Phenotypic quantification of Nphs1-deficient mice.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease in children and young adults. The most severe form of steroid-resistant nephrotic syndrome is congenital nephrotic syndrome Finnish type (CNSF), caused by biallelic loss-of-function variants in NPHS1, encoding nephrin. Since each of the 68 monogenic causes of steroid-resistant nephrotic syndrome represents a rare cause of the disease, tailoring therapeutic interventions to multiple molecular targets remains challenging, suggesting gene replacement therapy (GRT) as a viable alternative. To set the ground for a gene replacement study in vivo, we established rigorous, quantifiable, and reproducible phenotypic assessment of a conditional Nphs1 knockout mouse model. METHODS: By breeding a floxed Nphs1fl/- mouse (Nphs1tm1Afrn/J) previously studied for pancreatic ß-cell survival with a podocin promoter-driven Cre recombinase mouse model (Tg(NPHS2-Cre)295Lbh/J), we generated mice with podocyte-specific nephrin deficiency (Nphs1fl/fl NPHS2-Cre +). RESULTS: We observed a median survival to postnatal day P5 in nephrin-deficient mice, whereas heterozygous control mice and wild type (WT) control group showed 90% and 100% survival, respectively (at P50 days). Light microscopy analysis showed a significantly higher number of renal-tubular microcysts per kidney section in nephrin-deficient mice compared to the control groups (P < 0.0022). Transmission electron microscopy demonstrated reduced foot process (FP) density in nephrin-deficient mice compared to controls (P < 0.0001). Additionally, proteinuria quantitation using urine albumin-to-creatinine ratio (UACR) was significantly higher in nephrin-deficient mice compared to controls. CONCLUSIONS: This study represents the first comprehensive description of the kidney phenotype in a nephrin-deficient mouse model, laying the foundation for future gene replacement therapy endeavors.

Quantifiable and reproducible phenotypic assessment of a constitutive knockout mouse model for congenital nephrotic syndrome of the Finnish type.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of childhood chronic kidney disease. Congenital nephrotic syndrome of the Finnish type (CNF) (MIM# 256300) is caused by biallelic variants in the gene NPHS1, encoding nephrin, an integral component of the kidney filtration barrier. No causal treatments exist, and children inevitably require kidney replacement therapy. In preparation for gene replacement therapy (GRT) in CNF, we established a quantifiable and reproducible phenotypic assessment of the nephrin-deficient CNF mouse model: 129/Sv-Nphs1tm1Rkl/J. We assessed the phenotypic spectrum of homozygous mice (Nphs1tm1Rkl/Nphs1tm1Rkl) compared to heterozygous controls (Nphs1tm1Rkl/Nphs1WT) by the following parameters: 1. cohort survival, 2. podocyte foot process (FP) density per glomerular basement membrane (GBM) using transmission electron microscopy, 3. tubular microcysts in brightfield microscopy, and 4. urinary albumin/creatinine ratios. Nphs1tm1Rkl/Nphs1tm1Rkl mice exhibited: 1. perinatal lethality with median survival of 1 day, 2. FP effacement with median FP density of 1.00 FP/µm GBM (2.12 FP/µm in controls), 3. tubular dilation with 65 microcysts per section (6.5 in controls), and 4. increased albumin/creatinine ratio of 238 g/g (4.1 g/g in controls). We here established four quantifiable phenotyping features of a CNF mouse model to facilitate future GRT studies by enabling sensitive detection of phenotypic improvements.

Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

Recessive variants in MYO1C as a potential novel cause of proteinuric kidney disease.

BACKGROUND: Steroid-resistant nephrotic syndrome is the second leading cause of chronic kidney disease among patients < 25 years of age. Through exome sequencing, identification of > 65 monogenic causes has revealed insights into disease mechanisms of nephrotic syndrome (NS). METHODS: To elucidate novel monogenic causes of NS, we combined homozygosity mapping with exome sequencing in a worldwide cohort of 1649 pediatric patients with NS. RESULTS: We identified homozygous missense variants in MYO1C in two unrelated children with NS (c.292C > T, p.R98W; c.2273 A > T, p.K758M). We evaluated publicly available kidney single-cell RNA sequencing datasets and found MYO1C to be predominantly expressed in podocytes. We then performed structural modeling for the identified variants in PyMol using aligned shared regions from two available partial structures of MYO1C (4byf and 4r8g). In both structures, calmodulin, a common regulator of myosin activity, is shown to bind to the IQ motif. At both residue sites (K758; R98), there are ion-ion interactions stabilizing intradomain and ligand interactions: R98 binds to nearby D220 within the myosin motor domain and K758 binds to E14 on a calmodulin molecule. Variants of these charged residues to non-charged amino acids could ablate these ionic interactions, weakening protein structure and function establishing the impact of these variants. CONCLUSION: We here identified recessive variants in MYO1C as a potential novel cause of NS in children.

Recessive variants in MYO1C as a potential novel cause of proteinuric kidney disease.

Background: Steroid-resistant nephrotic syndrome is the second leading cause of chronic kidney disease among patients <25 years of age. Through whole exome sequencing, identification of >65 monogenic causes has rendered insights into disease mechanisms of nephrotic syndrome. Methods: To elucidate novel monogenic causes of NS, we combined homozygosity mapping with ES in a worldwide cohort of 1649 pediatric patients with NS. Results: We identified homozygous missense variants in MYO1C in two unrelated children with nephrotic syndrome (c.292C>T, p.R98W; c.2273 A>T, p.K758M). We evaluated publicly available kidney single-cell RNA sequencing datasets and found MYO1Cto be predominantly expressed in podocytes. We then performed structural modeling in molecular viewer PyMol using the super function aligning shared regions within both partial structures of MYO1C (4byf and 4r8g). In both structures, calmodulin, a common regulator of myosin activity, is shown to bind to the IQ motif. At both residue sites (K758; R98), there are ion-ion interactions stabilizing intradomain and ligand interactions: R98 binds to nearby D220 within the Myosin Motor Domain and K758 binds to E14 on a calmodulin molecule. Variants of these charged residues to non-charged amino acids could ablate these ionic interactions, weakening protein structure and function establishing the impact of these variants. Conclusion: We here identified recessive variants in MYO1C as a potential novel cause of nephrotic syndrome in children.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Quantitative phenotyping of Nphs1 knockout mice as a prerequisite for gene replacement studies.

Steroid-resistant nephrotic syndrome (SRNS) is the second most frequent cause of chronic kidney disease before the age of 25 yr. Nephrin, encoded by NPHS1, localizes to the slit diaphragm of glomerular podocytes and is the predominant structural component of the glomerular filtration barrier. Biallelic variants in NPHS1 can cause congenital nephrotic syndrome of the Finnish type, for which, to date, no causative therapy is available. Recently, adeno-associated virus (AAV) vectors targeting the glomerular podocyte have been assessed as a means for gene replacement therapy. Here, we established quantitative and reproducible phenotyping of a published, conditional Nphs1 knockout mouse model (Nphs1tm1.1Pgarg/J and Nphs2-Cre+) in preparation for a gene replacement study using AAV vectors. Nphs1 knockout mice (Nphs1fl/fl Nphs2-Cre+) exhibited 1) a median survival rate of 18 days (range: from 9 to 43 days; males: 16.5 days and females: 20 days); 2) an average foot process (FP) density of 1.0 FP/µm compared with 2.0 FP/µm in controls and a mean filtration slit density of 2.64 µm/µm2 compared with 4.36 µm/µm2 in controls; 3) a high number of proximal tubular microcysts; 4) the development of proteinuria within the first week of life as evidenced by urine albumin-to-creatinine ratios; and 5) significantly reduced levels of serum albumin and elevated blood urea nitrogen and creatinine levels. For none of these phenotypes, significant differences between sexes in Nphs1 knockout mice were observed. We quantitatively characterized five different phenotypic features of congenital nephrotic syndrome in Nphs1fl/fl Nphs2-Cre+ mice. Our results will facilitate future gene replacement therapy projects by allowing for sensitive detection of even subtle molecular effects.NEW & NOTEWORTHY To evaluate potential, even subtle molecular, therapeutic effects of gene replacement therapy (GRT) in a mouse model, prior rigorous quantifiable and reproducible disease phenotyping is necessary. Here, we, therefore, describe such a phenotyping effort in nephrin (Nphs1) knockout mice to establish the basis for GRT for congenital nephrotic syndrome. We believe that our findings set an important basis for upcoming/ongoing gene therapy approaches in the field of nephrology, especially for monogenic nephrotic syndrome.

Copy number variation analysis in 138 families with steroid-resistant nephrotic syndrome identifies causal homozygous deletions in PLCE1 and NPHS2 in two families.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of kidney failure in children and adults under the age of 20 years. Previously, we were able to detect by exome sequencing (ES) a known monogenic cause of SRNS in 25-30% of affected families. However, ES falls short of detecting copy number variants (CNV). Therefore, we hypothesized that causal CNVs could be detected in a large SRNS cohort. METHODS: We performed genome-wide single nucleotide polymorphism (SNP)-based CNV analysis on a cohort of 138 SRNS families, in whom we previously did not identify a genetic cause through ES. We evaluated ES and CNV data for variants in 60 known SRNS genes and in 13 genes in which variants are known to cause a phenocopy of SRNS. We applied previously published, predefined criteria for CNV evaluation. RESULTS: We detected a novel CNV in two genes in 2 out of 138 families (1.5%). The 9,673 bp homozygous deletion in PLCE1 and the 6,790 bp homozygous deletion in NPHS2 were confirmed across the breakpoints by PCR and Sanger sequencing. CONCLUSIONS: We confirmed that CNV analysis can identify the genetic cause in SRNS families that remained unsolved after ES. Though the rate of detected CNVs is minor, CNV analysis can be used when there are no other genetic causes identified. Causative CNVs are less common in SRNS than in other monogenic kidney diseases, such as congenital anomalies of the kidneys and urinary tract, where the detection rate was 5.3%. A higher resolution version of the Graphical abstract is available as Supplementary information.

Exome copy number variant detection, analysis and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and with new innovative methods can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the GREGoR consortium. Each family's CNV data was analyzed using the seqr platform and candidate CNVs classified using the 2020 ACMG/ClinGen CNV interpretation standards. We developed additional evidence criteria to address situations not covered by the current standards. The addition of CNV calling to exome analysis identified causal CNVs for 173 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb with estimates that 44% would not have been detected by standard chromosomal microarrays. The causal CNVs consisted of 141 deletions, 15 duplications, 4 suspected complex structural variants (SVs), 3 insertions and 10 complex SVs, the latter two groups being identified by orthogonal validation methods. We interpreted 153 CNVs as likely pathogenic/pathogenic and 20 CNVs as high interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Genetic stratification reveals COL4A variants and spontaneous remission in Egyptian children with proteinuria in the first 2 years of life.

AIM: The earlier the onset of proteinuria, the higher the incidence of genetic forms. Therefore, we aimed to analyse the spectrum of monogenic proteinuria in Egyptian children presenting at age <2 years. METHODS: The results of 27-gene panel or whole-exome sequencing were correlated with phenotype and treatment outcomes in 54 patients from 45 families. RESULTS: Disease-causing variants were identified in 29/45 (64.4%) families. Mutations often occurred in three podocytopathy genes: NPHS1, NPHS2 and PLCE1 (19 families). Some showed extrarenal manifestations. Additionally, mutations were detected in 10 other genes, including novel variants of OSGEP, SGPL1 and SYNPO2. COL4A variants phenocopied isolated steroid-resistant nephrotic syndrome (2/29 families, 6.9%). NPHS2 M1L was the single most common genetic finding beyond the age of 3 months (4/18 families, 22.2%). Biopsy results did not correlate with genotypes (n = 30). On renin-angiotensin-aldosterone system antagonists alone, partial and complete remission occurred in 3/24 (12.5%) patients with monogenic proteinuria each, whereas 6.3% (1/16) achieved complete remission on immunosuppression. CONCLUSION: Genotyping is mandatory to avoid biopsies and immunosuppression when proteinuria presents at age <2 years. Even with such a presentation, COL4A genes should be included. NPHS2 M1L was prevalent in Egyptian children (4 months-2 years) with proteinuria, demonstrating precision diagnostic utility.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

Inhibition of endoplasmic reticulum stress signaling rescues cytotoxicity of human apolipoprotein-L1 risk variants in Drosophila.

Risk variants of the apolipoprotein-L1 (APOL1) gene are associated with severe kidney disease, putting homozygous carriers at risk. Since APOL1 lacks orthologs in all major model organisms, a wide range of mechanisms frequently in conflict have been described for APOL1-associated nephropathies. The genetic toolkit in Drosophila allows unique in vivo insights into disrupted cellular homeostasis. To perform a mechanistic analysis, we expressed human APOL1 control and gain-of-function kidney risk variants in the podocyte-like garland cells of Drosophila nephrocytes and a wing precursor tissue. Expression of APOL1 risk variants was found to elevate endocytic function of garland cell nephrocytes that simultaneously showed early signs of cell death. Wild-type APOL1 had a significantly milder effect, while a control transgene with deletion of the short BH3 domain showed no overt phenotype. Nephrocyte endo-lysosomal function and slit diaphragm architecture remained unaffected by APOL1 risk variants, but endoplasmic reticulum (ER) swelling, chaperone induction, and expression of the reporter Xbp1-EGFP suggested an ER stress response. Pharmacological inhibition of ER stress diminished APOL1-mediated cell death and direct ER stress induction enhanced nephrocyte endocytic function similar to expression of APOL1 risk variants. We confirmed APOL1-dependent ER stress in the Drosophila wing precursor where silencing the IRE1-dependent branch of ER stress signaling by inhibition with Xbp1-RNAi abrogated cell death, representing the first rescue of APOL1-associated cytotoxicity in vivo. Thus, we uncovered ER stress as an essential consequence of APOL1 risk variant expression in vivo in Drosophila, suggesting a central role of this pathway in the pathogenesis of APOL1-associated nephropathies.

Reverse phenotyping facilitates disease allele calling in exome sequencing of patients with CAKUT.

PURPOSE: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. METHODS: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. RESULTS: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. CONCLUSION: We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Exome survey of individuals affected by VATER/VACTERL with renal phenotypes identifies phenocopies and novel candidate genes.

The acronym VATER/VACTERL refers to the rare nonrandom association of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L). For the clinical diagnosis, the presence of at least three CFs is required, individuals presenting with only two CFs have been categorized as VATER/VACTERL-like. The majority of VATER/VACTERL individuals displays a renal phenotype. Hitherto, variants in FGF8, FOXF1, HOXD13, LPP, TRAP1, PTEN, and ZIC3 have been associated with the VATER/VACTERL association; however, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes, both associated with a renal phenotype. In this study, we performed exome sequencing in 21 individuals and their families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype to identify potentially novel genetic causes. Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals (29%) in B9D1, FREM1, ZNF157, SP8, ACOT9, and TTLL11, respectively. The online tool GeneMatcher revealed another individual with a variant in ZNF157. Our study suggests six biallelic and X-chromosomal hemizygous VATER/VACTERL disease genes implicating all six genes in the expression of human renal malformations.

Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3 Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches.

INTRODUCTION: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways. METHODS: Here, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS. RESULTS: We first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation. CONCLUSION: Thus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

Recessive Mutations in SYNPO2 as a Candidate of Monogenic Nephrotic Syndrome.

INTRODUCTION: Most of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a "podocytopathy." METHODS: We performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients. RESULTS: We discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A-containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants. CONCLUSION: We show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome.

Mutations in PRDM15 Are a Novel Cause of Galloway-Mowat Syndrome.

BACKGROUND: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease. METHODS: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified. RESULTS: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes. CONCLUSIONS: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans.