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Background: The mammalian target of rapamycin inhibitor (mTORi) therapy after kidney transplantation is solely monitored pharmacokinetically, not necessarily reflecting PI3K-Akt-mTOR pathway blockade efficacy leading to potential under-or overimmunosuppression. Methods: In this cross-sectional study, phosphoflow cytometry was used to determine the efficacy of mTOR inhibition in peripheral T- and B-lymphocyte subsets by assessing p70S6 kinase (p70S6K) phosphorylation in renal transplant recipients upon treatment with a combination of either mTORi and calcineurin inhibitors (nâ =â 18), or mTORi with mycophenolic acid (nâ =â 9). Nine dialysis patients with end-stage renal disease and 17 healthy age-matched volunteers served as controls. Results: mTORi treatment reduced p70S6K phosphorylation in CD4+, CD8+ T, and CD19+ B cells compared with healthy controls (HCs). Subpopulation analysis of CD4+ T cells and CD19+ B cells revealed a significant reduction of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells (Pâ <â 0.05), CD24hiCD38hi transitional B cells (Pâ <â 0.001), CD24+CD38- memory B cells (Pâ <â 0.001), and CD24intCD38int-naive B cells (Pâ <â 0.05) upon mTORi treatment, whereas CD4+CD45RA-CD25++CD127- regulatory T cells and CD24-CD38hi plasmablasts were not affected. Compared with mTORi + mycophenolic acid therapy, mTORi + calcineurin inhibitor treatment exhibited an even stronger inhibition of p70S6K phosphorylation in CD4+CD45RA-CD25- Th cells and CD8+ T cells. However, trough levels of mTORi did not correlate with p70S6K phosphorylation. Conclusions: mTORi selectively inhibited p70S6K phosphorylation in select lymphocyte subtypes. Assessing p70S6K phosphorylation by phosphoflow cytometry may serve as an approach to understand cell subset specific effects of mTORi providing detailed pharmacodynamic information for individualizing immunosuppression.
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Adhesion G protein-coupled receptors (aGPCRs) comprise the second-largest class of GPCRs, the most common target for approved pharmacological therapies. aGPCRs play an important role in development and disease and have recently been associated with the kidney. Several aGPCRs are expressed in the kidney and some aGPCRs are either required for kidney development or their expression level is altered in diseased kidneys. Yet, general aGPCR function and their physiological role in the kidney are poorly understood. Here, we characterize in detail Gpr126 (Adgrg6) expression based on RNAscope® technology in zebrafish, mice, and humans during kidney development in adults. Gpr126 expression is enriched in the epithelial linage during nephrogenesis and persists in the adult kidney in parietal epithelial cells, collecting ducts, and urothelium. Single-cell RNAseq analysis shows that gpr126 expression is detected in zebrafish in a distinct ionocyte sub-population. It is co-detected selectively with slc9a3.2, slc4a4a, and trpv6, known to be involved in apical acid secretion, buffering blood or intracellular pH, and to maintain high cytoplasmic Ca2+ concentration, respectively. Furthermore, gpr126-expressing cells were enriched in the expression of potassium transporter kcnj1a.1 and gcm2, which regulate the expression of a calcium sensor receptor. Notably, the expression patterns of Trpv6, Kcnj1a.1, and Gpr126 in mouse kidneys are highly similar. Collectively, our approach permits a detailed insight into the spatio-temporal expression of Gpr126 and provides a basis to elucidate a possible role of Gpr126 in kidney physiology.
Assuntos
Receptores Acoplados a Proteínas G , Peixe-Zebra , Animais , Humanos , Camundongos , Proteínas de Ligação a DNA , Rim/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
PURPOSE: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC. METHODS: Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized. RESULTS: Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ2 = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca2+ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca2+ uptake, demonstrating loss of function. CONCLUSION: Rare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.
Assuntos
Nefrolitíase , Receptores Purinérgicos P2 , Humanos , Oxalato de Cálcio , Nefrolitíase/genética , Mutação de Sentido Incorreto/genética , Transportadores de Sulfato/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismoRESUMO
Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation.
Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Adulto , Testes Genéticos , Estudo de Associação Genômica Ampla , Humanos , Mutação , Doenças Renais Policísticas/genética , Rim Policístico Autossômico Dominante/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Hérnia Hiatal/genética , Microcefalia/genética , Mutação de Sentido Incorreto , Nefrose/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Pré-Escolar , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/deficiência , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Síndrome Nefrótica/genética , Podócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Pronefro/embriologia , Pronefro/metabolismo , Estabilidade Proteica , Fatores de Transcrição/química , Fatores de Transcrição/deficiência , Xenopus laevis/embriologia , Xenopus laevis/genética , Dedos de Zinco/genéticaRESUMO
Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1 and SLC4A1) constitute a monogenic causation in 58-70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1 have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three "classical" known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72 (2 families), previously implicated in V-ATPase trafficking in cells; and SLC4A2 (1 family), a paralog of the known distal renal tubular acidosis gene SLC4A1. Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2 revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2 as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2 as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72 mutations from amelogenesis imperfecta to distal renal tubular acidosis.
Assuntos
Acidose Tubular Renal , ATPases Vacuolares Próton-Translocadoras , Acidose Tubular Renal/genética , Proteína 1 de Troca de Ânion do Eritrócito , Criança , Antiportadores de Cloreto-Bicarbonato , Análise Mutacional de DNA , Fatores de Transcrição Forkhead , Humanos , Mutação , ATPases Vacuolares Próton-Translocadoras/genética , Sequenciamento do ExomaRESUMO
BACKGROUND: CD2-associated protein (CD2AP), a slit diaphragm-associated scaffolding protein involved in survival and regulation of the cytoskeleton in podocytes, is considered a "stabilizer" of the slit diaphragm complex that connects the slit diaphragm protein nephrin to the cytoskeleton of the cell. Tyrosine phosphorylation of slit diaphragm molecules can influence their surface expression, but it is unknown whether tyrosine phosphorylation events of CD2AP are also physiologically relevant to slit diaphragm stability. METHODS: We used isoelectric focusing, western blot analysis, and immunofluorescence to investigate phosphorylation of CD2AP, and phospho-CD2AP antibodies and site-directed mutagenesis to define the specific phosphorylated tyrosine residues. We used cross-species rescue experiments in Cd2apKD zebrafish and in Drosophila cindrRNAi mutants to define the physiologic relevance of CD2AP phosphorylation of the tyrosine residues. RESULTS: We found that VEGF-A stimulation can induce a tyrosine phosphorylation response in CD2AP in podocytes, and that these phosphorylation events have an important effect on slit diaphragm protein localization and functionality in vivo. We demonstrated that tyrosine in position Y10 of the SH3-1 domain of CD2AP is indispensable for CD2AP function in vivo. We found that the binding affinity of nephrin to CD2AP is significantly enhanced in the absence of Y10; however, unexpectedly, this increased affinity leads not to stabilization but to functional impairment of the glomerular filtration barrier. CONCLUSIONS: Our findings provide insight into CD2AP and its phosphorylation in the context of slit diaphragm functionality, and indicate a fine-tuned affinity balance of CD2AP and nephrin that is influenced by receptor tyrosine kinase stimulation.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/química , Tirosina/metabolismo , Animais , Drosophila melanogaster , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Fosforilação , Podócitos/metabolismo , Estabilidade Proteica , Fator A de Crescimento do Endotélio Vascular/farmacologia , Peixe-ZebraRESUMO
Recently, recessive mutations of MAGI2 were identified as a cause of steroid-resistant nephrotic syndrome (SRNS) in humans and mice. To further delineate the pathogenesis of MAGI2 loss of function, we generated stable knockout lines for the two zebrafish orthologues magi2a and magi2b by CRISPR/Cas9. We also developed a novel assay for the direct detection of proteinuria in zebrafish independent of transgenic background. Whereas knockout of magi2b did not yield a nephrotic syndrome phenotype, magi2a-/- larvae developed ascites, periorbital edema, and proteinuria, as indicated by increased excretion of low molecular weight protein. Electron microscopy demonstrated extensive podocyte foot process effacement. As in human SRNS, we observed genotype/phenotype correlation, with edema onset occurring earlier in zebrafish with truncating alleles (5-6 days post fertilization) versus hypomorphic alleles (19-20 days post fertilization). Paradoxically, corticosteroid treatment exacerbated the phenotype, with earlier onset of edema. In contrast, treatment with cyclosporine A or tacrolimus had no significant effect. Although RhoA signaling has been implicated as a downstream mediator of MAGI2 activity, targeting of the RhoA pathway did not modify the nephrotic syndrome phenotype. In the first CRISPR/Cas9 zebrafish knockout model of SRNS, we found that corticosteroids may have a paradoxical effect in the setting of specific genetic mutations.
Assuntos
Glucocorticoides/farmacologia , Imunossupressores/farmacologia , Proteínas de Membrana/genética , Síndrome Nefrótica/tratamento farmacológico , Proteinúria/tratamento farmacológico , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Progressão da Doença , Resistência a Medicamentos , Técnicas de Inativação de Genes , Glucocorticoides/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/genética , Proteinúria/patologia , Transdução de Sinais/efeitos dos fármacos , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Resultado do Tratamento , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Nephrolithiasis (NL) affects 1 in 11 individuals worldwide and causes significant patient morbidity. We previously demonstrated a genetic cause of NL can be identified in 11-29% of pre-dominantly American and European stone formers. Pakistan, which resides within the Afro-Asian stone belt, has a high prevalence of nephrolithiasis (12%) as well as high rate of consanguinity (> 50%). We recruited 235 Pakistani subjects hospitalized for nephrolithiasis from five tertiary hospitals in the Punjab province of Pakistan. Subjects were surveyed for age of onset, NL recurrence, and family history. We conducted high-throughput exon sequencing of 30 NL disease genes and variant analysis to identify monogenic causative mutations in each subject. We detected likely causative mutations in 4 of 30 disease genes, yielding a likely molecular diagnosis in 7% (17 of 235) of NL families. Only 1 of 17 causative mutations was identified in an autosomal recessive disease gene. 10 of the 12 detected mutations were novel mutations (83%). SLC34A1 was most frequently mutated (12 of 17 solved families). We observed a higher frequency of causative mutations in subjects with a positive NL family history (13/109, 12%) versus those with a negative family history (4/120, 3%). Five missense SLC34A1 variants identified through genetic analysis demonstrated defective phosphate transport. We examined the monogenic causes of NL in a novel geographic cohort and most frequently identified dominant mutations in the sodium-phosphate transporter SLC34A1 with functional validation.
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Perfilação da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Nefrolitíase/epidemiologia , Nefrolitíase/genética , Adolescente , Adulto , Idoso , Alelos , Animais , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Família , Feminino , Perfilação da Expressão Gênica/métodos , Genótipo , Geografia Médica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Paquistão/epidemiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Xenopus laevis , Adulto JovemRESUMO
BACKGROUND: Whole-exome sequencing (WES) finds a CKD-related mutation in approximately 20% of patients presenting with CKD before 25 years of age. Although provision of a molecular diagnosis could have important implications for clinical management, evidence is lacking on the diagnostic yield and clinical utility of WES for pediatric renal transplant recipients. METHODS: To determine the diagnostic yield of WES in pediatric kidney transplant recipients, we recruited 104 patients who had received a transplant at Boston Children's Hospital from 2007 through 2017, performed WES, and analyzed results for likely deleterious variants in approximately 400 genes known to cause CKD. RESULTS: By WES, we identified a genetic cause of CKD in 34 out of 104 (32.7%) transplant recipients. The likelihood of detecting a molecular genetic diagnosis was highest for patients with urinary stone disease (three out of three individuals), followed by renal cystic ciliopathies (seven out of nine individuals), steroid-resistant nephrotic syndrome (nine out of 21 individuals), congenital anomalies of the kidney and urinary tract (ten out of 55 individuals), and chronic glomerulonephritis (one out of seven individuals). WES also yielded a molecular diagnosis for four out of nine individuals with ESRD of unknown etiology. The WES-related molecular genetic diagnosis had implications for clinical care for five patients. CONCLUSIONS: Nearly one third of pediatric renal transplant recipients had a genetic cause of their kidney disease identified by WES. Knowledge of this genetic information can help guide management of both transplant patients and potential living related donors.
Assuntos
Sequenciamento do Exoma/métodos , Transplante de Rim/métodos , Medicina de Precisão/métodos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/cirurgia , Adolescente , Boston , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Predisposição Genética para Doença/epidemiologia , Testes Genéticos/métodos , Rejeição de Enxerto , Sobrevivência de Enxerto , Hospitais Pediátricos , Humanos , Transplante de Rim/efeitos adversos , Masculino , Prognóstico , Insuficiência Renal Crônica/fisiopatologia , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Análise de Sobrevida , Transplantados/estatística & dados numéricos , Resultado do TratamentoRESUMO
Genome-wide association studies (GWAS) have identified multiple chronic kidney disease (CKD)-associated single-nucleotide polymorphisms (SNPs) mainly localized to non-coding genomic regions. To understand which genes and which cell types are affected by these genetic variants, compartment-specific transcriptome, genome, and epigenome data were analyzed in an integrative manner in a recent study by Qiu et al. (Qiu et al., 2018).
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Estudo de Associação Genômica Ampla , Insuficiência Renal Crônica , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
BACKGROUND: Nephrotic syndrome (NS), a chronic kidney disease, is characterized by significant loss of protein in the urine causing hypoalbuminemia and edema. In general, â¼15% of childhood-onset cases do not respond to steroid therapy and are classified as steroid-resistant NS (SRNS). In â¼30% of cases with SRNS, a causative mutation can be detected in one of 44 monogenic SRNS genes. The gene LAMA5 encodes laminin-α5, an essential component of the glomerular basement membrane. Mice with a hypomorphic mutation in the orthologous gene Lama5 develop proteinuria and hematuria. METHODS: To identify additional monogenic causes of NS, we performed whole exome sequencing in 300 families with pediatric NS. In consanguineous families we applied homozygosity mapping to identify genomic candidate loci for the underlying recessive mutation. RESULTS: In three families, in whom mutations in known NS genes were excluded, but in whom a recessive, monogenic cause of NS was strongly suspected based on pedigree information, we identified homozygous variants of unknown significance (VUS) in the gene LAMA5. While all affected individuals had nonsyndromic NS with an early onset of disease, their clinical outcome and response to immunosuppressive therapy differed notably. CONCLUSION: We here identify recessive VUS in the gene LAMA5 in patients with partially treatment-responsive NS. More data will be needed to determine the impact of these VUS in disease management. However, familial occurrence of disease, data from genetic mapping and a mouse model that recapitulates the NS phenotypes suggest that these genetic variants may be inherited factors that contribute to the development of NS in pediatric patients.
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Sequenciamento do Exoma/métodos , Imunossupressores/uso terapêutico , Laminina/genética , Mutação , Síndrome Nefrótica/genética , Adolescente , Adulto , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/patologia , Linhagem , Fenótipo , Prognóstico , Adulto JovemRESUMO
BACKGROUND: Alport syndrome (AS) and atypical hemolytic-uremic syndrome (aHUS) are rare forms of chronic kidney disease (CKD) that can lead to a severe decline of renal function. Steroid-resistant nephrotic syndrome (SRNS) is more common than AS and aHUS and causes 10% of childhood-onset CKD. In recent years, multiple monogenic causes of AS, aHUS and SRNS have been identified, but their relative prevalence has yet to be studied together in a typical pediatric cohort of children with proteinuria and hematuria. We hypothesized that identification of causative mutations by whole exome sequencing (WES) in known monogenic nephritis and nephrosis genes would allow distinguishing nephritis from nephrosis in a typical pediatric group of patients with both proteinuria and hematuria at any level. METHODS: We therefore conducted an exon sequencing (WES) analysis for 11 AS, aHUS and thrombotic thrombocytopenic purpura-causing genes in an international cohort of 371 patients from 362 families presenting with both proteinuria and hematuria before age 25 years. In parallel, we conducted either WES or high-throughput exon sequencing for 23 SRNS-causing genes in all patients. RESULTS: We detected pathogenic mutations in 18 of the 34 genes analyzed, leading to a molecular diagnosis in 14.1% of families (51 of 362). Disease-causing mutations were detected in 3 AS-causing genes (4.7%), 3 aHUS-causing genes (1.4%) and 12 NS-causing genes (8.0%). We observed a much higher mutation detection rate for monogenic forms of CKD in consanguineous families (35.7% versus 10.1%). CONCLUSIONS: We present the first estimate of relative frequency of inherited AS, aHUS and NS in a typical pediatric cohort with proteinuria and hematuria. Important therapeutic and preventative measures may result from mutational analysis in individuals with proteinuria and hematuria.
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Sequenciamento do Exoma/métodos , Marcadores Genéticos , Mutação , Nefrite/diagnóstico , Nefrite/genética , Nefrose/diagnóstico , Nefrose/genética , Adolescente , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/genética , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Nefrite Hereditária/diagnóstico , Nefrite Hereditária/genética , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/genética , PrognósticoRESUMO
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.
Assuntos
Síndrome Nefrótica/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Síndrome Nefrótica/genética , Síndrome Nefrótica/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Xenopus/genética , Xenopus laevis , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Galloway-Mowat syndrome (GAMOS) is a phenotypically heterogeneous disorder characterized by neurodevelopmental defects combined with renal-glomerular disease, manifesting with proteinuria. To identify additional monogenic disease causes, we here performed whole exome sequencing (WES), linkage analysis, and homozygosity mapping in three affected siblings of an Indian family with GAMOS. Applying established criteria for variant filtering, we identify a novel homozygous splice site mutation in the gene WDR4 as the likely disease-causing mutation in this family. In line with previous reports, we observe growth deficiency, microcephaly, developmental delay, and intellectual disability as phenotypic features resulting from WDR4 mutations. However, the newly identified allele additionally gives rise to proteinuria and nephrotic syndrome, a phenotype that was never reported in patients with WDR4 mutations. Our data thus expand the phenotypic spectrum of WDR4 mutations by demonstrating that, depending on the specific mutated allele, a renal phenotype may be present. This finding suggests that GAMOS may occupy a phenotypic spectrum with other microcephalic diseases. Furthermore, WDR4 is an additional example of a gene that encodes a tRNA modifying enzyme and gives rise to GAMOS, if mutated. Our findings thereby support the recent observation that, like neurons, podocytes of the renal glomerulus are particularly vulnerable to cellular defects resulting from altered tRNA modifications.
Assuntos
Proteínas de Ligação ao GTP/genética , Hérnia Hiatal/genética , Microcefalia/genética , Mutação , Nefrose/genética , Adolescente , Criança , Pré-Escolar , Genes Recessivos , Humanos , Sequenciamento do ExomaRESUMO
BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of kidney disease in the first three decades of life. Previous gene panel studies showed monogenic causation in up to 12% of patients with CAKUT. METHODS: We applied whole-exome sequencing to analyze the genotypes of individuals from 232 families with CAKUT, evaluating for mutations in single genes known to cause human CAKUT and genes known to cause CAKUT in mice. In consanguineous or multiplex families, we additionally performed a search for novel monogenic causes of CAKUT. RESULTS: In 29 families (13%), we detected a causative mutation in a known gene for isolated or syndromic CAKUT that sufficiently explained the patient's CAKUT phenotype. In three families (1%), we detected a mutation in a gene reported to cause a phenocopy of CAKUT. In 15 of 155 families with isolated CAKUT, we detected deleterious mutations in syndromic CAKUT genes. Our additional search for novel monogenic causes of CAKUT in consanguineous and multiplex families revealed a potential single, novel monogenic CAKUT gene in 19 of 232 families (8%). CONCLUSIONS: We identified monogenic mutations in a known human CAKUT gene or CAKUT phenocopy gene as the cause of disease in 14% of the CAKUT families in this study. Whole-exome sequencing provides an etiologic diagnosis in a high fraction of patients with CAKUT and will provide a new basis for the mechanistic understanding of CAKUT.
Assuntos
Sequenciamento do Exoma/métodos , Predisposição Genética para Doença/epidemiologia , Linhagem , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Animais , Humanos , Incidência , Rim/anormalidades , Camundongos , Fenótipo , Prognóstico , Medição de Risco , Sensibilidade e Especificidade , Distribuição por Sexo , Sistema Urinário/anormalidades , Anormalidades Urogenitais/epidemiologia , Refluxo Vesicoureteral/epidemiologiaRESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/genética , Síndrome Nefrótica/genética , Podócitos/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Animais , Movimento Celular/genética , Células Cultivadas , Estudos de Coortes , Progressão da Doença , Drosophila melanogaster , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Programas de Rastreamento/métodos , Mutação de Sentido Incorreto , Síndrome Nefrótica/patologia , Linhagem , Proteínas de Ligação a Fosfato , Podócitos/patologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Sequenciamento do ExomaRESUMO
No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.
Assuntos
Resistência a Medicamentos/genética , Glucocorticoides/farmacologia , Síndrome Nefrótica/tratamento farmacológico , Mapas de Interação de Proteínas/genética , Proteína rhoA de Ligação ao GTP/genética , Adulto , Animais , Criança , Pré-Escolar , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/uso terapêutico , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Síndrome Nefrótica/genética , Linhagem , Podócitos , RNA Interferente Pequeno/metabolismo , Resultado do Tratamento , Sequenciamento do Exoma , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest.