Uncovering risk factors for kidney injury in children with a solitary functioning kidney.

Children with a solitary functioning kidney (SFK) have an increased risk of kidney injury. The exact risk of and risk factors for kidney injury remain unknown, which impedes personalized care. Here, we recruited a nationwide multicenter cohort of 944 patients with SFK to get more insight into this by consenting patients born in 1993-2020 and diagnosed with congenital or acquired SFK before adulthood. The median follow-up was 12.8 years and four indications of kidney injury were studied: urine protein-creatinine ratios, blood pressure, estimated glomerular filtration rate and use of anti-hypertensive/proteinuric medication. For each indicator except medication use, separate cut-off values for any injury and severe injury were used. Survival analyses indicated that at 18 years of age, any or severe kidney injury were present in 75% and 39% of patients with congenital SFK, respectively. Risk factors for kidney injury included kidney agenesis as cause of the SFK, anomalies in the SFK, and high body mass index at last follow-up. Kidney agenesis and being overweight were specifically associated with proteinuria and high blood pressure, whereas anomalies in the SFK were associated with reduced estimated glomerular filtration rates. The high prevalence of kidney injury in patients with SFK emphasizes the need for long-term follow-up, in which lifestyle is an important topic to address. More research into the etiological role of risk factors will help to translate our findings into individualized care strategies. Thus, our study shows that a significant proportion of children with SFK will develop kidney injury over time.

A Genome-Wide Association Study into the Aetiology of Congenital Solitary Functioning Kidney.

Congenital solitary functioning kidney (CSFK) is a birth defect that occurs in 1:1500 children and predisposes them to kidney injury. Its aetiology is likely multifactorial. In addition to known monogenic causes and environmental risk factors, common genetic variation may contribute to susceptibility to CSFK. We performed a genome-wide association study among 452 patients with CSFK and two control groups of 669 healthy children and 5363 unaffected adults. Variants in two loci reached the genome-wide significance threshold of 5 × 10-8, and variants in 30 loci reached the suggestive significance threshold of 1 × 10-5. Of these, an identified locus with lead single nucleotide variant (SNV) rs140804918 (odds ratio 3.1, p-value = 1.4 × 10-8) on chromosome 7 was most promising due to its close proximity to HGF, a gene known to be involved in kidney development. Based on their known molecular functions, both KCTD20 and STK38 could explain the suggestive significant association with lead SNV rs148413365 on chromosome 6. Our findings need replication in an independent cohort of CSFK patients before they can be established definitively. However, our analysis suggests that common variants play a role in CSFK aetiology. Future research could enhance our understanding of the molecular mechanisms involved.

mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

CDH12 as a Candidate Gene for Kidney Injury in Posterior Urethral Valve Cases: A Genome-wide Association Study Among Patients with Obstructive Uropathies.

BACKGROUND: Posterior urethral valves (PUVs) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair kidney development. OBJECTIVE: To identify genetic variants associated with kidney injury in patients with obstructive uropathy. DESIGN SETTING AND PARTICIPANTS: We included 487 patients born in 1981 or later who underwent pyeloplasty or valve resection before 18 yr of age in the discovery phase, 102 PUV patients in a first replication phase, and 102 in a second replication phase. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Signs of kidney injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m2, high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning at <45%. We used χ2 tests to calculate p values and odds ratios for >600 000 single-nucleotide polymorphisms (SNPs) in the discovery sample comparing patients with and without signs of kidney injury within 5 yr after surgery. We performed stratified analyses for PUV and UPJO and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples for the associated SNPs, and RNA and protein expression analyses for the associated gene in fetal tissues. RESULTS AND LIMITATIONS: Despite the small and nonhomogeneous sample, we observed suggestive associations for six SNPs in three loci, of which rs6874819 in the CDH12 gene was the most clear (p = 7.5 × 10-7). This SNP also seemed to be associated with time to kidney injury in the PUV discovery and replication samples. RNA expression analyses showed clear CDH12 expression in fetal kidneys, which was confirmed by protein immunolocalization. CONCLUSIONS: This study identified CDH12 as a candidate gene for kidney injury in PUV. PATIENT SUMMARY: We found that variants of the CDH12 gene increase the risk of kidney injury in patients with extra flaps of tissue in the urethra (posterior urethral valves). This is the first report on this gene in this context. Our study provides interesting new information about the pathways involved and important leads for further research for this condition.

Maternal risk associated with the VACTERL association: A case-control study.

BACKGROUND: The VACTERL association (VACTERL) includes at least three of these congenital anomalies: vertebral, anal, cardiac, trachea-esophageal, renal, and limb anomalies. Assisted reproductive techniques (ART), pregestational diabetes mellitus, and chronic lower obstructive pulmonary disorders (CLOPD) have been associated with VACTERL. We aimed to replicate these findings and were interested in additional maternal risk factors. METHODS: A case-control study using self-administered questionnaires was performed including 142 VACTERL cases and 2,135 population-based healthy controls. Multivariable logistic regression analyses were performed to estimate confounder adjusted odds ratios (aOR) and 95% confidence intervals (95%CI). RESULTS: Parents who used invasive ART had an increased risk of VACTERL in offspring (aOR 4.4 [95%CI 2.1-8.8]), whereas the increased risk for mothers with CLOPD could not be replicated. None of the case mothers had pregestational diabetes mellitus. Primiparity (1.5 [1.1-2.1]) and maternal pregestational overweight and obesity (1.8 [1.2-2.8] and 1.8 [1.0-3.4]) were associated with VACTERL. Consistent folic acid supplement use during the advised periconceptional period may reduce the risk of VACTERL (0.5 [0.3-1.0]). Maternal smoking resulted in an almost twofold increased risk of VACTERL. CONCLUSION: We identified invasive ART, primiparity, pregestational overweight and obesity, lack of folic acid supplement use, and smoking as risk factors for VACTERL.

Clinical Integration of Genome Diagnostics for Congenital Anomalies of the Kidney and Urinary Tract.

Revolutions in genetics, epigenetics, and bioinformatics are currently changing the outline of diagnostics and clinical medicine. From a nephrologist's perspective, individuals with congenital anomalies of the kidney and urinary tract (CAKUT) are an important patient category: not only is CAKUT the predominant cause of kidney failure in children and young adults, but the strong phenotypic and genotypic heterogeneity of kidney and urinary tract malformations has hampered standardization of clinical decision making until now. However, patients with CAKUT may benefit from precision medicine, including an integrated diagnostics trajectory, genetic counseling, and personalized management to improve clinical outcomes of developmental kidney and urinary tract defects. In this review, we discuss the present understanding of the molecular etiology of CAKUT and the currently available genome diagnostic modalities in the clinical care of patients with CAKUT. Finally, we discuss how clinical integration of findings from large-scale genetic, epigenetic, and gene-environment interaction studies may improve the prognosis of all individuals with CAKUT.

Impact of next generation sequencing on our understanding of CAKUT.

Congenital abnormalities of the kidney and urinary tract (CAKUT) form the leading cause of pediatric end-stage renal disease. Knowledge on the molecular mechanisms that underlie CAKUT leads to the improvement of DNA diagnostics and counseling regarding prognosis and recurrence risk estimation for CAKUT patients and their relatives. Implementation of next generation sequencing in research and diagnostic settings has led to the identification of the molecular basis of many developmental diseases. In this review, we summarize the efforts on next generation sequencing in CAKUT research and we discuss how next generation sequencing added to our understanding of CAKUT genetics. Although next generation sequencing has certainly proven to be a game changer in the field of disease gene identification and novel CAKUT-causing gene variants have been identified, most CAKUT cases still remain unsolved. Occurring with genetic and phenotypic heterogeneity along with incomplete penetrance, the identification of CAKUT etiology poses many challenges. We see great potential for combined -omics approaches that include next generation sequencing in the identification of CAKUT-specific biomarkers, which is necessary to optimize the care for CAKUT patients.

Changes in the urinary extracellular vesicle proteome are associated with nephronophthisis-related ciliopathies.

Nephronophthisis is one of the leading genetic causes of end-stage renal disease in childhood. Early diagnostics and prognostics for nephronophthisis are currently limited. We aimed to identify non-invasive protein biomarkers for nephronophthisis in urinary extracellular vesicles. Extracellular vesicles were isolated from urine of 12 patients with a nephronophthisis-related ciliopathy and 12 age- and gender-matched controls, followed by in-depth label-free LC-MS/MS proteomics analysis of gel fractionated extracellular vesicle proteins. Supervised cluster analysis of proteomic profiles separated patients from controls. We identified 156 differentially expressed proteins with fold change ≥4 in patients compared to controls (P < .05). Importantly, expression levels of discriminating proteins were correlated with chronic kidney disease stage, suggesting possible applications for urinary extracellular vesicle biomarkers in prognostics for nephronophthisis. Enrichment analysis of gene ontology terms revealed GO terms including signaling, actin cytoskeleton and endocytosis among the downregulated proteins in patients, whereas terms related to response to wounding and extracellular matrix organization were enriched among upregulated proteins. Our findings represent the first step towards a non-invasive diagnostic test for nephronophthisis. Further research is needed to determine specificity of the candidate biomarkers. In conclusion, proteomic profiles of urinary extracellular vesicles differentiate nephronophthisis-related ciliopathy patients from healthy controls. SIGNIFICANCE: Nephronophthisis is an important cause of end-stage renal disease in children and is associated with an average diagnostic delay of 3.5 years. This is the first study investigating candidate biomarkers for nephronophthisis using global proteomics analysis of urinary extracellular vesicles in patients with nephronophthisis compared to control individuals. We show that measuring protein markers in urinary extracellular vesicles is a promising approach for non-invasive early diagnostics of nephronophthisis.

PDE3A gene screening improves diagnostics for patients with Bilginturan syndrome (hypertension and brachydactyly syndrome).

Autosomal-dominant hypertension and brachydactyly syndrome (HTNB; Bilginturan syndrome) is known to cause stroke before age 50 when untreated. We report a novel PDE3A gene mutation in a mother and daughter affected with dominant brachydactyly of the hands and feet, a short stature, and hypertension. The hypertension was medically responsive to anti-hypertensive treatment. The 3-bp deletion in the PDE3A gene presented de novo in the mother. Here, we expand the list of PDE3A mutations identified in Bilginturan syndrome and emphasize the importance of standardized genetic testing of HTNB patients to improve diagnostics at an early age. We recommend extended phenotyping in patients with brachydactyly, a short stature or hypertension in clinical practice.

Clinical and genetic analyses of a Dutch cohort of 40 patients with a nephronophthisis-related ciliopathy.

BACKGROUND: Nephronophthisis is an autosomal recessive ciliopathy and important cause of end-stage renal disease (ESRD) in children and young adults. Diagnostic delay is frequent. This study investigates clinical characteristics, initial symptoms, and genetic defects in a cohort with nephronophthisis-related ciliopathy, to improve early detection and genetic counseling. METHODS: Forty patients from 36 families with nephronophthisis-related ciliopathy were recruited at university medical centers and online. Comprehensive clinical and genotypic data were recorded. Patients without molecular diagnosis were offered genetic analysis. RESULTS: Of 40 patients, 45% had isolated nephronophthisis, 48% syndromic diagnosis, and 7% nephronophthisis with extrarenal features not constituting a recognizable syndrome. Patients developed ESRD at median 13 years (range 5-47). Median age of symptom onset was 9 years in both isolated and syndromic forms (range 5-26 vs. 5-33). Common presenting symptoms were fatigue (42%), polydipsia/polyuria (33%), and hypertension (21%). Renal ultrasound showed small-to-normal-sized kidneys, increased echogenicity (65%), cysts (43%), and abnormal corticomedullary differentiation (32%). Renal biopsies in eight patients showed nonspecific signs of chronic kidney disease (CKD). Twenty-three patients (58%) had genetic diagnosis upon inclusion. Thirteen of those without a genetic diagnosis gave consent for genetic testing, and a cause was identified in five (38%). CONCLUSIONS: Nephronophthisis is genetically and phenotypically heterogeneous and should be considered in children and young adults presenting with persistent fatigue and polyuria, and in all patients with unexplained CKD. As symptom onset can occur into adulthood, presymptomatic monitoring of kidney function in syndromic ciliopathy patients should continue until at least age 30.

The KOUNCIL Consortium: From Genetic Defects to Therapeutic Development for Nephronophthisis.

Nephronophthisis (NPH) is the most common monogenic cause of renal failure in children. Treatment options are limited to dialysis and transplantation. Therapeutics to significantly delay or prevent end-stage renal disease (ESRD) in children are currently not available. In the Dutch-Anglo KOUNCIL (Kidney-Oriented UNderstanding of correcting CILiopathies) consortium, several groups and specialties united to perform scientific groundwork with the aim to develop genetic and therapeutic personalized care for NPH patients. At the start of this consortium, a genetic diagnosis for NPH was available for only 30-40% of patients, which improved to 50-60% during the course of the 4-year KOUNCIL project. Other major accomplishments of the consortium were (1) the establishment of a Dutch renal ciliopathy patient database with genotype and phenotype data; (2) composition of a proteomics-based integrated network of protein modules disrupted in NPH; (3) the development of non-invasive, urine-based assays that allow functional assessment of genomic variants in NPH and of therapeutic efficiency of drugs; and (4) chemical screening toward the identification of compounds that delay or prevent disease progression in NPH, which resulted in four potential medical interventions for NPH. In conclusion, the KOUNCIL consortium effectively channeled complementary approaches to broaden our understanding of NPH pathogenesis, resulted in 54 publications, improvement of genome diagnostics for NPH patients, awareness in the nephrology and clinical genetics communities for NPH, and new avenues for patient management.

Sox11 gene disruption causes congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital abnormalities of the kidney and the urinary tract (CAKUT) belong to the most common birth defects in human, but the molecular basis for the majority of CAKUT patients remains unknown. Here we show that the transcription factor SOX11 is a crucial regulator of kidney development. SOX11 is expressed in both mesenchymal and epithelial components of the early kidney anlagen. Deletion of Sox11 in mice causes an extension of the domain expressing Gdnf within rostral regions of the nephrogenic cord and results in duplex kidney formation. On the molecular level SOX11 directly binds and regulates a locus control region of the protocadherin B cluster. At later stages of kidney development, SOX11 becomes restricted to the intermediate segment of the developing nephron where it is required for the elongation of Henle's loop. Finally, mutation analysis in a cohort of patients suffering from CAKUT identified a series of rare SOX11 variants, one of which interferes with the transactivation capacity of the SOX11 protein. Taken together these data demonstrate a key role for SOX11 in normal kidney development and may suggest that variants in this gene predispose to CAKUT in humans.

The expanding phenotypic spectra of kidney diseases: insights from genetic studies.

Next-generation sequencing (NGS) has led to the identification of previously unrecognized phenotypes associated with classic kidney disease genes. In addition to improving diagnostics for genetically heterogeneous diseases and enabling a faster rate of gene discovery, NGS has enabled an expansion and redefinition of nephrogenetic disease categories. Findings from these studies raise the question of whether disease diagnoses should be made on clinical grounds, on genetic evidence or a combination thereof. Here, we discuss the major kidney disease-associated genes and gene categories for which NGS has expanded the phenotypic spectrum. For example, COL4A3-5 genes, which are classically associated with Alport syndrome, are now understood to also be involved in the aetiology of focal segmental glomerulosclerosis. DGKE, which is associated with nephrotic syndrome, is also mutated in patients with atypical haemolytic uraemic syndrome. We examine how a shared genetic background between diverse clinical phenotypes can provide insight into the function of genes and novel links with essential pathophysiological mechanisms. In addition, we consider genetic and epigenetic factors that contribute to the observed phenotypic heterogeneity of kidney diseases and discuss the challenges in the interpretation of genetic data. Finally, we discuss the implications of the expanding phenotypic spectra associated with kidney disease genes for clinical practice, genetic counselling and personalized care, and present our recommendations for the use of NGS-based tests in routine nephrology practice.

AGORA, a data- and biobank for birth defects and childhood cancer.

BACKGROUND: Research regarding the etiology of birth defects and childhood cancer is essential to develop preventive measures, but often requires large study populations. Therefore, we established the AGORA data- and biobank in the Netherlands. In this study, we describe its rationale, design, and ongoing data collection. METHODS: Children diagnosed with and/or treated for a structural birth defect or childhood cancer and their parents are invited to participate in the AGORA data- and biobank. Controls are recruited through random sampling from municipal registries. The parents receive questionnaires about demographics, family and pregnancy history, health status, prescribed medication, lifestyle, and occupational exposures before and during the index pregnancy. In addition, blood or saliva is collected from children and parents, while medical records are reviewed for diagnostic information. RESULTS: So far, we have collected data from over 6,860 families (3,747 birth defects, 905 childhood cancers, and 2,208 controls). The types of birth defects vary widely and comprise malformations of the digestive, respiratory, and urogenital tracts as well as facial, cardiovascular, kidney, skeletal, and central nervous system anomalies. The most frequently occurring childhood cancer types are acute lymphatic leukemia, Hodgkin and non-Hodgkin lymphoma, Wilms' tumor, and brain and spinal cord tumors. Our genetic and/or epidemiologic studies have been focused on hypospadias, anorectal malformations, congenital anomalies of the kidney and urinary tract (CAKUT), and orofacial clefts. CONCLUSION: The large AGORA data- and biobank offers great opportunities for investigating genetic and nongenetic risk factors for disorders in children and is open to collaborative initiatives. Birth Defects Research (Part A) 106:675-684, 2016. © 2016 Wiley Periodicals, Inc.

Maternal risk factors involved in specific congenital anomalies of the kidney and urinary tract: A case-control study.

BACKGROUND: Congenital anomalies of the kidney and urinary tract (CAKUT) comprise a heterogeneous group of birth defects with a variety of genetic and nongenetic factors suspected of involvement in the etiology. However, little is known about risk factors in specific CAKUT phenotypes. Therefore, we studied potential maternal risk factors in individual phenotypes within the CAKUT spectrum. METHODS: Questionnaire data were collected from parents of 562 children with CAKUT and 2139 healthy controls within the AGORA data- and biobank. Potential maternal risk factors investigated included folic acid use, overweight and obesity, smoking, alcohol consumption, subfertility, and diabetes mellitus. We performed logistic regression analyses to assess associations between these potential risk factors and CAKUT phenotypes. RESULTS: Increased risks of CAKUT were observed for folic acid use and maternal obesity, while fertility treatment by in vitro fertilization or intrauterine insemination and diabetes diagnosed during pregnancy also seem to be associated with CAKUT. Use of multivitamins reduced the risk (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.2-1.0) as opposed to use of folic acid supplements only (OR, 1.3; 95% CI, 1.0-1.8). Folic acid use was associated with duplex collecting systems (OR, 1.8; 95% CI, 1.0-3.4) and vesicoureteral reflux (OR, 1.8; 95% CI, 1.1-2.9) in particular. A relatively strong association was observed between diabetes during pregnancy and posterior urethral valves (OR, 2.6; 95% CI, 1.1-5.9). CONCLUSION: Use of folic acid only seems to be counterproductive for prevention of CAKUT, in contrast to multivitamin use. Furthermore, we observed differences in risk factor patterns among CAKUT phenotypes, which stress the importance of separate analyses for each phenotype. Birth Defects Research (Part A) 106:596-603, 2016. © 2016 Wiley Periodicals, Inc.

De novo 14q24.2q24.3 microdeletion including IFT43 is associated with intellectual disability, skeletal anomalies, cardiac anomalies, and myopia.

We report an 11-year-old girl with mild intellectual disability, skeletal anomalies, congenital heart defect, myopia, and facial dysmorphisms including an extra incisor, cup-shaped ears, and a preauricular skin tag. Array comparative genomic hybridization analysis identified a de novo 4.5-Mb microdeletion on chromosome 14q24.2q24.3. The deleted region and phenotype partially overlap with previously reported patients. Here, we provide an overview of the literature on 14q24 microdeletions and further delineate the associated phenotype. We performed exome sequencing to examine other causes for the phenotype and queried genes present in the 14q24.2q24.3 microdeletion that are associated with recessive disease for variants in the non-deleted allele. The deleted region contains 65 protein-coding genes, including the ciliary gene IFT43. Although Sanger and exome sequencing did not identify variants in the second IFT43 allele or in other IFT complex A-protein-encoding genes, immunocytochemistry showed increased accumulation of IFT-B proteins at the ciliary tip in patient-derived fibroblasts compared to control cells, demonstrating defective retrograde ciliary transport. This could suggest a ciliary defect in the pathogenesis of this disorder. © 2016 Wiley Periodicals, Inc.

Wnt5a Deficiency Leads to Anomalies in Ureteric Tree Development, Tubular Epithelial Cell Organization and Basement Membrane Integrity Pointing to a Role in Kidney Collecting Duct Patterning.

The Wnts can be considered as candidates for the Congenital Anomaly of Kidney and Urinary Tract, CAKUT diseases since they take part in the control of kidney organogenesis. Of them Wnt5a is expressed in ureteric bud (UB) and its deficiency leads to duplex collecting system (13/90) uni- or bilateral kidney agenesis (10/90), hypoplasia with altered pattern of ureteric tree organization (42/90) and lobularization defects with partly fused ureter trunks (25/90) unlike in controls. The UB had also notably less tips due to Wnt5a deficiency being at E15.5 306 and at E16.5 765 corresponding to 428 and 1022 in control (p<0.02; p<0.03) respectively. These changes due to Wnt5a knock out associated with anomalies in the ultrastructure of the UB daughter epithelial cells. The basement membrane (BM) was malformed so that the BM thickness increased from 46.3 nm to 71.2 nm (p<0.01) at E16.5 in the Wnt5a knock out when compared to control. Expression of a panel of BM components such as laminin and of type IV collagen was also reduced due to the Wnt5a knock out. The P4ha1 gene that encodes a catalytic subunit of collagen prolyl 4-hydroxylase I (C-P4H-I) in collagen synthesis expression and the overall C-P4H enzyme activity were elevated by around 26% due to impairment in Wnt5a function from control. The compound Wnt5a+/-;P4ha1+/- embryos demonstrated Wnt5a-/- related defects, for example local hyperplasia in the UB tree. A R260H WNT5A variant was identified from renal human disease cohort. Functional studies of the consequence of the corresponding mouse variant in comparison to normal ligand reduced Wnt5a-signalling in vitro. Together Wnt5a has a novel function in kidney organogenesis by contributing to patterning of UB derived collecting duct development contributing putatively to congenital disease.

Prioritization and burden analysis of rare variants in 208 candidate genes suggest they do not play a major role in CAKUT.

The leading cause of end-stage renal disease in children is attributed to congenital anomalies of the kidney and urinary tract (CAKUT). Familial clustering and mouse models support the presence of monogenic causes. Genetic testing is insufficient as it mainly focuses on HNF1B and PAX2 mutations that are thought to explain CAKUT in 5­15% of patients. To identify novel, potentially pathogenic variants in additional genes, we designed a panel of genes identified from studies on familial forms of isolated or syndromic CAKUT and genes suggested by in vitro and in vivo CAKUT models. The coding exons of 208 genes were analyzed in 453 patients with CAKUT using next-generation sequencing. Rare truncating, splice-site variants, and non-synonymous variants, predicted to be deleterious and conserved, were prioritized as the most promising variants to have an effect on CAKUT. Previously reported disease-causing mutations were detected, but only five were fully penetrant causal mutations that improved diagnosis. We prioritized 148 candidate variants in 151 patients, found in 82 genes, for follow-up studies. Using a burden test, no significant excess of rare variants in any of the genes in our cohort compared with controls was found. Thus, in a study representing the largest set of genes analyzed in CAKUT patients to date, the contribution of previously implicated genes to CAKUT risk was significantly smaller than expected, and the disease may be more complex than previously assumed.