ABSTRACT
The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.
ABSTRACT
PURPOSE: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations. METHODS: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts. RESULTS: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect. CONCLUSION: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.
Subject(s)
Leigh Disease , Moyamoya Disease , Stroke , Humans , Child , Moyamoya Disease/genetics , Leigh Disease/complications , Transcription Factors/genetics , Ubiquitin-Protein Ligases/genetics , Zinc , Genetic Predisposition to Disease , Adenosine Triphosphatases/geneticsABSTRACT
Recessive variants in NDUFAF3 are a known cause of complex I (CI)-related mitochondrial disorders (MDs). The seven patients reported to date exhibited severe neurologic symptoms and lactic acidosis, followed by a fatal course and death during infancy in most cases. We present a 10-year-old patient with a neurodevelopmental disorder, progressive exercise intolerance, dystonia, basal ganglia abnormalities, and elevated lactate concentration in blood. Trio-exome sequencing revealed compound-heterozygosity for a pathogenic splice-site and a likely pathogenic missense variant in NDUFAF3. Spectrophotometric analysis of fibroblast-derived mitochondria demonstrated a relatively mild reduction of CI activity. Complexome analyses revealed severely reduced NDUFAF3 as well as CI in patient fibroblasts. Accumulation of early sub-assemblies of the membrane arm of CI associated with mitochondrial complex I intermediate assembly (MCIA) complex was observed. The most striking additional findings were both the unusual occurrence of free monomeric CI holding MCIA and other assembly factors. Here we discuss our patient in context of genotype, phenotype and metabolite data from previously reported NDUFAF3 cases. With the atypical presentation of our patient, we provide further insight into the phenotypic spectrum of NDUFAF3-related MDs. Complexome analysis in our patient confirms the previously defined role of NDUFAF3 within CI biogenesis, yet adds new aspects regarding the correct timing of both the association of soluble and membrane arm modules and CI-maturation as well as respiratory supercomplex formation.
Subject(s)
Acidosis, Lactic , Mitochondrial Diseases , Humans , Child , Mitochondrial Diseases/genetics , Mitochondria/genetics , Mitochondria/metabolism , Exome Sequencing , Acidosis, Lactic/genetics , Phenotype , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolismABSTRACT
Congenital anomalies of the kidney and urinary tract (CAKUT) are the most prevalent cause of chronic kidney disease that manifests in children. To date ~23 different monogenic causes have been implicated in isolated forms of human CAKUT, but the vast majority remains elusive. In a previous study, we identified a homozygous missense variant in E26 transformation-specific (ETS) Variant Transcription Factor 4 (ETV4) causing CAKUT via dysregulation of the transcriptional function of ETV4, and a resulting abrogation of GDNF/RET/ETV4 signaling pathway. This CAKUT family remains the only family with an ETV4 variant reported so far. Here, we describe one additional CAKUT family with a homozygous truncating variant in ETV4 (p.(Lys6*)) that was identified by exome sequencing. The variant was found in an individual with isolated CAKUT displaying posterior urethral valves and renal dysplasia. The newly identified stop variant conceptually truncates the ETS_PEA3_N and ETS domains that regulate DNA-binding transcription factor activity. The variant has never been reported homozygously in the gnomAD database. To our knowledge, we here report the first CAKUT family with a truncating variant in ETV4, potentially causing the isolated CAKUT phenotype observed in the affected individual.
Subject(s)
Urinary Tract , Urogenital Abnormalities , Vesico-Ureteral Reflux , Child , Humans , Urogenital Abnormalities/genetics , Kidney/abnormalities , Urinary Tract/metabolism , Vesico-Ureteral Reflux/genetics , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolismABSTRACT
Background: Congenital anomalies of the kidneys and urinary tract (CAKUT) are the most common cause of chronic kidney disease among children and adults younger than 30 yr. In our previous study, whole-exome sequencing (WES) identified a known monogenic cause of isolated or syndromic CAKUT in 13% of families with CAKUT. However, WES has limitations and detection of copy number variations (CNV) is technically challenging, and CNVs causative of CAKUT have previously been detected in up to 16% of cases. Objective: To detect CNVs causing CAKUT in this WES cohort and increase the diagnostic yield. Design setting and participants: We performed a genome-wide single nucleotide polymorphism (SNP)-based CNV analysis on the same CAKUT cohort for whom WES was previously conducted. Outcome measurements and statistical analysis: We evaluated and classified the CNVs using previously published predefined criteria. Results and limitations: In a cohort of 170 CAKUT families, we detected a pathogenic CNV known to cause CAKUT in nine families (5.29%, 9/170). There were no competing variants on genome-wide CNV analysis or WES analysis. In addition, we identified novel likely pathogenic CNVs that may cause a CAKUT phenotype in three of the 170 families (1.76%). Conclusions: CNV analysis in this cohort of 170 CAKUT families previously examined via WES increased the rate of diagnosis of genetic causes of CAKUT from 13% on WES to 18% on WES + CNV analysis combined. We also identified three candidate loci that may potentially cause CAKUT. Patient summary: We conducted a genetics study on families with congenital anomalies of the kidney and urinary tract (CAKUT). We identified gene mutations that can explain CAKUT symptoms in 5.29% of the families, which increased the percentage of genetic causes of CAKUT to 18% from a previous study, so roughly one in five of our patients with CAKUT had a genetic cause. These analyses can help patients with CAKUT and their families in identifying a possible genetic cause.
ABSTRACT
Neurological symptoms are frequent and often a leading feature of childhood-onset mitochondrial disorders (MD) but the exact incidence of MD in unselected neuropediatric patients is unknown. Their early detection is desirable due to a potentially rapid clinical decline and the availability of management options. In 491 children with neurological symptoms, a comprehensive diagnostic work-up including exome sequencing was performed. The success rate in terms of a molecular genetic diagnosis within our cohort was 51%. Disease-causing variants in a mitochondria-associated gene were detected in 12% of solved cases. In order to facilitate the clinical identification of MDs within neuropediatric cohorts, we have created an easy-to-use bedside-tool, the MDC-NP. In our cohort, the MDC-NP predicted disease conditions related to MDs with a sensitivity of 0.83, and a specificity of 0.96.
Subject(s)
Genetic Predisposition to Disease , Mitochondrial Diseases/epidemiology , Mitochondrial Diseases/genetics , Nervous System Diseases/epidemiology , Nervous System Diseases/genetics , Age Factors , Alleles , Child , Cohort Studies , Genes, Mitochondrial , Genetic Association Studies , Genotype , Humans , Mitochondrial Diseases/diagnosis , Mutation , Nervous System Diseases/diagnosis , Phenotype , Prevalence , PrognosisABSTRACT
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.
Subject(s)
Anorectal Malformations/genetics , Esophageal Atresia/genetics , Genetic Predisposition to Disease , Heart Diseases/genetics , Tracheoesophageal Fistula/genetics , Anorectal Malformations/complications , Anorectal Malformations/pathology , Cytoskeletal Proteins/genetics , DNA-Binding Proteins/genetics , Esophageal Atresia/complications , Esophageal Atresia/pathology , Female , Genes, X-Linked/genetics , Genetic Association Studies , HSP90 Heat-Shock Proteins/genetics , Heart Diseases/complications , Heart Diseases/pathology , Hemizygote , Homeodomain Proteins/genetics , Humans , Kidney/abnormalities , Male , Receptors, Interleukin/genetics , Tracheoesophageal Fistula/complications , Tracheoesophageal Fistula/pathology , Transcription Factors/genetics , Exome SequencingABSTRACT
INTRODUCTION: Monogenic diseases play an important role in critically ill neonates and infants treated in the intensive care unit. This study aimed to determine the diagnostic yield of whole-exome sequencing (WES) for monogenic diseases and identify phenotypes more likely associated with a genetic etiology. METHODS: From March 2017 to 2020, a comprehensive diagnostic workup including WES in a single academic center was performed in 61 unrelated, critically ill neonates and infants with an unknown underlying disease within the first year of life. We conducted 59 trio-WES, 1 duo-WES, and 1 single-WES analyses. Symptoms were classified according to the Human Phenotype Ontology. RESULTS: The overall molecular genetic diagnostic rate within our cohort was 46% (28/61) and 50% (15/30) in the subgroup of preterm neonates. Identifying the genetic cause of disease facilitates individualized management in the majority of patients. A positive or negative predictive power of specific clinical features for a genetic diagnosis could not be observed. CONCLUSION: WES is a powerful noninvasive diagnostic tool in critically ill neonates and infants with a high diagnostic rate. We recommend initiating WES as early as possible due to the impact on management and family counseling. Recommendations regarding the clinical utility of WES in critically ill neonates and infants should not be based on the phenotype alone. Here, we present a clinical workflow for the application of WES for critically ill neonates and infants in an interdisciplinary setting.
Subject(s)
Critical Illness , Intensive Care Units , Genetic Testing , Humans , Infant , Phenotype , Exome SequencingABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , Hernia, Hiatal/genetics , Microcephaly/genetics , Mutation, Missense , Nephrosis/genetics , Transcription Factors/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Cell Line , Child, Preschool , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/deficiency , Female , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Gene Knockout Techniques , High-Throughput Nucleotide Sequencing , Humans , Infant , Infant, Newborn , Male , Models, Molecular , Nephrotic Syndrome/genetics , Podocytes/metabolism , Polymorphism, Single Nucleotide , Pronephros/embryology , Pronephros/metabolism , Protein Stability , Transcription Factors/chemistry , Transcription Factors/deficiency , Xenopus laevis/embryology , Xenopus laevis/genetics , Zinc Fingers/geneticsABSTRACT
Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.
Subject(s)
Electron Transport Complex I/metabolism , HSP40 Heat-Shock Proteins/genetics , Mutation , Optic Atrophy, Hereditary, Leber/genetics , Optic Atrophy, Hereditary, Leber/metabolism , Adolescent , Adult , Cell Line , Child, Preschool , Electron Transport Complex I/chemistry , Female , Gene Knockout Techniques , Genes, Recessive , HSP40 Heat-Shock Proteins/deficiency , HSP40 Heat-Shock Proteins/metabolism , Homozygote , Humans , Male , Middle Aged , Pedigree , Penetrance , Phenotype , Protein Subunits , Reactive Oxygen Species/metabolism , Young AdultABSTRACT
Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.
Subject(s)
DNA-Binding Proteins/genetics , Epigenesis, Genetic , Forkhead Transcription Factors/genetics , Mutation , Repressor Proteins/genetics , Transcription Factors/genetics , Urinary Tract/metabolism , Urogenital Abnormalities/genetics , Amphibian Proteins/antagonists & inhibitors , Amphibian Proteins/genetics , Amphibian Proteins/metabolism , Animals , Case-Control Studies , Child , Child, Preschool , DNA-Binding Proteins/metabolism , Family , Female , Forkhead Transcription Factors/metabolism , Heterozygote , Humans , Infant , Larva/genetics , Larva/growth & development , Larva/metabolism , Male , Mice , Mice, Knockout , Morpholinos/genetics , Morpholinos/metabolism , Pedigree , Protein Binding , Repressor Proteins/metabolism , Transcription Factors/metabolism , Urinary Tract/abnormalities , Urogenital Abnormalities/metabolism , Urogenital Abnormalities/pathology , Exome Sequencing , XenopusABSTRACT
Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first three decades of life, and in utero obstruction to urine flow is a frequent cause of secondary upper urinary tract malformations. Here, using whole-exome sequencing, we identified three different biallelic mutations in CHRNA3, which encodes the α3 subunit of the nicotinic acetylcholine receptor, in five affected individuals from three unrelated families with functional lower urinary tract obstruction and secondary CAKUT. Four individuals from two families have additional dysautonomic features, including impaired pupillary light reflexes. Functional studies in vitro demonstrated that the mutant nicotinic acetylcholine receptors were unable to generate current following stimulation with acetylcholine. Moreover, the truncating mutations p.Thr337Asnfs∗81 and p.Ser340∗ led to impaired plasma membrane localization of CHRNA3. Although the importance of acetylcholine signaling in normal bladder function has been recognized, we demonstrate for the first time that mutations in CHRNA3 can cause bladder dysfunction, urinary tract malformations, and dysautonomia. These data point to a pathophysiologic sequence by which monogenic mutations in genes that regulate bladder innervation may secondarily cause CAKUT.
Subject(s)
Autonomic Nervous System Diseases/etiology , Kidney/abnormalities , Mutation , Receptors, Nicotinic/genetics , Urinary Tract/abnormalities , Urogenital Abnormalities/etiology , Adult , Autonomic Nervous System Diseases/genetics , Autonomic Nervous System Diseases/pathology , Female , Follow-Up Studies , Humans , Kidney/pathology , Male , Pedigree , Prognosis , Urinary Tract/pathology , Urogenital Abnormalities/genetics , Urogenital Abnormalities/pathology , Young AdultABSTRACT
Approximately 500 monogenic causes of chronic kidney disease (CKD) have been identified, mainly in pediatric populations. The frequency of monogenic causes among adults with CKD has been less extensively studied. To determine the likelihood of detecting monogenic causes of CKD in adults presenting to nephrology services in Ireland, we conducted whole exome sequencing (WES) in a multi-centre cohort of 114 families including 138 affected individuals with CKD. Affected adults were recruited from 78 families with a positive family history, 16 families with extra-renal features, and 20 families with neither a family history nor extra-renal features. We detected a pathogenic mutation in a known CKD gene in 42 of 114 families (37%). A monogenic cause was identified in 36% of affected families with a positive family history of CKD, 69% of those with extra-renal features, and only 15% of those without a family history or extra-renal features. There was no difference in the rate of genetic diagnosis in individuals with childhood versus adult onset CKD. Among the 42 families in whom a monogenic cause was identified, WES confirmed the clinical diagnosis in 17 (40%), corrected the clinical diagnosis in 9 (22%), and established a diagnosis for the first time in 16 families referred with CKD of unknown etiology (38%). In this multi-centre study of adults with CKD, a molecular genetic diagnosis was established in over one-third of families. In the evolving era of precision medicine, WES may be an important tool to identify the cause of CKD in adults.
Subject(s)
Exome Sequencing , Genetic Predisposition to Disease , Genetic Testing/methods , Renal Insufficiency, Chronic/genetics , Adolescent , Adult , Age of Onset , Aged , Aged, 80 and over , Cohort Studies , Exome/genetics , Female , Humans , Ireland , Kidney , Male , Medical History Taking , Middle Aged , Mutation , Pedigree , Precision Medicine , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/therapy , Young AdultABSTRACT
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.
Subject(s)
Exome Sequencing/methods , Kidney Transplantation/methods , Precision Medicine/methods , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/surgery , Adolescent , Boston , Child , Child, Preschool , Cohort Studies , Female , Genetic Predisposition to Disease/epidemiology , Genetic Testing/methods , Graft Rejection , Graft Survival , Hospitals, Pediatric , Humans , Kidney Transplantation/adverse effects , Male , Prognosis , Renal Insufficiency, Chronic/physiopathology , Retrospective Studies , Risk Assessment , Severity of Illness Index , Survival Analysis , Transplant Recipients/statistics & numerical data , Treatment OutcomeABSTRACT
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.
Subject(s)
Exome Sequencing/methods , Genetic Markers , Mutation , Nephritis/diagnosis , Nephritis/genetics , Nephrosis/diagnosis , Nephrosis/genetics , Adolescent , Atypical Hemolytic Uremic Syndrome/diagnosis , Atypical Hemolytic Uremic Syndrome/genetics , Child , Child, Preschool , Cohort Studies , DNA Mutational Analysis , Diagnosis, Differential , Female , Humans , Infant , Infant, Newborn , Male , Nephritis, Hereditary/diagnosis , Nephritis, Hereditary/genetics , Nephrotic Syndrome/diagnosis , Nephrotic Syndrome/genetics , PrognosisABSTRACT
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.
Subject(s)
Exome Sequencing/methods , Genetic Predisposition to Disease/epidemiology , Pedigree , Urogenital Abnormalities/genetics , Vesico-Ureteral Reflux/genetics , Animals , Humans , Incidence , Kidney/abnormalities , Mice , Phenotype , Prognosis , Risk Assessment , Sensitivity and Specificity , Sex Distribution , Urinary Tract/abnormalities , Urogenital Abnormalities/epidemiology , Vesico-Ureteral Reflux/epidemiologyABSTRACT
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.
Subject(s)
Gene Expression Regulation , Membrane Proteins/genetics , Nephrotic Syndrome/genetics , Podocytes/metabolism , rab5 GTP-Binding Proteins/genetics , Animals , Cell Movement/genetics , Cells, Cultured , Cohort Studies , Disease Progression , Drosophila melanogaster , Female , Genetic Predisposition to Disease , Humans , Male , Mass Screening/methods , Mutation, Missense , Nephrotic Syndrome/pathology , Pedigree , Phosphate-Binding Proteins , Podocytes/pathology , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction/methods , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/pathology , Exome SequencingABSTRACT
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.
Subject(s)
Drug Resistance/genetics , Glucocorticoids/pharmacology , Nephrotic Syndrome/drug therapy , Protein Interaction Maps/genetics , rhoA GTP-Binding Protein/genetics , Adult , Animals , Child , Child, Preschool , DNA Mutational Analysis , Disease Models, Animal , Female , Gene Knockdown Techniques , Glucocorticoids/therapeutic use , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Infant , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Mutation , Nephrotic Syndrome/genetics , Pedigree , Podocytes , RNA, Small Interfering/metabolism , Treatment Outcome , Exome Sequencing , rhoA GTP-Binding Protein/metabolismABSTRACT
Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause (40-50%) of chronic kidney disease (CKD) in children. About 40 monogenic causes of CAKUT have so far been discovered. To date less than 20% of CAKUT cases can be explained by mutations in these 40 genes. To identify additional monogenic causes of CAKUT, we performed whole exome sequencing (WES) and homozygosity mapping (HM) in a patient with CAKUT from Indian origin and consanguineous descent. We identified a homozygous missense mutation (c.1336C>T, p.Arg446Cys) in the gene Von Willebrand factor A domain containing 2 (VWA2). With immunohistochemistry studies on kidneys of newborn (P1) mice, we show that Vwa2 and Fraser extracellular matrix complex subunit 1 (Fras1) co-localize in the nephrogenic zone of the renal cortex. We identified a pronounced expression of Vwa2 in the basement membrane of the ureteric bud (UB) and derivatives of the metanephric mesenchyme (MM). By applying in vitro assays, we demonstrate that the Arg446Cys mutation decreases translocation of monomeric VWA2 protein and increases translocation of aggregated VWA2 protein into the extracellular space. This is potentially due to the additional, unpaired cysteine residue in the mutated protein that is used for intermolecular disulfide bond formation. VWA2 is a known, direct interactor of FRAS1 of the Fraser-Complex (FC). FC-encoding genes and interacting proteins have previously been implicated in the pathogenesis of syndromic and/or isolated CAKUT phenotypes in humans. VWA2 therefore constitutes a very strong candidate in the search for novel CAKUT-causing genes. Our results from in vitro experiments indicate a dose-dependent neomorphic effect of the Arg446Cys homozygous mutation in VWA2.
Subject(s)
Biomarkers, Tumor/genetics , Fraser Syndrome/genetics , Mutation, Missense , Urogenital Abnormalities/genetics , Vesico-Ureteral Reflux/genetics , Amino Acid Sequence , Amino Acid Substitution , Animals , Animals, Newborn , Biomarkers, Tumor/chemistry , Calcium-Binding Proteins , Child , Consanguinity , Conserved Sequence , Exons , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental , Homozygote , Humans , Male , Mice , Models, Animal , Models, Molecular , Pedigree , Sequence Homology, Amino Acid , Urogenital System/growth & development , Urogenital System/metabolismABSTRACT
Congenital anomalies of the kidneys and urinary tract (CAKUT) comprise a large spectrum of congenital malformations ranging from severe manifestations, such as renal agenesis, to potentially milder conditions, such as vesicoureteral reflux. CAKUT causes approximately 40% of ESRD that manifests within the first three decades of life. Several lines of evidence indicate that CAKUT is often caused by recessive or dominant mutations in single (monogenic) genes. To date, approximately 40 monogenic genes are known to cause CAKUT if mutated, explaining 5%-20% of patients. However, hundreds of different monogenic CAKUT genes probably exist. The discovery of novel CAKUT-causing genes remains challenging because of this pronounced heterogeneity, variable expressivity, and incomplete penetrance. We here give an overview of known genetic causes for human CAKUT and shed light on distinct renal morphogenetic pathways that were identified as relevant for CAKUT in mice and humans.