Kirsten Y. Renkema

Gain of glycosylation in integrin α3 causes lung disease and nephrotic syndrome

Integrins are transmembrane αβ glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3β1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3β1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with β1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the β1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3β1, causing a fatal multiorgan disorder.

Common variants in DGKK are strongly associated with risk of hypospadias

Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase κ, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 × 10−11 and OR = 2.3, P = 2.9 × 10−9, respectively) and in the Dutch (OR = 3.9, P = 2.4 × 10−5 and OR = 3.8, P = 3.4 × 10−5) and Swedish (OR = 2.5, P = 2.6 × 10−8 and OR = 2.2, P = 2.7 × 10−6) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.

Next-generation sequencing for research and diagnostics in kidney disease

The advent of next-generation sequencing technologies has enabled genetic nephrology research to move beyond single gene analysis to the simultaneous investigation of hundreds of genes and entire pathways. These new sequencing approaches have been used to identify and characterize causal factors that underlie inherited heterogeneous kidney diseases such as nephronophthisis and congenital anomalies of the kidney and urinary tract. In this Review, we describe the development of next-generation sequencing in basic and clinical research and discuss the implementation of this novel technology in routine patient management. Widespread use of targeted and nontargeted approaches for gene identification in clinical practice will require consistent phenotyping, appropriate disease modelling and collaborative efforts to combine and integrate data analyses. Next-generation sequencing is an exceptionally promising technique that has the potential to improve the management of patients with inherited kidney diseases. However, identifying the molecular mechanisms that lead to renal developmental disorders and ciliopathies is difficult. A major challenge in the near future will be how best to integrate data obtained using next-generation sequencing with personalized medicine, including use of high-throughput disease modelling as a tool to support the clinical diagnosis of kidney diseases.

Genetic, environmental, and epigenetic factors involved in CAKUT

Congenital anomalies of the kidney and urinary tract (CAKUT) refer to a spectrum of structural renal malformations and are the leading cause of end-stage renal disease in children. The genetic diagnosis of CAKUT has proven to be challenging due to genetic and phenotypic heterogeneity and incomplete genetic penetrance. Monogenic causes of CAKUT have been identified using different approaches, including single gene screening, and gene panel and whole exome sequencing. The majority of the identified mutations, however, lack substantial evidence to support a pathogenic role in CAKUT. Copy number variants or single nucleotide variants that are associated with CAKUT have also been identified. Numerous studies support the influence of epigenetic and environmental factors on kidney development and the natural history of CAKUT, suggesting that the pathogenesis of this syndrome is multifactorial. In this Review we describe the current knowledge regarding the genetic susceptibility underlying CAKUT and the approaches used to investigate the genetic basis of CAKUT. We outline the associated environmental risk factors and epigenetic influences on CAKUT and discuss the challenges and strategies used to fully address the involvement and interplay of these factors in the pathogenesis of the disease.

Novel perspectives for investigating congenital anomalies of the kidney and urinary tract (CAKUT)

Congenital anomalies of the kidney and urinary tract (CAKUT) are the commonest cause of chronic kidney disease in children. Structural anomalies within the CAKUT spectrum include renal agenesis, kidney hypo-/dysplasia, multicystic kidney dysplasia, duplex collecting system, posterior urethral valves and ureter abnormalities. While most CAKUT cases are sporadic, familial clustering of CAKUT is common, emphasizing a strong genetic contribution to CAKUT origin. Animal experiments demonstrate that alterations in genes crucial for kidney development can cause experimental CAKUT, while expression studies implicate mislocalization and/or aberrant levels of the encoded proteins in human CAKUT. Further insight into the pathogenesis of CAKUT will improve strategies for early diagnosis, follow-up and treatment. Here, we outline a collaborative approach to identify and characterize novel factors underlying human CAKUT. This European consortium will share the largest collection of CAKUT patients available worldwide and undertake multidisciplinary research into molecular and genetic pathogenesis, with extension into translational studies to improve long-term patient outcomes.

Genes in the Ureteric Budding Pathway: Association Study on Vesico-Ureteral Reflux Patients

Vesico-ureteral reflux (VUR) is the retrograde passage of urine from the bladder to the urinary tract and causes 8.5% of end-stage renal disease in children. It is a complex genetic developmental disorder, in which ectopic embryonal ureteric budding is implicated in the pathogenesis. VUR is part of the spectrum of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). We performed an extensive association study for primary VUR using a two-stage, case-control design, investigating 44 candidate genes in the ureteric budding pathway in 409 Dutch VUR patients. The 44 genes were selected from the literature and a set of 567 single nucleotide polymorphisms (SNPs) capturing their genetic variation was genotyped in 207 cases and 554 controls. The 14 SNPs with p<0.005 were included in a follow-up study in 202 cases and 892 controls. Of the total cohort, ∼50% showed a clear-cut primary VUR phenotype and ∼25% had both a duplex collecting system and VUR. We also looked for association in these two extreme phenotype groups. None of the SNPs reached a significant p-value. Common genetic variants in four genes (GREM1, EYA1, ROBO2 and UPK3A) show a trend towards association with the development of primary VUR (GREM1, EYA1, ROBO2) or duplex collecting system (EYA1 and UPK3A). SNPs in three genes (TGFB1, GNB3 and VEGFA) have been shown to be associated with VUR in other populations. Only the result of rs1800469 in TGFB1 hinted at association in our study. This is the first extensive study of common variants in the genes of the ureteric budding pathway and the genetic susceptibility to primary VUR.

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.

Non-invasive sources of cells with primary cilia from pediatric and adult patients.

BackgroundCiliopathies give rise to a multitude of organ-specific pathologies; obtaining relevant primary patient material is useful for both diagnostics and research. However, acquisition of primary ciliated cells from patients, particularly pediatric patients, presents multiple difficulties. Biopsies and blood samples are invasive, and patients (and their parents) may be reluctant to travel to medical centers, especially for research purposes. We sought to develop non-invasive methods of obtaining viable and ciliated primary cells from ciliopathy patients which could be obtained in the home environment.FindingsWe introduce two methods for the non-invasive acquisition of primary ciliated cells. In one approach, we collected spontaneously shed deciduous (milk) teeth from children. Fibroblast-like cells were observed after approximately 2 weeks of culture of fragmented teeth. Secondly, urine samples were collected from children or adults. Cellular content was isolated and after approximately 1 week, renal epithelial cells were observed. Both urine and tooth-derived cells ciliate and express ciliary proteins visible with immunofluorescence. Urine-derived renal epithelial cells (URECs) are amenable to 3D culturing, siRNA knockdown, and ex vivo drug testing.ConclusionsAs evidence continues to accumulate showing that the primary cilium has a central role in development and disease, the need for readily available and ciliated patient cells will increase. Here, we introduce two methods for the non-invasive acquisition of cells with primary cilia. We believe that these cells can be used for further ex vivo study of ciliopathies and in the future, for personalized medicine.

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.

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.