contrast with region (Figure 1C), and its substitution by arginine was pre- the distribution of CAKUT-associated mutations identified dicted to be damaging with high confidence (PolyPhen-2 in the present study. [http://genetics.bwh.harvard.edu/pph2/], score 0.99; SIFT [http://sift.jcvi.org/], score: 0). Clinical Characteristics To investigate the potential role of PAX2 variationinother The clinical characteristics for the FSGS families and individ- FSGS families, Sanger sequencing of the entire coding region uals with CAKUT are outlined in Tables 2 and 3. In the index of the gene was performed using genomic DNA from 175 family FE-EQ, ages of disease onset ranged from 17 years in additional unrelated subjects with familial disease. We also FG-EQ IV(8) to 68 years in FG-EQ III(8). ESRD occurred in two performed PAX2 mutation screening in a second cohort of of five affected individuals, at ages 40 and 58 years. Urography 85individualswithCAKUT.Thisanalysisidentified seven was performed in these two individuals, with evidence of bi- heterozygous single nucleotide substitutions in each of the lateral renal pelvis dilatation in one. All five affected individuals familial FSGS and CAKUT groups (4% and 8% of cases, re- had ultrasonographic examinations that identified no other spectively) (Table 1). None of the variants found were present structural abnormalities. No ocular or auditory abnormalities in sequences of approximately 7592 nominally normal indi- were documented. FG-EQ IV(7) had a slightly elevated 24-hour viduals available in public databases, including the Exome Se- urine collection at age 39 years, but a repeat collection was nor- quencing Project, the 1000 Genomes Project, or dbSNP137. mal.Hisstatuswasdefined as indeterminate. Paraffin-embedded Two of the variants isolated in FSGS families (p.Arg1043 and kidney biopsy tissue from individual FG-EQ III(8) was exam- p.Thr164Asn) were previously identified in patients with CAKUT, ined. Periodic acid–Schiff staining revealed several segmentally with p.Thr164Asn considered benign.13 In the FSGS families, sclerosed glomeruli; electron microscopy showed diffuse podo- the mutation of interest was documented in all affected indi- cytopathy as evidenced by degenerative changes, microvillous viduals where DNA was available. Incomplete penetrance was transformation, vacuolization, and lysosome accumulation but documented in one family (Supplemental Figure 1). Direct with focal foot process effacement (Figure 3, A and B). These sequencing demonstrated the de novo origin of the PAX2 mu- electron microscopy findings are similar to those observed in tation in the three patients with CAKUT for which parental biopsy samples from individuals with other genetic causes, such DNAs were available and short tandem repeat genotyping con- as ACTN4-associated disease.14 firmed paternity (Table 1). Examination of the burden of cod- Clinical re-evaluation of the affected members of family ing nonsynonymous variants in the entire PAX2 gene in familial FG-KV heterozygous for the nonsense mutation revealed a FSGS revealed significant enrichment for variants compared more severe phenotype, compatible with undiagnosed PRS. with 6503 controls in the Exome Sequencing Project (P,0.05 This same mutation was previously described in PRS.15 by two-tailed chi-squared test with Yates correction). A statistically significant difference was also found when we compared the burden of CAKUT variants to control variants (Supple- In Silico Structural Analysis mental Table 4). Specifically, the number of rare variants (de- Detailed molecular modeling was performed to explore the fined as minor allele frequency #1%) divided by the total effects of the identified PAX2 missense mutations. The domain

1944 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH

Table 1. List of PAX2 variants in FSGS families and CAKUT cohorts Family or DNA Amino Acid Exon Domain Polyphen2 Prediction Individual ID Change Change FSGS families FG-BF c.491C.A 4 p.Thr164Asn In between paired and octapeptide Probably damaging FG-DG c.239C.T 3 p.Pro80Leu Paired: linker subdomain Probably damaging FG-EQ c.565G.A 5 p.Gly189Arg Octapeptide Probably damaging FG-GE c.398C.T 3 p.Ser133Phe Paired: C terminus subdomain Probably damaging FG-IX c.167G.A 2 p.Arg56Gln Paired: N terminus subdomain Possibly damaging FG-JO c.448A.G 4 p.Thr150Ala In between paired and octapeptide domain Benign FG-KV c.310C.T 3 p.Arg1043 Paired: C terminus subdomain NA CAKUT individuals CKT-11Ca c.887T.C 8 p.Leu296Pro Transactivation Probably damaging CKT-34C c.415A.G 4 p.Ile139Val Paired: C terminus subdomain and Both predicted c.985A.G 8 p.Thr329Ala transactivation to be benign CKT-35C c.5A.G 1 p.Asp2Gly Paired domain: before N terminus subdomain Probably damaging CKT-39Ca c.892C.T 8 p.Pro298Ser Transactivation Benign CKT-46C c.887T.C 8 p.Leu296Pro Transactivation Probably damaging CKT-54C c.1240T.C 11 p.Tyr414His Transactivation Probably damaging CKT-89Ca c.884C.T 8 p.Ala295Val Transactivation Possibly damaging Nucleotide and amino acid sequence changes are reported using the following National Center for Biotechnology Information RefSeq accession numbers (NM_003987 and NP_003978). NA, not available. aPatients with CAKUT for whom de novo mutations could be conﬁrmed.

structure of PAX2 can be inferred with high reliability because of its high sequence homology with other PAX family proteins for which crystallographic structures are available. The PAX2 protein comprises an N-terminal DNA-binding paired box domain consisting of two subdomains separated by a linker region, which is followed by a highly conserved octapeptide that is involved in functional modulation of the protein by specific interactions with Groucho/TLE/Grg proteins, converting PAX2 from a transcriptional activator to a potent repressor. The C-terminal half of the protein contains a partial so-called homeodomain that can function as a second DNA-binding motif, or as a protein–protein interaction motif, and a transactivation domain, which regulates – Figure 2. PAX2 domain structure and localization of FSGS-associated and CAKUT- and gene transcription.13,16 20 PRS-causing mutations. (A) Location of mutations identified in the study are shown in Three of the FSGS missense mutations, the schematic PAX2 domain structure. PAX2 is characterized by an N-terminal paired p.Arg56Gln, p.Pro80Leu, and p.Ser133Phe, domain consisting of the N terminus (residues 16–76, violet) and C terminus (88–142, affected residues located in the paired blue) separated by a small linker region (cyan). The relative locations of the other domain near the N terminus of the protein. – domains, including the octapeptide motif (residues 185 192, green), homeodomain Although no structural data for PAX2 are (yellow), and transactivation domain (red), are also indicated. Mutations identified in available, crystallographic structure of this the familial FSGS cohort are shown above the cartoon, while those identified in the CAKUT cohort are reported below the cartoon. The asterisk indicates co-occurring highly conserved domain has been deter- mutations in the same individual. (B) Location of missense changes and small in-frame mined for PAX5 in complex with DNA and indels reported in the PAX2 variant database (www.lovd.nl/PAX2). Variants with un- the transcription cofactor ETS-1 (PDB ID certain clinical impact are not shown. Mutations identified in patients with PRS are 1K78), allowing the use of this structure to indicated with red characters, while those occurring in patients with renal dysplasia/ predict the effect of PAX2 substitutions hypoplasia and isolated ocular involvement are in green and blue, respectively. without the need to generate a homology

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1945 BASIC RESEARCH www.jasn.org

Table 2. Clinical features of FSGS families with PAX2 mutation–associated disease Ages at Patients Ages at Family Self-Reported Persons Ultrasonography Patients with Disease with Development Diagnosis ID Ethnicity Affected (n) Findings Biopsy (n) Onset (yr) ESRD (n) of ESRD (yr) FG-BF White 8 2 1 Unknown Increased FSGS 1 echogenicity FG-DO African 7–11 2 Unknown Unknown Unknown Proteinuria Unknown American FG-EQ European 17–68 6 2 40, 58 Dilated renal FSGS 2 pelvis, small kidneys FG-GE Unknown Unknown 2 1 Unknown Slightly small Proteinuria 1 kidney, calyceal diverticulum FG-IX Middle Eastern 36 4 Unknown Unknown Unknown FSGS Unknown FG-JO East Indian 31–32 5 4 30–36 Unknown FSGS 3 FG-KV European 15–24 3 1 42 Unknown FSGS, undiagnosed 3 American PRS

Table 3. Clinical features of patients with CAKUT who have PAX2 mutation–associated disease Individual ID Age at Diagnosis (yr) Clinical Features CKT-11 8 Solitary kidney with moderate hydronephrosis and hydrocele CKT-34 2 Unilateral UPJO. Associated nonrenal manifestations, including dysmorphic facial syndrome, ventriculomegaly, and seizures. Result of genetic testing for Opitz G syndrome was negative. CKT-35 4 Unilateral UPJO and bilateral VUR CKT-39 4 Solitary kidney and mild unilateral ventriculomegaly CKT-46 3 Horseshoe, small ectopic left kidney. Multiple congenital abnormalities and dysmorphic features; hemifacial microsomia. CKT-54 5 Unilateral UVJO/UPJO CKT-89 3 Left UPJO, right VUR UPJO, ureteropelvic junction obstruction; VUR, vesicoureteral reflux; UVJO, ureterovesical junction obstruction. model (Figure 4A).21 The sequence of this domain in PAX5 activity.23 Such transcriptional repression has been shown to differs from that of PAX2 by just three residues (97, 122 and be mediated by its interaction with the Groucho/TLE/Grg 123), which are far from those residues affected by the muta- family of corepressors.19,20 On the basis of its location, we tions identified here. All three FSGS mutations in this region hypothesized that the p.Gly189Arg substitution might influ- were predicted to affect PAX2 binding to DNA. Specifically, ence PAX2 interaction with TLE proteins, thus perturbing both the p.Arg56Gln and p.Ser133Phe substitutions were ex- switching between its active and inhibited conformation. To pected to disrupt interactions at these sites that directly in- explore this hypothesis, a homology model of the PAX2 octa- teract with DNA through an ion pair and H-bond, respectively. peptide domain bound to TLE1 was constructed (Figure 4, B Furthermore, the p.Arg56Gln substitution was also predicted and C). Structural analysis of this model supports the idea to interrupt the electrostatic interaction with ETS-1 cofactor at that, by introducing a cationic side chain in correspondence this site (Figure 4A). According to the structural model, the of a region of negative electrostatic potential of the corepres- third mutation, p.Pro80Leu, located in the linker region be- sor, the arginine residue improves the electrostatic interaction tween the N-terminal and C-terminal regions of the paired between PAX2 and TLE1. The p.Gly189Arg substitution is also domain, does not interact directly with DNA; however, because predicted to affect the conformational freedom of the octa- of the peculiar conformational properties of proline, this sub- peptide when in solution. This region of PAX2 is probably stitution is expected to alter the flexibility and conformation of unstructured when not associated with TLE proteins. There- the linker (Figure 4A).22 fore, the p.Gly189Arg substitution would reduce the degree of One of the identified PAX2 missense mutations in the FSGS conformational disorder in the free octapeptide and the en- group, p.Gly189Arg, was located in the octapeptide region. tropic cost of peptide immobilization and helical structuring Experimental evidence supports the view that this motif on the TLE1 surface. This would provide an additional con- plays a negative modulatory role on PAX2 transcriptional tribution to an increased binding affinity for the mutant.

1946 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH

domain), and Gly189Arg (octapeptide motif) amino acid substitutions were selected for further study. All mutants were efficiently expressed in HEK 293T cells, with levels that were similar to those of the wild-type protein, basally or in presence of TLE4, a well known repressor of PAX2 function (Supplemental Figure 3). The transactivation ability of each mutant, alone or in combination with TLE4, was measured by luciferase assays performed using transiently transfected HEK 293T cells (Figure 5A). As predicted by molecular modeling, the mutants exhibited different behavior in transactivation properties. Cells transiently expressing the PAX2G189R mutant fi Figure 3. Pathologic ndings in a renal biopsy specimen from an affected individual in showed an efficient induction of luciferase fi family FG-EQ demonstrated FSGS. (A) Representative image of a paraf nsectionfrom expression basally, which was equivalent to individual FG-EQ III(8) stained with periodic acid–Schiff. Glomeruli are enlarged, and that observed for the wild-type protein, indi- several show segmental sclerosis (asterisk) and focal adhesion of the tuft to Bowman’s capsule (arrow). Periodic acid-Schiff–positive protein casts are present (arrowhead). Scale cating unaffected DNA-binding capability to m the PRS4 consensus binding site. In contrast, bar=20 m. (B) Representative electron micrograph from the same kidney shown in part R56Q P80L fi cells expressing the PAX2 ,PAX2 ,and A, demonstrating damage to the glomerular ltration barrier and signs of diffuse po- S133F docyte injury characterized by extensive podocyte foot process effacement (arrowheads), PAX2 mutants were characterized by a microvillous transformation (arrow), and lysosome and vacuole formation within the cy- significantly weaker induction, which was toplasm (asterisk). Scale bar=5 mm(directmagnification: 35000). consistent with the predicted perturbing effect of mutations on DNA binding. However, TLE4 was documented to dramatic reduce G189R The remaining FSGS associated mutations, p.Thr150Ala the transactivation activity of the PAX2 mutant compared and p.Thr164Asn, are located in the region between the paired with wild-type (Figure 5A). This finding, consistent with our in domain and the octapeptide, where no structural data are silico structural analyses, supports the idea of a more stable inter- available for modeling. For this reason, no predictions can be action of this mutant with proteins of the TLE family. To confirm attempted on the structural effects of these lesions. However, it this hypothesis, coimmunoprecipitation assays were performed is interesting to note that phosphorylation prediction servers in HEK 293T cells coexpressing myc-tagged TLE4 together with PhosphoMotif and KinasePhos 2.0 indicate both Thr150 and hemagglutinin (HA)-tagged wild-type PAX2 or the PAX2G189R Thr164 as possible phosphorylation sites.24,25 mutant, which documented a dramatically enhanced interaction Withrespectto themissense variantsidentifiedinthe CAKUT of the mutant with TLE4 (Figure 5B). cohort, only one is located in the paired domain (p.Ile139Val) To demonstrate that the weak transactivation activity of the (Figure 4A). This variant is found in an individual harboring a PAX2R56Q, PAX2P80L,andPAX2S133F proteins was due to de- mutation in the transactivation domain as well, and it is diffi- creased DNA binding, chromatin from National Institutes of cult to know the extent of each variants’ contribution to dis- Health 3T3 cells expressing HA-tagged wild-type PAX2 or ease. In PAX2, Ile139 is part of the hydrophobic core of the each mutants, in the presence or absence of TLE4, was prepared C-terminal subdomain of the paired box domain. Valine is less 48 hours after transfection for chromatin immunoprecipitation bulky than isoleucine, and therefore the p.Ile139Val substitu- assays (Figure 5C). Consistent with the previous findings, bind- tion could form a small cavity in this core but how this affects ing to the PRS4 promoter sequence was documented for the the biochemical and DNA binding properties of the PAX2 pro- wild-type protein and PAX2G189R mutant, while a reduced bind- tein is difficult to predict. ing to DNA was observed for the other mutants. No predictions are possible for the other CAKUTmutations Overall, these data provide evidence for diverse perturbing identified in this study located in the transactivation domain effects of the FSGS-associated PAX2 mutations on protein because no structural data are available. Interestingly, similar to function, affecting proper binding to DNA (p.Arg56Gln, what was observed for Thr150 and Thr164, residue Thr329 p.Pro80Leu, and p.Ser133Phe) or enhanced interaction with (affected by the substitution p.Thr329Ala) was predicted as a repressors (p.Gly189Arg). possible phosphorylation site. DISCUSSION Functional Studies To characterize the functional behavior of FSGS-associated PAX2 Our genetic, in silico analysis and functional data suggest that mutants, the Arg56Gln, Pro80Leu and Ser133Phe (DNA-binding PAX2 missense variants may lead to an expanded phenotypic

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1947 BASIC RESEARCH www.jasn.org

spectrum that includes FSGS, through haploinsufficiency and/or dominant negative effects. Our data support the view that mutations in PAX2 might account for a significant proportion (approximately 4%) of families designated to have heredi- tary FSGS. Mutations in this key kidney development transcription factor have also been reported to cause congenital abnormalities of the kidney and urinary tract. Our screening demonstrates that PAX2 mutations accounts for disease in 8% of a CAKUT cohort. We expand the phenotypic spectrum associated with PAX2 mutations to include not only CAKUT but also autosomal dominant adult-onset FSGS in the absence of other syndromic features. Given the known role of PAX2 in kidney development, it is not surprising that mutations in this gene can lead to congenital and structural disease. It is possible that some of the families in our cohort designated as having familial primary FSGS have this pathologic lesion due to subtle developmental abnormalities, such as reduced nephron mass, although we are unable to quantitate this. Further- more, the presence of nephromegaly, which can be seen with reduced nephron mass, is also observed in primary FSGS. Nonetheless, this possibility demonstrates the limitations and heterogeneous nature of the pathologic diagnosis we call FSGS. It also highlights the potential benefits of clarifying diagnosis in such a heterogeneous disorder through genetic analysis to avoid unnecessary immunosuppressive Figure 4. Structural analysis and molecular modeling predict that FSGS mutations agents. Of note, most PRS-causing PAX2 located in the paired domain of PAX2 affect DNA binding, while the lesion in the mutations result in truncated proteins, fi octapeptide region increases PAX2 af nity for TLE corepressors. (A) In the upper panel, which is in contrast with the preponderance the structure of a complex between the paired domain of PAX5 (green), DNA, and the of missense mutations in our familial FSGS ETS-1 cofactor (magenta) is shown. PAX5 in this domain differs from PAX2 by just three residues, which are located far from those affected by the FSGS paired domain mu- cohort. We speculate that these hypomorphic tations. The FSGS paired domain missense mutations are indicated in red. H-bonds or mutations may have a role in leading to less fi ion pairs formed by these residues are shown as thin cyan lines. (B) In the middle panel, severe disease, as de ned by phenotype and location of FSGS, CAKUT, and PRS missense mutations in the paired domain are shown. relatively late ages of clinical presentation. Residues affected by FSGS and CAKUT mutations identified in this study are shown in This may also help to explain the “incom- red and blue, respectively. Residues affected by missense mutations previously reported plete penetrance” observed in one FSGS in the PAX2 variants database are shown in cyan. (C) In the left lower panel, the homology model of the complex between the PAX2 p.Gly189Arg octapeptide and TLE1. TLE1 is shown in gray, with its hydrophobic residues interacting with the octapeptide represented as light blue spheres and side chains forming H-bonds with the octa- complex between the octapeptide (green) peptide as light blue sticks. The backbone of the octapeptide is shown as a green and TLE1. The surface of TLE1 is colored ribbon, and its hydrophobic and hydrophilic side chains are reported as blue and according to the electrostatic potential (from yellow sticks, respectively. The mutated residue (p.Gly189Arg) is in magenta. (D) In the red to blue for potential values increasing from right lower panel, electrostatic interactions of p.Arg189 in the homology model of the 210 to 10 kT/e).

1948 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH

Figure 5. Functional characterization of FSGS-associated PAX2 mutations revealed results consistent with structural analysis and molecular modeling predictions. (A) Transactivation assays. Induction of the luciferase reporter (PRS4-luc) in response to cotransfection with wild-type (WT) PAX2 or individual mutants, basally (left). The PAX2G189R mutant exhibits a transactivation activity similar to that of the wild-type protein, while a signiﬁcantly reduced increase in reporter expression is documented in cells expressing the other mutants. Luciferase induction observed in the presence of TLE4 is also shown, reported as the fold inhibition relative to wild-type protein (right). Coexpression of the repressor protein TLE4 results in an enhanced inhibition of luciferase induction in cells expressing the PAX2G189R mutant, compared with wild-type PAX2 and the other FSGS-associated mutants. Values are expressed as the means6SEM of six in- dependent experiments. *P#0.05; **P#0.01. All the experiments were normalized to a renilla internal control vector. (B) Coimmu- noprecipitation assays. Coimmunoprecipitation of Myc-tagged TLE4 with HA-tagged PAX2 (wild-type or G189R mutant) using lysates from transiently transfected HEK 293T cells is shown. Anti-HA immunoprecipitates (top panels) and total cell lysates (bottom panels) were analyzed by Western blotting with the indicated antibodies. Note that the PAX2G189R mutant forms a more stable complex with TLE4 compared with wild-type PAX2. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (C) Chromatin immunoprecipitation assays. National Institutes of Health 3T3 cells were transiently transfected with the PRS4-luciferase construct in the presence of HA- tagged PAX2 proteins, with or without Myc-tagged TLE4. Primer pairs against the PRS4 element were used for realtime quantitative PCR. Relative amounts of PCR products are expressed as fold of enrichment normalized versus input and versus background (mouse IgG antibodies). Wild-type PAX2 and the PAX2G189R mutant more efﬁciently binds to PRS4 compared with the PAX2R56Q,PAX2P80L, and PAX2S133F mutants. For all experiments, equivalence of PAX2 and TLE4 expression levels is shown in Supplemental Figure 3. family, where carriers of the variant may have subtle undetect- targets that allow for communication between the ureteric bud able clinical characteristics (Supplemental Figure 1). If true, and surrounding metanephric mesenchyme, the latter of this would be analogous to some individuals with PKD2 mu- which ultimately epithelializes to form podocytes.8 Evidence tations who have a milder renal cystic phenotype that can often reported in the literature suggests that PAX2 strongly represses go undetected.26 the expression of WT1, a nuclear protein expressed in podo- We also consider an alternate mechanism by which these cytes, by interacting with Groucho/TLE/Grg proteins through variants may lead to segmental scarring. During kidney its octapeptide motif, and activates it in their absence, while development, PAX2 is important in activating downstream WT1 is a PAX2 repressor.19,20 We hypothesize that

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1949 BASIC RESEARCH www.jasn.org dysregulation of PAX2 targets, such as WT1, may lead to FSGS recent reference human genome (UCSC hg19), with the Burrows- by disrupting the development and/or function of the podo- Wheeler Aligner (v.0.5.9-rcl).33 The Genome Analysis Toolkit was cyte.20,27–29 used to further process the aligned read data, and the same program The functional data presented here support structural was used to genotype the individuals from the processed read predictions that different missense mutations lead to FSGS data.34,35 Variants were filtered against dbSNP 137, the 1000 Ge- through different mechanisms. Loss of function of a PAX2 nomes Project, and the Exome Sequencing Project. We further used allele has been previously described to be the inciting event the Integrative Genomics Viewer to manually identify some of the in renal disease, in part due to the predominance of nonsense novel variants as artifacts.36 Variants were filtered by comparison to mutations reported in PAX2 related autosomal dominant con- the other related affected individual sent for exome sequencing— ditions. Further support of PAX2 gene dosage being critical for variants that appeared in both affected individuals remained in the normal development was obtained in mouse models where analysis, even at low coverage, given the unlikely possibility of this knockout of or nonsense mutations in the PAX2 gene led happening by chance. If a variant was seen at low coverage in one to a phenotype analogous to PRS.30–32 We provide evidence sample but not in the other because of noncapture, it was kept in the that missense mutations can lead to disease through loss of analysis as well. Variants of interest were confirmed by Sanger se- function but by diverse mechanism(s) as well, which could quencing. Functional annotation of candidate genes was performed have implications for individualizing drug therapy.27 by retrieving information from several data sources containing an- notations on Gene Ontology terms (GO project), Online Mendelian Inheritance in Man reports, human phenotypes (Human Phenotype CONCISE METHODS Ontology project), mouse phenotypes (Mouse Genome Database– MGD phenotypes), protein-protein interactions (STRING), path- Patients ways (KEGG PATHWAY), gene expression data (Gene Atlas), protein Individuals belonging to 176 families with FSGS and 85 individuals domains (Pfam, InterPro), and literature (National Center for Bio- with CAKUTwere included in this study. Inherited cases were defined technology Information’s PubMed). Candidate genes were then pri- as families with two or more affected individuals. All families studied oritized with GeneDistiller, using functional relationships to genes had an inheritance pattern consistent with autosomal dominance. already known to be implicated in similar disease phenotypes (i.e., Familial FSGS affected status was defined as having either a reported ACTN4, APOL1, CD2AP, INF2, MYO1E, NEDE, NPHS1, NPHS2, history of proteinuria with urine albumin-to-creatinine ratio .250 PLCE1, TRPC6, and WT1) as ranking parameters.37 mg/g, nephrotic syndrome, or biopsy-proven FSGS in a family with at least one other case of documented FSGS or nephrotic syndrome. Sanger Sequencing CAKUT-affected status was defined as having fetal or postnatal ultra- Sanger sequencing was performed on all FSGS and CAKUT samples sonographic evidence of the following: renal agenesis, renal dysplasia using a Big Dye 3.1 terminator cycle sequencing kit (Life Technologies, (undifferentiated renal tissue), renal hypoplasia, duplex kidney, Grand Island, NY) and analyzed with an ABI Prism 3730 XL DNA horseshoe kidney, ureteropelvic junction obstruction with and with- analyzer (Applied Biosystems, Foster City, CA). Primer sequences are out megaloureter (ureter is refluxing or obstructed), duplication of available on request. Sequence chromatograms were analyzed using the ureter, ureteral agenesis, vesicoureteral reflux, and ectopic ureter the Sequencher software (Gene Codes, Ann Arbor, MI). Specific (abnormally located terminal portion of the ureter, often ending in variants identified in family probands were sequenced in all available the urethra). Included CAKUT individuals did not have associated affected family members to investigate whether the variant segregated symptoms consistent with PRS. We obtained blood or saliva for DNA with disease (Supplemental Figure 1). If an affected individual did not isolation as well as clinical information after receiving informed con- harbor the variant of interest, it was excluded as disease-causing. sent from participants in accordance with the Institutional Review Board at the Beth Israel Deaconess Medical Center and Children’s Parental Status Hospital Boston. Clinical information was obtained from telephone DNA belonging to three individuals with CAKUT along with their interviews, questionnaires, and physician reports. Genomic DNAwas parents was available for DNA profiling to establish biologic parental extracted from blood or saliva samples using standard procedures. status. PowerPlex 16TM HS PCR Amplification Kit (Promega, Madison, WI) was used to amplify and genotype 16 loci (15 STR Exome Sequencing and Sequence Data Analysis loci and amelogenin) in nine human genomic DNAsamples according Targeted enrichment and parallel sequencing was performed on to protocol. genomic DNA belonging to two affected individuals from a family with FSGS. Exome capture was performed using NimbleGen SeqCap Molecular Modeling and Structural Analysis EZ Exome v2 (NimbleGen, Madison, WI), which is estimated to cover The PAX2 octapeptide complex with the WD-repeat domain of TLE1 98% of the human coding genome corresponding to the Consensus was modeled by homology to the structure of TLE1 bound to an eh1 Conserved Domain Sequences database and 710 micro RNAs. motif peptide of human Goosecoid (PDB ID 2CE8).21 The sequence Enriched libraries were sequenced by 74–base pair, single end read of this peptide (MFSIDNILA) is representative of the consensus oc- sequencing on an Illumina GAII machine (Illumina Inc., San Diego, tapeptide sequence (F/Y) XIXXILX (where X can be any amino acid), CA). Next-generation sequencing reads were aligned to the most typical of Engrailed/Goosecoid/Nkx, and PAX proteins, with the

1950 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH addition of an N-terminal methionine. Most differences between this IP buffer except for containing 0.1% Triton X-100). Proteins were sequence and the octapeptide of PAX2 (YSINGILG) (D→N, N→G, eluted from protein A-Sepharose by boiling in SDS-PAGE sample A→G) involve residues facing the water phase, and not implicated in buffer, and Western blot was performed. any specific interactions. The only exception is substitution F→Y, which is involved in hydrophobic interactions with protein residues. Chromatin Immunoprecipitation National Institutes of Health 3T3 cells were transfected with each of However, this substitution is rather conservative and is easily accom- the PAX2 constructs and PRS4-Luc, with or without TLE4. Forty- modated in the protein pore where this side chain inserts. The octa- eight hours after transfection, cells were fixed with 1% formaldehyde peptide sequence was mutated to that of wild-type PAX2 by using the in culture medium (10 minutes). Cross-linking was stopped by the program UCSF Chimera, and the system energy was minimized with addition of glycine to 0.125 M. Cell pellets were washed in PBS, the “repair” function of the program FoldX 3.0.24,38 The same ap- suspended in cell lysis buffer (5 mM PIPES [pH 8.0], 85 mM KCl, proach was also used to introduce the p.Gly189Arg substitution. The 0.5% NP40, and protease inhibitors), incubated at 4°C for 5 minutes, electrostatic potential generated by TLE1 on its surface was calculated and centrifuged at 6000 rpm for 5 minutes. The nuclei were resus- with the program UCSF Chimera.38 pended in sonication buffer (50 mM Tris-HCl [pH 8.1], 10 mM EDTA, The position of mutated amino acid residues in the paired box 1% SDS, and protease inhibitors), incubated at 4°C for 10 minutes, domain, and their possible structural and functional effects, were and then sonicated on ice with five 20-second pulses. Sonicated lysates analyzed using the crystallographic structure of PAX5 in complex with were cleared by centrifugation at 4°C for 15 minutes. Chromatin was DNA and the transcription cofactor ETS-1 (PDB ID 1K78). The diluted in IP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM sequence of the paired domain of PAX5 differs from that of PAX2 by EDTA, 16.7 mM Tris-HCl [pH 8.1], and 167 mM NaCl) and pre- just three residues (97, 122, and 123), all relatively far from those cleared with 80 ml protein G-agarose. Each immunoprecipitation affected by the mutations, so that the generation of a homology model was performed using 5 mg anti-HA antibody (sc-8053;SantaCruzBio- was not necessary.39 Phosphorylation sites were predicted with the technology). After overnight incubation at 4°C, 60 ml protein G-agarose servers PhosphoMotif and KinasePhos 2.0.39,40 were added. Following 2-hour incubation, the beads were sequentially washed two times in IP dilution buffer, two times in high-salt buffer DNA Constructs (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20 mM Tris-HCl [pH 8.1], Full-length HA-tagged PAX2 and Myc-DDK tagged TLE4 cDNAs 500 mM NaCl), two times in LiCl buffer (100 mM Tris-HCl [pH 8.1], were provided by OriGene Technologies. The FSGS-associated 500 mM LiCl, 1% NP-40, 1% Na deoxycholate), and finally two times PAX2 mutations resulting in the p.Arg56Gln (c.167G.A), p.Pro80- in Tris-EDTA. Bound complexes were then eluted by vortexing beads Leu (c.239C.T), p.Ser133Phe (c.398C.T) and p.Gly189Arg twice for 15 min at 25°C in 250 ml of elution buffer (50 mM Na bi- (c.565G.A) amino acid substitutions (NP_003978.2, NM_003987) carbonate and 1% SDS). NaCl, 5 M, was added to a final concentration were introduced by site-directed mutagenesis (Agilent Technologies). of 0.2 M to the pooled eluates, and cross-links reversed by incubating The reporter plasmid, PRS4-Luc, containing five tandem repeats of samples at 65°C overnight. The samples were digested with proteinase the PAX2 DNA-binding site, cloned upstream of the herpes simplex K for 1 hour at 56°C, and DNA isolation was performed using column virus thymidine kinase promoter, was provided by Dr. G.R. Dressler purification. Precipitated DNA was reconstituted in sterile water, and (University of Michigan Medical School, Ann Arbor, MI). quantitative real-time PCR of precipitated genomic DNA relative to inputs was performed using the primers 59-GCTACCGGACTCA- Transactivation Assays GATCTCG-39 (PRS4-forward) and 59-TGCGAAGTGGACCTCG- HEK293 cells (ATCC) wereculturedin DMEM supplemented with10% GACC-39 (PRS4-reverse). FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin in 5% CO2/95% air at 37°C. Cells were seeded in 12-well plates and transfected using Histochemistry PAX2 250 ng of reporter plasmid and 500 ng of wild-type or each of the Formalin-fixed human kidney tissue was paraffin-processed and sec- m TLE4 four mutant cDNAs, with or without 1 gof plasmid, using tioned at 4 mm. After processing for antigen retrieval (pressure cooker in ’ Fugene 6, according to the manufacturer sprotocol(Promega).Twenty- citrate buffer [pH 6]), sections were stained with Periodic-acid Schiff. fi fl four hours after transfection, re y luciferase and renilla luciferase Images of representative glomeruli were taken with an Olympus BX53 activities were assayed using the Dual Luciferase Reporter Assay Kit microscope equipped with an Olympus DP72 camera. (Promega) in a Lumat LB9501 luminometer (E&G Berthold). Electron Microscopy Immunoprecipitation of PAX2/TLE4 Complex Ultrathin sections of resin-embedded kidney tissue were cut at 80 nm, HEK 293T cells were transfected with each of the PAX2 WTor mounted on 200 mesh copper grids, treated with uranyl acetate and p.Gly189Arg constructs, with or without TLE4 as indicated in the lead citrate, and examined in a JEOL 1010 transmission electron Figure 5 legend. Forty-eight hours after transfection, lysates were pre- microscope (Tokyo, Japan). pared in immunoprecipitation protocol (IP) buffer (20 mM Tris-HCl [pH 8.0], 100 mM NaCl, 0.5% Triton X-100, and protease inhibitor ACKNOWLEDGMENTS mixture). Lysates were incubated with anti-HA monoclonal antibodies for 16 hours at 4°C. Antibodies were captured with protein A-Sepharose M.B. is supported by a training fellowship from the Kidney Research for 2 hours at 4°C and washed five times with IP-wash buffer (same as Scientist Core Education and National Training Program, Canadian

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1951 BASIC RESEARCH www.jasn.org

Society of Nephrology, and Canadian Institutes of Health Research. 11. Schimmenti LA, Cunliffe HE, McNoe LA, Ward TA, French MC, Shim This work was also supported by grants from the US National In- HH, Zhang YH, Proesmans W, Leys A, Byerly KA, Braddock SR, Masuno stitutes of Health (DK54931 to M.R.P., NHLBI/NHGRI Exome M, Imaizumi K, Devriendt K, Eccles MR: Further delineation of renal- coloboma syndrome in patients with extreme variability of phenotype Project grant R01-HL094963), the NephCure Foundation (M.R.P), and identical PAX2 mutations. Am J Hum Genet 60: 869–878, 1997 and Istituto Superiore di Sanità (ricerca corrente 2012) (M.T.). 12. Amiel J, Audollent S, Joly D, Dureau P, Salomon R, Tellier AL, Augé J, We thank the families for their participation. The authors thank Bouissou F, Antignac C, Gubler MC, Eccles MR, Munnich A, Vekemans Andrea Uscinski Knob, Najwah Hayman, Dr. Stephen Fadem, M, Lyonnet S, Attié-Bitach T: PAX2 mutations in renal-coloboma syn- Dr. Ramin Sam and Dr. Charles Diskin for their assistance in obtaining drome: Mutational hotspot and germline mosaicism. Eur J Hum Genet 8: 820–826, 2000 clinical information. We thank Drs. Christine and Jonathan Seidman 13. Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, for assistance with exome capture, Dr. Kostantinos Giannakakis for Binenbaum G, Jensen UB, Cochat P, DeCramer S, Dixon J, Drouin R, providing archived kidney biopsy tissue, Dr. Catherine Grgicak for Falk MJ, Feret H, Gise R, Hunter A, Johnson K, Kumar R, Lavocat MP, performing DNA STR profiling in her laboratory and Dr. Gregory Martin L, Morinière V, Mowat D, Murer L, Nguyen HT, Peretz-Amit G, Dressler for providing the PRS4-luciferase reporter construct. The Pierce E, Place E, Rodig N, Salerno A, Sastry S, Sato T, Sayer JA, Schaafsma GC, Shoemaker L, Stockton DW, Tan WH, Tenconi R, authors also thank the NHLBI GO Exome Sequencing Project and its Vanhille P, Vats A, Wang X, Warman B, Weleber RG, White SM, Wilson- ongoing studies which produced and provided exome variant calls for Brackett C, Zand DJ, Eccles M, Schimmenti LA, Heidet L: Update of comparison: the Lung GO Sequencing Project (HL-102923), the WHI PAX2 mutations in renal coloboma syndrome and establishment of a Sequencing Project (HL-102924), the Broad GO Sequencing Project locus-specific database. Hum Mutat 33: 457–466, 2012 (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the 14. Henderson JM, Alexander MP, Pollak MR: Patients with ACTN4 mutations demonstrate distinctive features of glomerular injury. JAmSoc Heart GO Sequencing Project (HL-103010). Nephrol 20: 961–968, 2009 15. Cheong HI, Cho HY, Kim JH, Yu YS, Ha IS, Choi Y: A clinico-genetic study of renal coloboma syndrome in children. Pediatr Nephrol 22: DISCLOSURES 1283–1289, 2007 None. 16. Bouchard M, Schleiffer A, Eisenhaber F, Busslinger M: Pax genes: Evolution and function. In: Encyclopedia of Life Sciences. New York, Wiley, 2005 REFERENCES 17. Lechner MS, Dressler GR: Mapping of Pax-2 transcription activation domains. J Biol Chem 271: 21088–21093, 1996 18. Dörfler P, Busslinger M: C-terminal activating and inhibitory domains 1. D’Agati VD, Kaskel FJ, Falk RJ: Focal segmental glomerulosclerosis. determine the transactivation potential of BSAP (Pax-5), Pax-2 and NEnglJMed365: 2398–2411, 2011 Pax-8. EMBO J 15: 1971–1982, 1996 2. Meyrier A: Focal and segmental glomerulosclerosis: Multiple pathways 19. Cai Y, Brophy PD, Levitan I, Stifani S, Dressler GR: Groucho suppresses are involved. Semin Nephrol 31: 326–332, 2011 Pax2 transactivation by inhibition of JNK-mediated phosphorylation. 3. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong HQ, Mathis EMBO J 22: 5522–5529, 2003 BJ, Rodríguez-Pérez JC, Allen PG, Beggs AH, Pollak MR: Mutations in 20. Wagner KD, Wagner N, Guo JK, Elger M, Dallman MJ, Bugeon L, ACTN4, encoding alpha-actinin-4, cause familial focal segmental glo- Schedl A: An inducible mouse model for PAX2-dependent glomerular merulosclerosis. Nat Genet 24: 251–256, 2000 disease: Insights into a complex pathogenesis. Curr Biol 16: 793–800, 4. Reiser J, Polu KR, Möller CC, Kenlan P, Altintas MM, Wei C, Faul C, 2006 Herbert S, Villegas I, Avila-Casado C, McGee M, Sugimoto H, Brown D, 21. Jennings BH, Pickles LM, Wainwright SM, Roe SM, Pearl LH, Ish- Kalluri R, Mundel P, Smith PL, Clapham DE, Pollak MR: TRPC6 is a Horowicz D: Molecular recognition of transcriptional repressor motifs glomerular slit diaphragm-associated channel required for normal renal by the WD domain of the Groucho/TLE corepressor. Mol Cell 22: 645– function. Nat Genet 37: 739–744, 2005 655, 2006 5. Brown EJ, Schlöndorff JS, Becker DJ, Tsukaguchi H, Tonna SJ, Uscinski 22. Reiersen H, Rees AR: The hunchback and its neighbours: Proline as an AL, Higgs HN, Henderson JM, Pollak MR: Mutations in the formin gene environmental modulator. Trends Biochem Sci 26: 679–684, 2001 INF2 cause focal segmental glomerulosclerosis. Nature Genet 42, 72– 23. Chi N, Epstein JA: Getting your Pax straight: Pax proteins in de- 76, 2010 velopment and disease. Trends Genet 18: 41–47, 2002 6. Barua M, Brown EJ, Charoonratana VT, Genovese G, Sun H, Pollak MR: 24. Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L: The Mutations in the INF2 gene account for a significant proportion of fa- FoldX web server: An online force field. Nucleic Acids Res 33[Web milial but not sporadic focal and segmental glomerulosclerosis. Kidney Server issue]: W382–W388, 2005 Int 83: 316–322, 2013 25. Wong YH, Lee TY, Liang HK, Huang CM, Wang TY, Yang YH, Chu CH, 7. Nakanishi K, Yoshikawa N: Genetic disorders of human congenital Huang HD, Ko MT, Hwang JK: KinasePhos 2.0: A web server for iden- anomalies of the kidney and urinary tract (CAKUT). Pediatr Int 45: 610– tifying protein kinase-specific phosphorylation sites based on se- 616, 2003 quences and coupling patterns. Nucleic Acids Res 35[Web Server 8. Marlier AC, Lloyd G: Genetic abnormalities of renal development and issue]: W588–W594, 2007 morphogenesis. In: Genetic Diseases of the Kidney, edited by Lifton 26. Harris PC, Rossetti S: Determinants of renal disease variability in RPS, Somlo S, Giebisch GH, Seldin DW, Burlington, MA, Academic ADPKD. Adv Chronic Kidney Dis 17: 131–139, 2010 Press, 2009, pp 468–471. 27. Lipska BS, Iatropoulos P, Maranta R, Caridi G, Ozaltin F, Anarat A, Balat 9. Harshman LA, Brophy PD: PAX2 in human kidney malformations and A, Gellermann J, Trautmann A, Erdogan O, Saeed B, Emre S, disease. Pediatr Nephrol 27: 1265–1275, 2012 Bogdanovic R, Azocar M, Balasz-Chmielewska I, Benetti E, Caliskan S, 10. Eccles MR, Schimmenti LA: Renal-coloboma syndrome: A multi-system Mir S, Melk A, Ertan P, Baskin E, Jardim H, Davitaia T, Wasilewska A, developmental disorder caused by PAX2 mutations. Clin Genet 56: 1– Drozdz D, Szczepanska M, Jankauskiene A, Higuita LM, Ardissino G, 9, 1999 Ozkaya O, Kuzma-Mroczkowska E, Soylemezoglu O, Ranchin B,

1952 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1942–1953, 2014 www.jasn.org BASIC RESEARCH

Medynska A, Tkaczyk M, Peco-Antic A, Akil I, Jarmolinski T, Firszt- 34. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Adamczyk A, Dusek J, Simonetti GD, Gok F, Gheissari A, Emma F, Krmar Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome RT, Fischbach M, Printza N, Simkova E, Mele C, Ghiggeri GM, Schaefer F; Analysis Toolkit: A MapReduce framework for analyzing next-generation PodoNet Consortium: Genetic screening in adolescents with steroid- DNA sequencing data. Genome Res 20: 1297–1303, 2010 resistant nephrotic syndrome. Kidney Int 84: 206–213, 2013 35. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, 28. Gebeshuber CA, Kornauth C, Dong L, Sierig R, Seibler J, Reiss M, Philippakis AA, del Angel G, Rivas MA, Hanna M, McKenna A, Fennell Tauber S, Bilban M, Wang S, Kain R, Böhmig GA, Moeller MJ, Gröne TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, HJ, Englert C, Martinez J, Kerjaschki D: Focal segmental glomerulo- Daly MJ: A framework for variation discovery and genotyping using sclerosis is induced by microRNA-193a and its downregulation of WT1. next-generation DNA sequencing data. Nat Genet 43: 491–498, 2011 Nat Med 19: 481–487, 2013 36. Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, 29. Dressler GR, Wilkinson JE, Rothenpieler UW, Patterson LT, Williams- Getz G, Mesirov JP: Integrative genomics viewer. Nat Biotechnol 29: Simons L, Westphal H: Deregulation of Pax-2 expression in transgenic 24–26, 2011 mice generates severe kidney abnormalities. Nature 362: 65–67, 1993 37. Seelow D, Schwarz JM, Schuelke M: GeneDistiller—distilling candidate 30. Torres M, Gómez-Pardo E, Dressler GR, Gruss P: Pax-2 controls multiple genes from linkage intervals. PLoS ONE 3: e3874, 2008 steps of urogenital development. Development 121: 4057–4065, 1995 38.PettersenEF,GoddardTD,HuangCC,CouchGS,GreenblattDM, 31. Favor J, Sandulache R, Neuhäuser-Klaus A, Pretsch W, Chatterjee B, Meng EC, Ferrin TE: UCSF Chimera—a visualization system for ex- Senft E, Wurst W, Blanquet V, Grimes P, Spörle R, Schughart K: The ploratory research and analysis. JComputChem25: 1605–1612, 2004 mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a 39. Garvie CW, Hagman J, Wolberger C: Structural studies of Ets-1/Pax5 family with renal-coloboma syndrome and results in developmental complex formation on DNA. Mol Cell 8: 1267–1276, 2001 defects of the brain, ear, eye, and kidney. Proc Natl Acad Sci U S A 93: 40. Amanchy R, Periaswamy B, Mathivanan S, Reddy R, Tattikota SG, 13870–13875, 1996 Pandey A: A curated compendium of phosphorylation motifs. Nat 32. Alur RP, Vijayasarathy C, Brown JD, Mehtani M, Onojafe IF, Sergeev YV, Biotechnol 25: 285–286, 2007 Boobalan E, Jones M, Tang K, Liu H, Xia CH, Gong X, Brooks BP: Papil- lorenal syndrome-causing missense mutations in PAX2/Pax2 result in hypomorphic alleles in mouse and human. PLoS Genet 6: e1000870, 2010 33. Li H, Durbin R: Fast and accurate short read alignment with Burrows- This article contains supplemental material online at http://jasn.asnjournals. Wheeler transform. Bioinformatics 25: 1754–1760, 2009 org/lookup/suppl/doi:10.1681/ASN.2013070686/-/DCSupplemental.

J Am Soc Nephrol 25: 1942–1953, 2014 PAX2 Mutations Associate with FSGS 1953

Supplementary Figure 1. Pedigrees of FSGS families with PAX2 mutations. Affected individuals are indicated in gray. Individuals heterozygous for the PAX2 mutation are denoted by “+” while individuals without the mutation are denoted by “‐’. Individuals where no DNA was available have no notation. Numbers indicate number of individuals of that gender. Diamond represent individuals of unknown gender. Clinical re‐evaluation of the affected members of family FG‐KV heterozygous for the nonsense mutation revealed a more severe phenotype, compatible with undiagnosed PRS in one of the affected children. This same mutation, p.Arg104X, was previously described in PRS.

Supplementary Figure 2. Truncating PAX2 mutations reported in the PAX2 variants database.

Supplementary Figure 3. Expression analysis of wild‐type PAX2 and FSGS‐ associated mutants.

Gene Chromosome Position Reference Nucleotide Amino Acid Coverage base change Change MACF1 1 39838241 ACAG A p.Ala2334del 22/40 FAM123C 2 131520076 A G p.Lys144Arg 5/8 FYCO1 3 46008554 G A p.Pro758Ser 4/9 MINA 3 97668751 CCTT C p.Lys331‐ 11/33 WWTR1 3 149374727 C T p.Glu123Lys 1/6 TNFSF10 3 172224420 T G p.Glu236Asp 19/57 IL1RAP 3 190321970 G C p.Asp40His 12/38 TMEM175 4 947002 C T p.His150Tyr 9/20 METTL14 4 119625153 A G p.Asn230Asp 17/29 NDUFS4 5 52942235 C T p.Thr113Met 13/34 MDN1 6 90428894 C T p.Met2006Ile 14/54 PDE1C 7 31867930 T G p.Thr421Pro 15/41 AASS 7 121721588 G A p.Pro749Leu 8/22 C8orf44 8 67590022 CG C ‐ 13/23 PABPC1 8 101724941 C T p.Arg227Gln 12/24 C9orf93 9 15623341 T C p.Leu251Pro 21/52 KCNT1 9 138662242 G A p.Arg528His 7/19 PLAU 10 75673375 T C p.Ile144Thr 4/17 PAX2 10 102541071 G A p.Gly189Arg 12/19 VWCE 11 61032639 T C p.Thr136Ala 2/9 CAPRIN2 12 30872016 C A p.Gln431His 4/40 RBM19 12 114261045 T G p.Gln956Pro 7/10 TXNDC11 16 11781806 T C p.Tyr687Cys 0/10 ZNF585B 19 37676572 C A p.Val211Leu 0/8 BCOR X 39923603 C T p.Arg1145Gln 9/10

Supplementary Table 1. List of variants after analysis of exome data from individuals FG‐EQ III(8) and IV(8). Of the 25 variants found, 22 are nonsynonymous substitutions while 3 and indels. Chromosome coordinates are given by Hg19 reference. Coverage lists data for FG‐EQ III(8)/FG‐EQ IV(8).

Gene Overall MGD Expression Interaction GO HPO Pfam Interpro Pathways symbol score phenotypes correlation PAX2 12780 12739 14 4 16 7 MACF1 49 7 10 8 6 18 NDUFS4 43 2 4 22 10 5 PLAU 40 6 2 28 4 BCOR 30 6 8 10 2 4 WWTR1 29 9 16 4 TNFSF10 23 8 12 3 RBM19 19 7 6 6 PABPC1 15 8 7 FYCO1 14 4 10 ZNF585B 13 5 2 6 MINA 13 3 10 IL1RAP 13 6 2 1 4 AASS 12 2 3 2 5 MDN1 10 4 6 PDE1C 10 5 0 5 CAPRIN2 8 5 3 TXNDC11 3 3 KCNT1 3 3 VWCE 2 2 TMEM175 2 2 C8orf44 2 2 METTL14 1 1 C9orf93 0 FAM123C 0

Supplementary Table 2. Functional annotation of 25 candidate genes from several data sources including Gene Ontology (GO), Human Phenotype Ontology (HPO), Mouse Genome Database (MGD), Pfam, and Interpro. The Pfam database provides alignments and hidden Markov models for protein domains. Interpro is a database of protein families, domains and functional sites.

Protein function Gene (from NCBI GENE, UniProtKB and specific references, where indicated) Microtubule‐actin crosslinking factor. MACF1 Cytoskeletal linker protein Family with sequence similarity 123C. Also called AMER3 (APC membrane recruitment 3). Strongly expressed in FAM123C the central as well as the peripheral nervous system, thus suggesting important roles of this gene during neurogenesis (Devel. Dyn., 2010, 239, 1867–1878; BMC Evolutionary Biology 2010, 10:280) FYVE and coiled‐coil domain containing 1. FYCO1 Plays a role in microtubule plus end‐directed transport of autophagic vesicles MYC induced nuclear antigen. MINA Involved in cellular proliferation. May play an important role in cell growth and survival. WW domain‐containing transcription regulator protein 1. WWTR1 Highly expressed in kidney. Tumor necrosis factor (ligand) superfamily, member 10. Cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This TNFSF10 protein preferentially induces apoptosis in transformed and tumor cells, but does not appear to kill normal cells although it is expressed at a significant level in most normal tissues. Interleukin 1 receptor accessory protein. Interleukin 1 induces synthesis of acute phase and proinflammatory IL1RAP proteins during infection, tissue damage, or stress, by forming a complex at the cell membrane with an interleukin 1 receptor and this accessory protein. Transmembrane protein 175. TMEM175 Uncharacterized function. Methyltransferase‐like protein 14. METTL14 mRNA (2'‐O‐methyladenosine‐N6‐)‐methyltransferase activity NADH dehydrogenase (ubiquinone) Fe‐S protein 4, 18kDa. Accessory subunit of the mitochondrial membrane respiratory chain NADH NDUFS4 dehydrogenase (Complex I), or NADH:ubiquinone oxidoreductase, the first multi‐subunit enzyme complex of the mitochondrial respiratory chain MDN1, midasin homolog. MDN1 Nuclear chaperone required for maturation and nuclear export of pre‐60S ribosome subunits (by similarity) Phosphodiesterase 1C, calmodulin‐dependent 70kDa. Cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of the PDE1C cyclic nucleotides cAMP and cGMP to the corresponding nucleoside 5‐ prime‐monophosphates. Aminoadipate‐semialdehyde synthase. AASS A bifunctional enzyme that catalyzes the first two steps in the mammalian lysine degradation pathway. The N‐terminal and the C‐terminal portions of this enzyme contain lysine‐ketoglutarate reductase and saccharopine dehydrogenase activity, respectively, resulting in the conversion of lysine to alpha‐aminoadipic semialdehyde. C8orf44 Chromosome 8 open reading frame 44. Uncharacterized protein Poly(A) binding protein, cytoplasmic 1. PABPC1 This protein shuttles between the nucleus and cytoplasm and binds to the 3’ poly(A) tail of eukaryotic messenger RNAs via RNA‐recognition motifs Chromosome 9 open reading frame 9. C9orf93 Uncharacterized protein Potassium channel subfamily T member 1. Outwardly rectifying potassium channel subunit KCNT1 Activated by high intracellular sodium or chloride levels and upon stimulation of G‐protein coupled receptors, Plasminogen activator, urokinase. PLAU Serine protease involved in degradation of the extracellular matrix and possibly tumor cell migration and proliferation. Paired box 2. PAX2 Transcription factor with a critical role in the development of the urogenital tract, the eyes, and the CNS. von Willebrand factor C and EGF domains. VWCE May be a regulatory element in the beta‐catenin signaling pathway and a target for chemoprevention of hepatocellular carcinoma Caprin family member 2. May regulate the transport and translation of mRNAs, modulating for instance the expression of proteins involved in synaptic plasticity in CAPRIN2 neurons. Involved in regulation of growth as erythroblasts shift from a highly proliferative state towards their terminal phase of differentiation. May be involved in apoptosis. RNA binding motif protein 19. Nucleolar protein that contains six RNA‐binding motifs. May be involved in RBM19 regulating ribosome biogenesis. Plays a role in embryo pre‐implantation development (by similarity). Thioredoxin domain containing 11. May act as a redox regulator involved in DUOX proteins folding. The TXNDC11 interaction with DUOX1 and DUOX2 suggest that it belongs to a multiprotein complex constituting the thyroid H2O2 generating system. Zinc finger protein 585B. ZNF585B May be involved in transcriptional regulation. BCL6 corepressor. Transcriptional corepressor. May specifically inhibit gene expression when BCOR recruited to promoter regions by sequence specific DNA‐binding proteins such as BCL6 and MLLT3.

Supplementary Table 3. List of variants after analysis of exome data from individuals FG‐EQ III(8) and IV(8). Chromosome coordinates are given using Hg19 reference.

Number of alleles with Number of alleles without p‐value rare coding NS variants rare coding NS variants FSGS families 9 345 <0.0001 CAKUT 8 162 <0.0001 ESP (control) 57 12949 ‐

Supplementary Table 4. Burden of coding nonsynonymous and synonymous variants in FSGS and CAKUT groups compared to a control group. There is an enrichment of variants between the patient and control group, which is statistically significant when looking at the familial FSGS and CAKUT cohort. The abbreviations NS and ESP stand for non‐synonymous and NHLBI Exome sequencing project, respectively.