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Background Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. Methods Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. Results Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. Conclusion We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. Graphical Abstract A higher resolution version of the Graphical abstract is available as Supplementary information

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5-2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility. ispartof: NATURE COMMUNICATIONS vol:13 issue:1 ispartof: location:England status: published

Background Podocyte dysfunction is the main pathologic mechanism driving the development of FSGS and other morphologic types of steroid-resistant nephrotic syndrome (SRNS). Despite significant progress, the genetic causes of most cases of SRNS have yet to be identified. Methods Whole-genome sequencing was performed on 320 individuals from 201 families with familial and sporadic NS/FSGS with no pathogenic mutations in any known NS/FSGS genes. Results Two variants in the gene encoding regulator of calcineurin type 1 (RCAN1) segregate with disease in two families with autosomal dominant FSGS/SRNS. In vitro, loss of RCAN1 reduced human podocyte viability due to increased calcineurin activity. Cells expressing mutant RCAN1 displayed increased calcineurin activity and NFAT activation that resulted in increased susceptibility to apoptosis compared with wild-type RCAN1. Treatment with GSK-3 inhibitors ameliorated this elevated calcineurin activity, suggesting the mutation alters the balance of RCAN1 regulation by GSK-3β, resulting in dysregulated calcineurin activity and apoptosis. Conclusions These data suggest mutations in RCAN1 can cause autosomal dominant FSGS. Despite the widespread use of calcineurin inhibitors in the treatment of NS, genetic mutations in a direct regulator of calcineurin have not been implicated in the etiology of NS/FSGS before this report. The findings highlight the therapeutic potential of targeting RCAN1 regulatory molecules, such as GSK-3β, in the treatment of FSGS.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10(−11)). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10(−15)). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

The essential role of membrane associated guanylate kinase 2 (MAGI2) in podocytes is indicated by the phenotypes of severe glomerulosclerosis of both MAGI2 knockout mice and in patients with congenital nephrotic syndrome (CNS) caused by mutations in MAGI2. Here, we show that MAGI2 forms a complex with the Rap1 guanine nucleotide exchange factor, RapGEF2, and that this complex is lost when expressing MAGI2 CNS variants. Co-expression of RapGEF2 with wild-type MAGI2, but not MAGI2 CNS variants, enhanced activation of the small GTPase Rap1, a central signaling node in podocytes. In mice, podocyte-specific RapGEF2 deletion resulted in spontaneous glomerulosclerosis, with qualitative glomerular features comparable to MAGI2 knockout mice. Knockdown of RapGEF2 or MAGI2 in human podocytes caused similar reductions in levels of Rap1 activation and Rap1-mediated downstream signaling. Furthermore, human podocytes expressing MAGI2 CNS variants show severe abnormalities of cellular morphology and dramatic loss of actin cytoskeletal organization, features completely rescued by pharmacological activation of Rap1 via a non-MAGI2 dependent upstream pathway. Finally, immunostaining of kidney sections from patients with congenital nephrotic syndrome and MAGI2 mutations showed reduced podocyte Rap1-mediated signaling. Thus, MAGI2-RapGEF2-Rap1 signaling is essential for normal podocyte function. Hence, disruption of this pathway is an important cause of the renal phenotype induced by MAGI2 CNS mutations.

Background and Aims Historically, paediatric patients with nephrotic syndrome (NS) have been crudely categorised by their response to steroids: steroid resistant (SRNS), steroid sensitive (SSNS) and secondary SRNS. However, growing evidence that these categories do not correspond to single diseases has led to a demand that they be re-defined based on biological mechanisms involved in the disease process. Indeed, next generation sequencing has identified causative mutations in up to 30% of paediatric NS patients (‘monogenic’ NS); however, specific disease mechanisms in the remaining patients remain unknown. We propose that the remaining phenotype variation in NS may be explained by DNA methylation, the reversible but heritable addition of methyl groups to DNA that change how the underlying DNA sequence is interpreted.

BackgroundLowe syndrome and Dent-2 disease are caused by mutations in theOCRLgene, which encodes for an inositol 5-phosphatase. The renal phenotype associated withOCRLmutations typically comprises a selective proximal tubulopathy, which can manifest as Fanconi syndrome in the most extreme cases.MethodsHere, we report a 12-year-old male with nephrotic-range proteinuria and focal segmental glomerulosclerosis on renal biopsy. As a glomerular pathology was suspected, extensive investigation of tubular function was not performed.ResultsSurprisingly, whole exome sequencing identified a genetic variant inOCRL(c1467-2A>G) that introduced a novel splice mutation leading to skipping of exon 15. In situ hybridisation of adult human kidney tissue and zebrafish larvae showedOCRLexpression in the glomerulus, supporting a role for OCRL in glomerular function. In cultured podocytes, we found that OCRL associated with the linker protein IPIP27A and CD2AP, a protein that is important for maintenance of the podocyte slit diaphragm.ConclusionTaken together, this work suggests a previously under-appreciated role for OCRL in glomerular function and highlights the importance of investigating tubular function in patients with persistent proteinuria.

After renal transplantation, recurrence of the original disease is the second most common cause of graft loss, after rejection. The most dramatic manifestation of this phenomenon is in patients with nephrotic syndrome (NS). NS is a descriptive term describing a clinical picture centred on proteinuria arising from damage to the glomerular filtration barrier (GFB). There are many different drivers of that damage, ranging from immune dysregulation to genetic disorders and chronic disease/infections. The main categories in childhood are “idiopathic” (presumed immune mediated) and genetic NS, with further stratification of the idiopathic group according to steroid responses. A significant proportion of patients with NS progress to established renal failure, requiring transplantation, and one of the most difficult clinical scenarios faced by nephrologists is the recurrence of the original disease in up to 50% of patients, usually rapidly post-transplant. This is thought to be the archetypal “circulating factor” disease, in which as yet unknown circulating plasma “factor(s)” in the recipient target the donor kidney. The ability to predict in advance which patients will suffer recurrence would enhance our ability to counsel patients and families, and potentially identify those patients before transplant for tailored immunosuppressive preparation. Until very recently, stratification based on clinical categorisations has been poor in being able to predict those patients in whom disease will recur, and laboratory biomarkers are yet to be adequately refined. However, by mapping our growing understanding of disease mechanisms to clinical phenotypes, and with greatly improved genetic diagnostics, we have made progress in being able to stratify patients more specifically, and allow better predictive algorithms to be developed. Using our knowledge of podocyte biology, circulating factor-induced specific biomarkers are also being tested. This review is aimed at outlining those advances, and suggesting how we can move further forward in both clinical and biological markers of disease type.

Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9–30% in selected series. Using whole exome sequencing we sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1 , NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL , COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism. ispartof: Journal of Clinical Investigation vol:127 issue:3 pages:942-953 ispartof: location:United States status: published

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function., Steroid-sensitive nephrotic syndrome (SRNS) can cause CKD and necessitate kidney transplant. Here the authors identify FAT1 mutations by homozygosity mapping and whole-exome sequencing in families with SRNS and provide functional mouse and zebrafish evidence that FAT1 is required for normal glomerular and tubular function and that FAT1 mutations can cause SRNS.

Background Mutations in podocyte and basement membrane genes are associated with a growing spectrum of glomerular disease affecting adults and children. Investigation of familial cases has helped to build understanding of both normal physiology and disease. Methods We investigated a consanguineous family with a wide clinical phenotype of glomerular disease using clinical, histological, and new genetic studies. Results We report striking variability in severity of nephropathy within an X-linked Alport syndrome (XLAS) family. Four siblings each carried a mutant COL4A5 allele, p.(Gly953Val) and p.(Gly1033Arg). Two boys had signs limited to hematuria and mild/moderate proteinuria. In striking contrast, a sister presented with end-stage renal disease (ESRD) at 8 years of age and an infant brother presented with nephrotic syndrome, progressing to ESRD by 3 years of age. Both were subsequently found to have homozygous variants in MYO1E, p.(Lys118Glu) and p.(Thr876Arg). MYO1E is a gene implicated in focal segmental glomerulosclerosis and it encodes a podocyte-expressed non-muscle myosin. Bioinformatic modeling demonstrated that the collagen IV-alpha3,4,5 extracellular network connected via known protein–protein interactions to intracellular myosin 1E. Conclusions COL4A5 and MYO1E mutations may summate to perturb common signaling pathways, resulting in more severe disease than anticipated independently. We suggest screening for MYO1E and other non-COL4 ‘podocyte gene’ mutations in XLAS when clinical nephropathy is more severe than expected for an individual’s age and sex.

Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.

Background Familial juvenile hyperuricaemic nephropathy is a rare inherited nephropathy with genetic heterogeneity. Categorised by genetic defect, mutations in uromodulin (UMOD), renin (REN) and hepatocyte nuclear factor-1β (HNF-1β) genes as well as linkage to chromosome 2p22.1-21 have previously been identified. Knowledge of the genetics of this phenotype has provided important clues to developmental pathways in the kidney. Case presentation We report a novel phenotype, with the typical features of hyperuricemia and renal deterioration, but with the additional unexpected feature of unilateral renal hypoplasia. Mutation analyses of the existing known genes and genetic loci were negative indicating a new monogenic cause. Interestingly two cousins of the index case did not share the latter feature, suggesting a modifier gene effect. Conclusion Unilateral renal hypo/aplasia is usually sporadic and relatively common, with no genetic cause to date identified. This reported pedigree reveals the possibility that a new, unknown renal developmental gene may be implicated in the FJHN phenotype.

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q(10) (CoQ(10)) biosynthesis. Mutations in ADCK4 resulted in reduced COQ(10). levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ(10) biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ(10) addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ(10) treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ(10) biosynthesis may be treatable with CoQ(10).