Human Congenital Anomalies Of The Kidney And Urinary Tract Research Articles

Heterozygous variants in the teashirt zinc finger homeobox 3 (TSHZ3) gene in human congenital anomalies of the kidney and urinary tract.

Around 180 genes have been associated with congenital anomalies of the kidney and urinary tract (CAKUT) in mice, and represent promising novel candidate genes for human CAKUT. In whole-exome sequencing data of two siblings with genetically unresolved multicystic dysplastic kidneys (MCDK), prioritizing variants in murine CAKUT-associated genes yielded a rare variant in the teashirt zinc finger homeobox 3 (TSHZ3) gene. Therefore, the role of TSHZ3 in human CAKUT was assessed. Twelve CAKUT patients from 9/301 (3%) families carried five different rare heterozygous TSHZ3 missense variants predicted to be deleterious. CAKUT patients with versus without TSHZ3 variants were more likely to present with hydronephrosis, hydroureter, ureteropelvic junction obstruction, MCDK, and with genital anomalies, developmental delay, overlapping with the previously described phenotypes in Tshz3-mutant mice and patients with heterozygous 19q12-q13.11 deletions encompassing the TSHZ3 locus. Comparable with Tshz3-mutant mice, the smooth muscle layer was disorganized in the renal pelvis and thinner in the proximal ureter of the nephrectomy specimen of a TSHZ3 variant carrier compared to controls. TSHZ3 was expressed in the human fetal kidney, and strongly at embryonic day 11.5-14.5 in mesenchymal compartments of the murine ureter, kidney, and bladder. TSHZ3 variants in a 5' region were more frequent in CAKUT patients than in gnomAD samples (p < 0.001). Mutant TSHZ3 harboring N-terminal variants showed significantly altered SOX9 and/or myocardin binding, possibly adversely affecting smooth muscle differentiation. Our results provide evidence that heterozygous TSHZ3 variants are associated with human CAKUT, particularly MCDK, hydronephrosis, and hydroureter, and, inconsistently, with specific extrarenal features, including genital anomalies.

Expression of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) Candidate Genes EDA2R, PCDH9, and TRAF7 in Normal Human Kidney Development and CAKUT.

Approximately half of the cases of chronic kidney disease (CKD) in childhood are caused by congenital anomalies of the kidney and urinary tract (CAKUT). Specific genes were identified as having significant importance in regard to the underlying genetic factors responsible for the CAKUT phenotype, and in our research, we focused on analyzing and comparing the expression levels of ectodysplasin A2 receptor (EDA2R), protocadherin9 (PCDH9), and TNF receptor-associated factor 7 (TRAF7) proteins in the cortex and medulla of healthy control kidneys during developmental phases 2, 3, and 4. We also performed an analysis of the area percentages of the mentioned proteins in the cortical and medullary sections of healthy embryonic and fetal kidneys compared to those affected by CAKUT, including duplex kidneys (DK), horseshoe kidneys (HK), hypoplastic kidneys (HYP), and dysplastic kidneys (DYS). We found that the CAKUT candidate gene proteins EDA2R, PCDH9, and TRAF7 are all expressed during normal human kidney development stages. In DYS, the expression of EDA2R was higher than in normal kidneys, likely due to EDA2R's role in apoptosis, which was upregulated in specific cases and could possibly contribute to the formation of DYS. The expression of PCDH9 was lower in HK, which can be attributed to the possible role of PCDH9 in cell migration suppression. Decreased PCDH9 expression is linked to increased cell migration, potentially contributing to the development of HK. The level of TRAF7 expression was reduced in all examined kidney disorders compared to normal kidneys, suggesting that this reduction might be attributed to the crucial role of TRAF7 in the formation of endothelium and ciliogenesis, both of which are essential for normal kidney development. Further research is required to ascertain the function of these proteins in both the typical development of the kidney and in CAKUT.

Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT)

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knock-out of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift towards a stromal cell identity. Histology of Foxd2 knock-out mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Prioritization of Monogenic Congenital Anomalies of the Kidney and Urinary Tract Candidate Genes with Existing Single-Cell Transcriptomics Data of the Human Fetal Kidney

Introduction: Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease in the first 3 decades of life. Over 40 genes have been identified as causative for isolated human CAKUT. However, many genes remain unknown, and the prioritization of potential CAKUT candidate genes is challenging. To develop an independent approach to prioritize CAKUT candidate genes, we hypothesized that monogenic CAKUT genes are most likely co-expressed along a temporal axis during kidney development and that genes with coinciding high expression may represent strong novel CAKUT candidate genes. Methods: We analyzed single-cell mRNA (sc-mRNA) transcriptomics data of human fetal kidney for temporal sc-mRNA co-expression of 40 known CAKUT genes. A maximum of high expression in consecutive timepoints of kidney development was found for four of the 40 genes (EYA1, SIX1, SIX2, and ITGA8) in nephron progenitor cells a, b, c, d (NPCa–d). We concluded that NPCa–d are relevant for CAKUT pathogenesis and intersected two lists of CAKUT candidate genes resulting from unbiased whole-exome sequencing (WES) with the 100 highest expressed genes in NPCa–d. Results: Intersection of the 100 highest expressed genes in NPCa–d with WES-derived CAKUT candidate genes identified an overlap with the candidate genes KIF19, TRIM36, USP35, CHTF18, in each of which a biallelic variant was detected in different families with CAKUT. Conclusion: Sc-mRNA expression data of human fetal kidney can be utilized to prioritize WES-derived CAKUT candidate genes. KIF19, TRIM36, USP35, and CHTF18 may represent strong novel candidate genes for CAKUT.

A novel homozygous missense variant in TBC1D31 in a consanguineous family with congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) is the leading cause of chronic kidney disease in the first three decades of life. Until now, more than 180 monogenic causes of isolated or syndromic CAKUT have been described. In addition, copy number variants (CNV) have also been implicated, however, all of these causative factors only explain a small fraction of patients with CAKUT, suggesting that additional yet-to-be-discovered novel genes are present. Herein, we report three siblings (two of them are monozygotic twin) of a consanguineous family with CAKUT. Whole-exome sequencing identified a homozygous variant in TBC1D31. Three dimensional protein modeling as well as molecular dynamics simulations predicted it as pathogenic. We therefore showed for the first time an association between a homozygous TBC1D31 variant with CAKUT in humans, expanding its genetic spectrum.

Downregulation of fibrosis related hsa-miR-29c-3p in human CAKUT

Congenital anomalies of the kidney and urinary tract (CAKUT) represent structural and functional urinary system malformations and take place as one of the most common congenital malformations with an incidence of 1:500. Ureteral obstruction-induced hydronephrosis is associated with renal fibrosis and chronic kidney diseases in the pediatric CAKUT. We aimed to construct interaction network of previously bioinformatically associated miRNAs with CAKUT differentially expressed genes in order to prioritize those associated with fibrotic process and to experimentally validate the expression of selected miRNAs in CAKUT patients compared to control group. We constructed interaction network of hsa-miR-101-3p, hsa-miR-101-5p and hsa-miR-29c-3p that showed significant association with fibrosis. The top enriched molecular pathway was extracellular matrix-receptor interaction (adjusted p = .0000263). We experimentally confirmed expression of three miRNAs (hsa-miR-29c-3p, hsa-miR-101-3p and hsa-miR-101-5p) in obstructed ureters (ureteropelvic junction obstruction and primary obstructive megaureter) and vesicoureteral reflux. The hsa-miR-29c-3p was shown to have lower expression in both patient groups compared to controls. Relative levels of hsa-miR-101-5p and hsa-miR-101-3p showed significant positive correlations in both groups of patients. Statistically significant correlation was observed between hsa-miR-101 (-3p and -5p) and hsa-miR-29c-3p only in the obstructed group. The significant downregulation of anti-fibrotic hsa-miR-29c-3p in obstructive CAKUT could explain activation of genes involved in fibrotic processes. As miRNAs are promising candidates in therapeutic approaches our results need further measurement of fibrotic markers or assessment of extent of fibrosis and functional evaluation of hsa-miR-29c.

ASH2L Controls Ureteric Bud Morphogenesis through the Regulation of RET/GFRA1 Signaling Activity in a Mouse Model.

Causes of congenital anomalies of the kidney and urinary tract (CAKUT) remain unclear. The authors investigated whether and how inactivation of Ash2l -which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide histone H3 lysine K4 (H3K4) methylation-might contribute to CAKUT. In a mouse model, inactivation of Ash2l in the ureteric bud (UB) lineage led to CAKUT-like phenotypes. Removal of ASH2L led to deficient H3K4 trimethylation, which slowed cell proliferation at the UB tip, delaying budding and impairing branching morphogenesis. The absence of ASH2L also downregulated the expression of Ret , Gfra1 , and Wnt11 genes involved in RET/GFRA1 signaling. These findings identify ASH2L-mediated H3K4 methylation as an upstream epigenetic regulator of signaling crucial for UB morphogenesis and indicate that deficiency or dysregulation of these processes may lead to CAKUT. Ureteric bud (UB) induction and branching morphogenesis are fundamental to the establishment of the renal architecture and are key determinants of nephron number. Defective UB morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor rearranged during transfection (RET) and coreceptor GFRA1 seems to be particularly important in UB development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT. To investigate whether and how inactivation of Ash2l , which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the UB lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with Cleavage Under Targets and Tagmentation sequencing. UB-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the UB tip, delaying budding and impairing UB branching morphogenesis. These effects were associated with downregulation of Ret , Gfra1 , and Wnt11 , which participate in RET/GFRA1 signaling. These experiments identify ASH2L-dependent H3K4 methylation in the UB lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in UB morphogenesis, which, if deficient, may lead to CAKUT.

Maternal risk factors for congenital anomalies of the kidney and urinary tract: A case-control study

Congenital anomalies of the kidney and urinary tract (CAKUT) are significant causes of pediatric morbidity and mortality. The spectrum of CAKUT can be part of a syndrome, but most of these abnormalities occur as isolated and sporadic forms. The etiology of human CAKUT is unknown in the majority of cases. This case-control study aimed to investigate the association between maternal characteristics and the occurrence of CAKUT and specific CAKUT phenotypes. In this case-control study, 29,653 newborns were evaluated consecutively in a tertiary neonatal unit using the Latin American Collaborative Study of Congenital Malformations (ECLAMC) registry. Newborns without congenital anomalies were matched to CAKUT cases by sex, date, and place of birth at a ratio of 3:1. For analysis purposes, the cases were stratified into four subgroups: upper tract abnormalities (UTA), including ureteropelvic junction obstruction, vesicoureteral reflux, primary megaureter and others (n=239), lower urinary tract obstruction (LUTO) (n=79), cystic diseases (n=59) and agenesis/hypodysplasia (n=28). Multivariable logistic regression analyses were used to calculate crude and adjusted odds ratios (ORs) with 95% confidence intervals (CIs) for associations between the maternal risk factors and the presence of CAKUT. The prevalence of non-syndromic CAKUT in our sample was 13 per 1000 live births. Data records allowed the analysis of 405 cases and 1208 controls. After adjustment by the binary regression logistic, three covariates remained associated as risk factors for the entire spectrum of CAKUT: consanguinity (Odds ratio [OR], 7.1, 95%CI, 2.4-20.4), family history of CAKUT (OR, 6.4, 95%CI, 1.9-21.3), and maternal chronic hypertension (OR, 14.69, 95%CI, 3.2-67.5) (Figure). These risk factors persisted consistently across the various CAKUT phenotypes with minor variations. Consanguinity was the only factor consistently associated with almost all CAKUT phenotypes. Maternal hypertension was associated with all phenotypes except for the agenesis/hypodysplasia group. The prevalence of CAKUT cases was 15 times higher in hypertensive mothers (3%) compared to normotensive mothers (0.2%). Our study suggests that an increased risk of CAKUT is associated with consanguinity, a positive family history of CAKUT, and maternal hypertension. However, the prevalence of these risk factors in our cohort was rare and most cases presented as sporadic forms.

Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes–Brocks syndrome 2

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient’s features overlapped Townes–Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1−/− murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

A homozygous HOXA11 variation as a potential novel cause of autosomal recessive congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) is the leading cause of end-stage kidney disease in children. Until now, more than 50 monogenic causes for CAKUT have been described, all of which only explain 10% to 20% of all patients with CAKUT, suggesting the presence of additional genes that cause CAKUT when mutated. Herein, we report two siblings of a consanguineous family with CAKUT, both of which rapidly progressed to chronic kidney disease in early childhood. Whole-exome sequencing followed by homozygosity mapping identified a homozygous variation in HOXA11. We therefore showed for the first time an association between a homozygous HOXA11 variation with CAKUT in humans, expanding the genetic spectrum of the disease.

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations

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.

Mouse Models of Congenital Kidney Anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects, which cause the majority of chronic kidney diseases in children. CAKUT covers a wide range of malformations that derive from deficiencies in embryonic kidney and lower urinary tract development, including renal aplasia, hypodysplasia, hypoplasia, ectopia, and different forms of ureter abnormalities. The majority of the genetic causes of CAKUT remain unknown. Research on mutant mice has identified multiple genes that critically regulate renal differentiation. The data generated from this research have served as an excellent resource to identify the genetic bases of human kidney defects and have led to significantly improved diagnostics. Furthermore, genetic data from human CAKUT studies have also revealed novel genes regulating kidney differentiation.

COL4A1 mutations as a potential novel cause of autosomal dominant CAKUT in humans.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney disease (~ 45%) that manifests before 30years of age. The genetic locus containing COL4A1 (13q33-34) has been implicated in vesicoureteral reflux (VUR), but mutations in COL4A1 have not been reported in CAKUT. We hypothesized that COL4A1 mutations cause CAKUT in humans. We performed whole exome sequencing (WES) in 550 families with CAKUT. As negative control cohorts we used WES sequencing data from patients with nephronophthisis (NPHP) with no genetic cause identified (n = 257) and with nephrotic syndrome (NS) due to monogenic causes (n = 100). We identified a not previously reported heterozygous missense variant in COL4A1 in three siblings with isolated VUR. When examining 549 families with CAKUT, we identified nine additional different heterozygous missense mutations in COL4A1 in 11 individuals from 11 unrelated families with CAKUT, while no COL4A1 mutations were identified in a control cohort with NPHP and only one in the cohort with NS. Most individuals (12/14) had isolated CAKUT with no extrarenal features. The predominant phenotype was VUR (9/14). There were no clinical features of the COL4A1-related disorders (e.g., HANAC syndrome, porencephaly, tortuosity of retinal arteries). Whereas COL4A1-related disorders are typically caused by glycine substitutions in the collagenous domain (84.4% of variants), only one variant in our cohort is a glycine substitution within the collagenous domain (1/10). We identified heterozygous COL4A1 mutations as a potential novel autosomal dominant cause of CAKUT that is allelic to the established COL4A1-related disorders and predominantly caused by non-glycine substitutions.

Noncoding rare variants of TBX6 in congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a wide range of congenital structural renal defects. CAKUT is the leading cause of chronic renal failure and end-stage renal disease in children. Studies in humans and animal models have confirmed the large genetic contribution to CAKUT. The previous evidence suggested that human TBX6 coding mutations might cause CAKUT via gene-dosage insufficiency. However, the potential involvement of TBX6 noncoding mutations in CAKUT remains to be elucidated. Here, we described DNA sequencing and copy-number analysis of TBX6 in 269 Chinese subjects with CAKUT. Interestingly, we identified two heterozygous noncoding variants of TBX6 in sporadic subjects with CAKUT: one is c.769-7delT, from a subject with duplex renal and collecting system, and the other is a 3' untranslated region (3'-UTR) variant (c.1392C>T) from a subject with unilateral renal hypoplasia. These two TBX6 noncoding variants are novel and extremely rare, respectively, in human populations archived in the ExAC database. The mini-gene splicing assay showed that the TBX6 c.769-7delT variant significantly reduced the splicing efficiency of TBX6 intron 5 when compared to the wild-type control. In this work, we identified a novel splicing variant of TBX6 in human CAKUT. Our experimental observations suggested that the TBX6 noncoding variant can affect gene expression and may potentially be involved in human CAKUT.

Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies of the Kidney and Urinary Tract.

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. 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. 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%). 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.

Targeted Exome Sequencing Identifies PBX1 as Involved in Monogenic Congenital Anomalies of the Kidney and Urinary Tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.

ETV4 Mutation in a Patient with Congenital Anomalies of the Kidney and Urinary Tract

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common reason for chronic kidney disease in children. Although more than 30 monogenic causes have been implicated in isolated forms of human CAKUT so far, the vast majority remains elusive. To identify novel monogenic causes of CAKUT we applied homozygosity mapping, together with whole exome sequencing, in a patient from consanguineous descent with isolated CAKUT. We identified a homozygous missense mutation (p.Arg415His) of the Ets Translocation Variant Gene 4 (ETV4). The transcription factor ETV4 is a downstream target of the GDNF/RET signaling pathway that plays a crucial role in kidney development. We show by means of electrophoretic mobility shift assay that the Arg415His mutant causes loss of the DNA binding affinity of ETV4 and fails to activate transcription in a cell-based luciferase reporter assay. We furthermore investigated the impact of the mutant protein on cell migration rate. Unlike wildtype ETV4, the Arg415His mutant failed to rescue cell migration defects observed in two ETV4 knock-down cell-lines. We therefore identified and functionally characterized a recessive mutation in ETV4 in a human patient with CAKUT. We hypothesize that the pathomechanism of this mutation could be via loss of the transcriptional function of ETV4, and a resulting abrogation of GDNF/RET/ETV4 signaling pathway.

Transcriptome-wide based identification of miRs in congenital anomalies of the kidney and urinary tract (CAKUT) in children: the significant upregulation of tissue miR-144 expression.

BackgroundThe genetic cause of most congenital anomalies of the kidney and urinary tract (CAKUT) cases remains unknown, therefore the novel approaches in searching for the common disease denominators are required. miRs regulate gene expression in humans and therefore have potentially therapeutic and biomarker properties. No studies thus far have attempted to explore the miRs in human CAKUT. We applied a new strategy to identify most specific miRs associated with CAKUT, in pediatric patients.MethodsData from the whole genome expression, gathered from ureter tissue samples of 19 patients and 7 controls, were used for the bioinformatic prediction of miRs activity in CAKUT. We integrated microarray gene expression data and miR target predictions from multiple prediction algorithms using Co-inertia analysis (CIA) in conjunction with correspondence analysis and between group analysis, to produce a ranked list of miRs associated with CAKUT. The CIA included five different sequence based miR target prediction algorithms and the Co-expression Meta-analysis of miR Targets. For the experimental validation of expression of miRs identified by the CIA we used tissue from 36 CAKUT patients and 9 controls. The results of gene ontology (GO) analysis on co-expressed targets of miRs associated with CAKUT were used for the selection of putative biological processes relevant to CAKUT.ResultsWe identified 7 miRs with a potential role in CAKUT. The top ranked miRs from miRCos communities 4, 1 and 7 were chosen for experimental validation of expression in CAKUT tissue. The 5.7 fold increase of hsa-miR-144 expression in human tissue from CAKUT patients compared to controls (p = 0.005) was observed. From the GO we selected 7 biological processes that could contribute to CAKUT, which genes are potentially influenced by hsa-miR-144. The hsa-miR-200a, hsa-miR-183 and hsa-miR-375 weren’t differentially expressed in CAKUT.ConclusionsThis study shows that integrative approach applied here was useful in identification of the miRs associated with CAKUT. The hsa-miR-144, first time identified in CAKUT, could be connected with biological processes crucial for normal development of kidney and urinary tract. Further functional analysis must follow to reveal the impact of hsa-miR-144 on CAKUT occurrence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0955-0) contains supplementary material, which is available to authorized users.

Targeted sequencing of 96 renal developmental microRNAs in 1213 individuals from 980 families with congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of chronic kidney diseases in children and young adults, accounting for ∼50% of cases. These anomalies represent maldevelopment of the genitourinary system and can be genetically explained in only 10-16% of cases by mutations or by copy number variations in protein coding sequences. Knock-out mouse models, lacking components of the microRNA (miRNA) processing machinery (i.e. Dicer, Drosha, Dgcr8), exhibit kidney malformations resembling human CAKUT. Given the Dicer-null mouse phenotype, which implicates a central role for miRNAs gene regulation during kidney development, we hypothesized that miRNAs expressed during kidney development may cause CAKUT in humans if mutated. To evaluate this possibility we carried out Next-Generation sequencing of 96 stem-loop regions of 73 renal developmental miRNA genes in 1248 individuals with non-syndromic CAKUT from 980 families. We sequenced 96 stem-loop regions encoded by 73 miRNA genes that are expressed during kidney development in humans, mice and rats. Overall, we identified in 31/1213 individuals from 26 families with 17 different single nucleotide variants. Two variants did not segregate with the disease and hence were not causative. Thirteen variants were likely benign variants because they occurred in control populations and/or they affected nucleotides of weak evolutionary conservation. Two out of 1213 unrelated individuals had potentially pathogenic variants with unknown biologic relevance affecting miRNAs MIR19B1 and MIR99A. Our results indicate that mutations affecting mature microRNAs in individuals with CAKUT are rare and thus most likely not a common cause of CAKUT in humans.

Copy number variation analysis identifies novel CAKUT candidate genes in children with a solitary functioning kidney

Copy number variations associate with different developmental phenotypes and represent a major cause of congenital anomalies of the kidney and urinary tract (CAKUT). Because rare pathogenic copy number variations are often large and contain multiple genes, identification of the underlying genetic drivers has proven to be difficult. Here we studied the role of rare copy number variations in 80 patients from the KIMONO-study cohort for which pathogenic mutations in three genes commonly implicated in CAKUT were excluded. In total, 13 known or novel genomic imbalances in 11 of 80 patients were absent or extremely rare in 23,362 population controls. To identify the most likely genetic drivers for the CAKUT phenotype underlying these rare copy number variations, we used a systematic in silico approach based on frequency in a large dataset of controls, annotation with publicly available databases for developmental diseases, tolerance and haploinsufficiency scores, and gene expression profile in the developing kidney and urinary tract. Five novel candidate genes for CAKUT were identified that showed specific expression in the human and mouse developing urinary tract. Among these genes, DLG1 and KIF12 are likely novel susceptibility genes for CAKUT in humans. Thus, there is a significant role of genomic imbalance in the determination of kidney developmental phenotypes. Additionally, we defined a systematic strategy to identify genetic drivers underlying rare copy number variations.