Monogenic Causes Of Steroid-resistant Nephrotic Syndrome Research Articles

Whole exome sequencing identifies monogenic forms of nephritis in a previously unsolved cohort of children with steroid-resistant nephrotic syndrome and hematuria.

Alport syndrome (AS), atypical hemolytic-uremic syndrome (aHUS), and fibronectin-glomerulopathy (FG) are rare forms of glomerular diseases that manifest in a combination of proteinuria, hematuria, and hypertension, referred to as nephritic syndrome. Due to phenotypic overlays, steroid-resistant nephrotic syndrome (SRNS) and nephritic syndrome have been difficult to discern diagnostically. SRNS is more common than nephritic syndrome and is the second leading cause of childhood-onset CKD. Fourteen monogenic causes of AS, aHUS, and FG and 60 monogenic causes of SRNS have been identified. As whole exome sequencing (WES) allows for unequivocal molecular genetic diagnostics, we hypothesize to be able to identify causative mutations in genes known to cause nephritic syndrome in patient cohorts with a clinical diagnosis of SRNS. We identified patients with hematuria and steroid-resistant proteinuria in an international patient cohort that we had submitted to WES and who were unsolved for known monogenic causes of SRNS. These 70 patients from 65 individual families were subsequently analyzed for causative mutations in 14 AS, aHUS, or FG causing genes. WES data were compared to a control cohort of 76 patients from 75 families that were diagnosed with nephronophthisis-related ciliopathies (NPHP-RC) and to a control cohort of 83 individuals from 75 families with SRNS, but without hematuria. We detected likely pathogenic genetic variants in 3 of 65 families (4.6%) in 2 of the 14 genes analyzed. We confirmed that in cohorts of childhood-onset SRNS, patients with nephritic syndrome can be discerned by WES. The findings highlight the importance of clinical genetic testing for therapeutic and preventative measures in patients with proteinuria. A higher resolution version of the Graphical abstract is available as Supplementary information.

Genetic Study in Korean Pediatric Patients with Steroid-Resistant Nephrotic Syndrome or Focal Segmental Glomerulosclerosis

Steroid-resistant nephrotic syndrome (SRNS) is one of the major causes of end-stage renal disease (ESRD) in childhood and is mostly associated with focal segmental glomerulosclerosis (FSGS). More than 50 monogenic causes of SRNS or FSGS have been identified. Recently, the mutation detection rate in pediatric patients with SRNS has been reported to be approximately 30%. In this study, genotype-phenotype correlations in a cohort of 291 Korean pediatric patients with SRNS/FSGS were analyzed. The overall mutation detection rate was 43.6% (127 of 291 patients). WT1 was the most common causative gene (23.6%), followed by COQ6 (8.7%), NPHS1 (8.7%), NUP107 (7.1%), and COQ8B (6.3%). Mutations in COQ6, NUP107, and COQ8B were more frequently detected, and mutations in NPHS2 were less commonly detected in this cohort than in study cohorts from Western countries. The mutation detection rate was higher in patients with congenital onset, those who presented with proteinuria or chronic kidney disease/ESRD, and those who did not receive steroid treatment. Genetic diagnosis in patients with SRNS provides not only definitive diagnosis but also valuable information for decisions on treatment policy and prediction of prognosis. Therefore, further genotype-phenotype correlation studies are required.

Genetic tests in children with steroid-resistant nephrotic syndrome

Steroid-resistant nephrotic syndrome (SRNS) is a common cause of chronic kidney disease in children, and a considerable number of patients progress to end-stage renal disease. SRNS is a highly heterogeneous disorder, both clinically and genetically, and more than 50 monogenic causes of SRNS, including isolated and syndromic forms, have been identified. Recent large-cohort studies indicate that at least 30% of childhood-onset SRNS cases are genetic. The benefits of definitive molecular diagnosis by genetic testing include the avoidance of unnecessary and potentially harmful diagnostic procedures (e.g., kidney biopsy) and treatment (e.g., steroid and immunosuppressants), detection of rare and potentially treatable mutations (e.g., coenzyme Q10 biosynthesis pathway defect), prediction of prognosis (e.g., posttransplant recurrence), and providing precise genetic counseling. Furthermore, the identification of novel disease-causing genes could provide new insights into the pathogenic mechanisms of SRNS. Therefore, whenever accessible and affordable, genetic testing is recommended for all pediatric patients with SRNS, and should certainly be performed in patients with a higher probability of genetic predisposition based on genotype-phenotype correlation data. The genetic testing approach should be determined for each patient, and clinicians should, therefore, be aware of the advantages and disadvantages of methods currently available, which include Sanger sequencing, gene panel testing, and whole-exome or whole-genome sequencing. Importantly, the need for precise and thorough phenotyping by clinicians, even in the era of genomics, cannot be overemphasized. This review provides an update on recent advances in genetic studies, a suggested approach for the genetic testing of pediatric patients with SRNS.

TBC1D8B Mutations Implicate RAB11-Dependent Vesicular Trafficking in the Pathogenesis of Nephrotic Syndrome

Mutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology. We used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes. We identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function. Novel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.

Genetic Testing for Steroid-Resistant-Nephrotic Syndrome in an Outbred Population.

Background: Steroid-resistant nephrotic syndrome (SRNS) is a leading cause of end-stage kidney disease in children and young adults. Despite advances in genomic science that have led to the discovery of >50 monogenic causes of SRNS, there are no clear guidelines for genetic testing in clinical practice.Methods: Using high throughput sequencing, we evaluated 492 individuals from 181 families for mutations in 40 known SRNS genes. Causative mutations were defined as missense, truncating, and obligatory splice site variants with a minor allele frequency <1% in controls. Non-synonymous variants were considered pathogenic if determined to be deleterious by at least two in silico models. We further evaluated for differences in age at disease onset, family history of SRNS or chronic kidney disease, race, sex, renal biopsy findings, and extra-renal manifestations in subgroups with and without disease causing variants.Results: We identified causative variants in 40 of 181 families (22.1%) with SRNS. Variants in INF2, COL4A3, and WT1 were the most common, accounting for over half of all causative variants. Causative variants were identified in 34 of 86 families (39.5%) with familial disease and 6 of 95 individuals (6.3%) with sporadic disease (χ2 p < 0.00001). Family history was the only significant clinical predictor of genetic SRNS.Conclusion: We identified causative mutations in almost 40% of all families with hereditary SRNS and 6% of individuals with sporadic disease, making family history the single most important clinical predictors of monogenic SRNS. We recommend genetic testing in all patients with SRNS and a positive family history, but only selective testing in those with sporadic disease.

Treatment of steroid-resistant nephrotic syndrome in the genomic era.

The pathogenesis of steroid-resistant nephrotic syndrome (SRNS) is not completely known. Recent advances in genomics have elucidated some of the molecular mechanisms and pathophysiology of the disease. More than 50 monogenic causes of SRNS have been identified; however, these genes are responsible for only a small fraction of SRNS in outbred populations. There are currently no guidelines for genetic testing in SRNS, but evidence from the literature suggests that testing should be guided by the genetic architecture of the disease in the population. Notably, most genetic forms of SRNS do not respond to current immunosuppressive therapies; however, a small subset of patients with monogenic SRNS will achieve partial or complete remission with specific immunomodulatory agents, presumably due to non-immunosuppressive effects of these agents. We suggest a pragmatic approach to the therapy of genetic SRNS, as there is no evidence-based algorithm for the management of the disease.

Mutations in multiple components of the nuclear pore complex cause nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) almost invariably progresses to end-stage renal disease. Although more than 50 monogenic causes of SRNS have been described, a large proportion of SRNS remains unexplained. Recently, it was discovered that mutations of NUP93 and NUP205, encoding 2 proteins of the inner ring subunit of the nuclear pore complex (NPC), cause SRNS. Here, we describe mutations in genes encoding 4 components of the outer rings of the NPC, namely NUP107, NUP85, NUP133, and NUP160, in 13 families with SRNS. Using coimmunoprecipitation experiments, we showed that certain pathogenic alleles weakened the interaction between neighboring NPC subunits. We demonstrated that morpholino knockdown of nup107, nup85, or nup133 in Xenopus disrupted glomerulogenesis. Re-expression of WT mRNA, but not of mRNA reflecting mutations from SRNS patients, mitigated this phenotype. We furthermore found that CRISPR/Cas9 knockout of NUP107, NUP85, or NUP133 in podocytes activated Cdc42, an important effector of SRNS pathogenesis. CRISPR/Cas9 knockout of nup107 or nup85 in zebrafish caused developmental anomalies and early lethality. In contrast, an in-frame mutation of nup107 did not affect survival, thus mimicking the allelic effects seen in humans. In conclusion, we discovered here that mutations in 4 genes encoding components of the outer ring subunits of the NPC cause SRNS and thereby provide further evidence that specific hypomorphic mutations in these essential genes cause a distinct, organ-specific phenotype.

GAPVD1 and ANKFY1 Mutations Implicate RAB5 Regulation in Nephrotic Syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of CKD. The discovery of monogenic causes of SRNS has revealed specific pathogenetic pathways, but these monogenic causes do not explain all cases of SRNS. To identify novel monogenic causes of SRNS, we screened 665 patients by whole-exome sequencing. We then evaluated the in vitro functional significance of two genes and the mutations therein that we discovered through this sequencing and conducted complementary studies in podocyte-like Drosophila nephrocytes. We identified conserved, homozygous missense mutations of GAPVD1 in two families with early-onset NS and a homozygous missense mutation of ANKFY1 in two siblings with SRNS. GAPVD1 and ANKFY1 interact with the endosomal regulator RAB5. Coimmunoprecipitation assays indicated interaction between GAPVD1 and ANKFY1 proteins, which also colocalized when expressed in HEK293T cells. Silencing either protein diminished the podocyte migration rate. Compared with wild-type GAPVD1 and ANKFY1, the mutated proteins produced upon ectopic expression of GAPVD1 or ANKFY1 bearing the patient-derived mutations exhibited altered binding affinity for active RAB5 and reduced ability to rescue the knockout-induced defect in podocyte migration. Coimmunoprecipitation assays further demonstrated a physical interaction between nephrin and GAPVD1, and immunofluorescence revealed partial colocalization of these proteins in rat glomeruli. The patient-derived GAPVD1 mutations reduced nephrin-GAPVD1 binding affinity. In Drosophila, silencing Gapvd1 impaired endocytosis and caused mistrafficking of the nephrin ortholog. Mutations in GAPVD1 and probably in ANKFY1 are novel monogenic causes of NS. The discovery of these genes implicates RAB5 regulation in the pathogenesis of human NS.

Spectrum of mutations in Chinese children with steroid-resistant nephrotic syndrome.

The aim of this study was to elucidate whether genetic screening test results of pediatric patients with steroid-resistant nephrotic syndrome (SRNS) vary with ethnicity. Using high-throughput DNA sequencing, 28 nephrotic syndrome-related genes were analyzed in 110 chil-dren affected by SRNS and 10 children with isolated proteinuria enrolled by 5 centers in China (67 boys, 53 girls). Their age at disease onset ranged from 1day to 208months (median, 48.8months). Patients were excluded if their age at onset of disease was over 18years or if they were diagnosed as having Alport syndrome. A genetic etiology was identified in 28.3% of our cohort and the likelihood of establishing a genetic diagnosis decreased as the age at onset of nephrotic syndrome increased. The most common mutated genes were ADCK4 (6.67%), NPHS1 (5.83%), WT1 (5.83%), and NPHS2 (3.33%), and the difference in the frequencies of ADCK4 and NPHS2 mutations between this study and a study on monogenic causes of SRNS in the largest international cohort of 1,783 different families was significant. A case of congenital nephrotic syndrome was attributed to a homozygous missense mutation in ADCK4, and a de novo missense mutation in TRPC6 was detected in a case of infantile nephrotic syndrome. Our results showed that, in the first and the largest multicenter cohort of Chinese pediatric SRNS reported to date, ADCK4 is the most common causative gene, whereas there is a low prevalence of NPHS2 mutations. Our data indicated that the genetic testing results for pediatric SRNS patients vary with different ethnicities, and this information will help to improve management of the disease in clinical practice.

A small molecule screening to detect potential therapeutic targets in human podocytes.

A small molecule screening to detect potential therapeutic targets in human podocytes. Am J Physiol Renal Physiol 312: F157-F171, 2017. First published October 19, 2016; doi:10.1152/ajprenal.00386.2016. Steroid-resistant nephrotic syndrome (SRNS) inevitably progresses to end-stage kidney disease, requiring dialysis or transplantation for survival. However, treatment modalities and drug discovery remain limited. Mutations in over 30 genes have been discovered as monogenic causes of SRNS. Most of these genes are predominantly expressed in the glomerular epithelial cell, the podocyte, placing it at the center of the pathogenesis of SRNS. Podocyte migration rate (PMR) represents a relevant intermediate phenotype of disease in monogenic causes of SRNS. We therefore adapted PMR in a high-throughput manner to screen small molecules as potential therapeutic targets for SRNS. We performed a high-throughput drug screening of a National Institutes of Health Clinical Collection (NCC) library (n = 725 compounds) measuring PMR by videomicroscopy. We used the Woundmaker to perform individual 96-well scratch wounds and screened compounds using a quantitative kinetic live cell imaging migration assay using IncuCyte ZOOM technology. Using a normal distribution for the average PMR in wild-type podocytes with a vehicle control (DMSO), we applied a 90% confidence interval to define "distinct" compounds (5% faster/slower PMR) and found that 12 of 725 compounds (at 10 μM) reduced PMR. Clusters of drugs that alter PMR included actin/tubulin modulators such as the azole class of antifungals and antineoplastic vinca-alkaloids. We hereby identify compounds that alter PMR. The PMR assay provides a new avenue to test therapeutics for nephrotic syndrome. Positive results may reveal novel pathways in the study of glomerular diseases such as SRNS.

Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome.

Nucleoporins (NUPs) are essential components of the nuclear pore complex (NPC).1 Only few diseases have been attributed to NPC dysfunction.2-4 Steroid resistant nephrotic syndrome (SRNS), a frequent cause of chronic kidney disease, is caused by dysfunction of glomerular podocytes.5 Here we identify in 8 families with SRNS mutations of NUP93, its interaction partner NUP205, or exportin5 (XPO5) as a hitherto unrecognized monogenic cause of SRNS. NUP93 mutations caused disrupted NPC assembly. NUP93 knockdown reduced the presence of NUP205 in the NPC and, reciprocally, a NUP205 mutation abrogated NUP93 interaction. We demonstrate that NUP93 and XPO5 interact with the signaling protein SMAD4, and that NUP93 mutations abrogated interaction with SMAD4. Significantly, NUP93 mutations interfered with BMP7-induced SMAD transcriptional reporter activity. We hereby demonstrate that mutations of NUPs cause a distinct renal disease, and reveal SMAD signaling as a novel disease mechanism of SRNS, opening a potential new avenue for treatment.

Specific podocin mutations determine age of onset of nephrotic syndrome all the way into adult life

In steroid-resistant nephrotic syndrome (SRNS) Machuca et al. report that mutations of the recessive podocin gene cause adult-onset SRNS if the R229Q genetic variant occurs in a compound heterozygous state with another podocin mutation. Learning to tell apart the specific allele combinations of podocin mutations will be important for prognosis, genetic counseling in living related kidney donation, accurate etiologic classification within treatment studies, and the understanding of podocin function.