X-linked variations in SHROOM4 are implicated in congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems.

BACKGROUND: SHROOM4 is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in SHROOM4 have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system. METHODS: Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and knockdown (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of SHROOM4 during embryonic development. RESULTS: In this study, we identified putative disease-causing SNVs and CNVs in SHROOM4 in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed Shroom4 expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type SHROOM4 mRNA and morpholino. CONCLUSION: The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest SHROOM4 as a developmental gene for different organ systems.

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.

Rare Variants in BNC2 Are Implicated in Autosomal-Dominant Congenital Lower Urinary-Tract Obstruction.

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

Biallelic and monoallelic variants in PLXNA1 are implicated in a novel neurodevelopmental disorder with variable cerebral and eye anomalies.

PURPOSE: To investigate the effect of PLXNA1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and to functionally characterize the zebrafish homologs plxna1a and plxna1b during development. METHODS: We assembled ten patients from seven families with biallelic or de novo PLXNA1 variants. We describe genotype-phenotype correlations, investigated the variants by structural modeling, and used Morpholino knockdown experiments in zebrafish to characterize the embryonic role of plxna1a and plxna1b. RESULTS: Shared phenotypic features among patients include global developmental delay (9/10), brain anomalies (6/10), and eye anomalies (7/10). Notably, seizures were predominantly reported in patients with monoallelic variants. Structural modeling of missense variants in PLXNA1 suggests distortion in the native protein. Our zebrafish studies enforce an embryonic role of plxna1a and plxna1b in the development of the central nervous system and the eye. CONCLUSION: We propose that different biallelic and monoallelic variants in PLXNA1 result in a novel neurodevelopmental syndrome mainly comprising developmental delay, brain, and eye anomalies. We hypothesize that biallelic variants in the extracellular Plexin-A1 domains lead to impaired dimerization or lack of receptor molecules, whereas monoallelic variants in the intracellular Plexin-A1 domains might impair downstream signaling through a dominant-negative effect.

Exome survey of individuals affected by VATER/VACTERL with renal phenotypes identifies phenocopies and novel candidate genes.

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.

Increased psychosocial risk, depression and reduced quality of life living with autosomal dominant polycystic kidney disease.

BACKGROUND: The psychosocial impact of living with autosomal dominant polycystic kidney disease (ADPKD) is poorly understood. In this study, we assessed the overall quality of life (QOL), mood, perceived social support and psychosocial risk of having a diagnosis of ADPKD in a patient cohort from a major UK nephrology centre serving a large catchment population. METHODS: A postal questionnaire was sent to 349 patients registered at the Sheffield Kidney Institute with chronic kidney disease but not on renal replacement therapy (RRT). The questionnaire incorporated three validated forms: kidney disease quality-of-life short form (KDQOL SF1.3) to assess QOL; nine-item patient health questionnaire (PHQ9) to screen for depression; multidimensional scale of perceived social support (MSPSS) to evaluate perceived social support; as well as a novel genetic psychosocial risk instrument (GPRI-ADPKD) designed to study the specific psychosocial impact of coping with a diagnosis of ADPKD. RESULTS: The overall response rate was 53%. Patients with a lower estimated glomerular filtration rate (<30 mL/min) or larger kidneys (mean length on ultrasound ≥17 cm) reported reduced QOL and increased psychosocial risk. Clinically significant depression was reported in 22% and 62% felt guilty about passing ADPKD on to their children. In multivariate analysis, female gender was associated with overall poorer psychosocial well-being, whereas increasing age, lower kidney function, larger kidneys and loss of a first degree relative from ADPKD were additional risk factors for QOL, depression or psychosocial risk, respectively. CONCLUSIONS: Our results reveal a significantly poorer QOL and increasing psychosocial risk with markers of disease progression in patients, particularly women, with ADPKD prior to starting RRT. The future management strategy of ADPKD should address these issues and provide for better individual and family support throughout the patient journey.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association.

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.

Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for 40-50% of chronic kidney disease that manifests in the first two decades of life. Thus far, 31 monogenic causes of isolated CAKUT have been described, explaining ~12% of cases. To identify additional CAKUT-causing genes, we performed whole-exome sequencing followed by a genetic burden analysis in 26 genetically unsolved families with CAKUT. We identified two heterozygous mutations in SRGAP1 in 2 unrelated families. SRGAP1 is a small GTPase-activating protein in the SLIT2-ROBO2 signaling pathway, which is essential for development of the metanephric kidney. We then examined the pathway-derived candidate gene SLIT2 for mutations in cohort of 749 individuals with CAKUT and we identified 3 unrelated individuals with heterozygous mutations. The clinical phenotypes of individuals with mutations in SLIT2 or SRGAP1 were cystic dysplastic kidneys, unilateral renal agenesis, and duplicated collecting system. We show that SRGAP1 is expressed in early mouse nephrogenic mesenchyme and that it is coexpressed with ROBO2 in SIX2-positive nephron progenitor cells of the cap mesenchyme in developing rat kidney. We demonstrate that the newly identified mutations in SRGAP1 lead to an augmented inhibition of RAC1 in cultured human embryonic kidney cells and that the SLIT2 mutations compromise the ability of the SLIT2 ligand to inhibit cell migration. Thus, we report on two novel candidate genes for causing monogenic isolated CAKUT in humans.

Genome-wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities.

BACKGROUND: Anorectal malformations (ARM) have a prevalence of around 1 in 2500 live births. In around 50% of patients, the malformation is isolated, while in the remainder it arises within the context of complex genetic abnormalities or a defined genetic syndrome. Recent studies have implicated rare copy number variations (CNVs) in both isolated and nonisolated ARM, and identified plausible candidate genes. METHODS: In the present study, array-based molecular karyotyping was performed to identify causative CNVs in 32 sporadic ARM patients with comorbid abnormalities of the central nervous system (CNS). This phenotype was selected to enrich for rare CNVs, since previous research has implicated rare CNVs in both CNS abnormalities and ARM. RESULTS: In five patients, a probable disease-causing CNV was identified (del6q14.3q16.3, del14q32.2, del17q12q21.2, and two patients with del22q11.21). In three of these patients, the CNVs were de novo. For the remaining two patients, no parental DNA was available. Deletions at 22q11.21 and 6q14.3 have been associated with both CNS abnormalities and ARM. In contrast, deletions at 14q32.2 have only been described in patients with CNS abnormalities, and the del17q12q21.2 is a novel CNV. Expression studies in mice suggest that NEUROD2 and RARA, which reside within the newly identified del17q12q21.2 region, are candidate genes for the formation of microcephaly and ARM. CONCLUSION: The present data suggest that CNVs are a frequent cause of the ARM with CNS abnormalities phenotype, and that array-analysis is indicated in such patients.

Mild recessive mutations in six Fraser syndrome-related genes cause isolated congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately 40% of children with ESRD in the United States. Hitherto, mutations in 23 genes have been described as causing autosomal dominant isolated CAKUT in humans. However, >90% of cases of isolated CAKUT still remain without a molecular diagnosis. Here, we hypothesized that genes mutated in recessive mouse models with the specific CAKUT phenotype of unilateral renal agenesis may also be mutated in humans with isolated CAKUT. We applied next-generation sequencing technology for targeted exon sequencing of 12 recessive murine candidate genes in 574 individuals with isolated CAKUT from 590 families. In 15 of 590 families, we identified recessive mutations in the genes FRAS1, FREM2, GRIP1, FREM1, ITGA8, and GREM1, all of which function in the interaction of the ureteric bud and the metanephric mesenchyme. We show that isolated CAKUT may be caused partially by mutations in recessive genes. Our results also indicate that biallelic missense mutations in the Fraser/MOTA/BNAR spectrum genes cause isolated CAKUT, whereas truncating mutations are found in the multiorgan form of Fraser syndrome. The newly identified recessive biallelic mutations in these six genes represent the molecular cause of isolated CAKUT in 2.5% of the 590 affected families in this study.

Mutations in 12 known dominant disease-causing genes clarify many congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease. CAKUT can be caused by monogenic mutations; however, data are lacking on their frequency. Genetic diagnosis has been hampered by genetic heterogeneity and lack of genotype-phenotype correlation. To determine the percentage of cases with CAKUT that can be explained by mutations in known CAKUT genes, we analyzed the coding exons of the 17 known dominant CAKUT-causing genes in a cohort of 749 individuals from 650 families with CAKUT. The most common phenotypes in this CAKUT cohort were vesicoureteral reflux in 288 patients, renal hypodysplasia in 120 patients, and unilateral renal agenesis in 90 patients. We identified 37 different heterozygous mutations (33 novel) in 12 of the 17 known genes in 47 patients from 41 of the 650 families (6.3%). These mutations include (number of families): BMP7 (1), CDC5L (1), CHD1L (5), EYA1 (3), GATA3 (2), HNF1B (6), PAX2 (5), RET (3), ROBO2 (4), SALL1 (9), SIX2 (1), and SIX5 (1). Furthermore, several mutations previously reported to be disease-causing are most likely benign variants. Thus, in a large cohort over 6% of families with isolated CAKUT are caused by a mutation in 12 of 17 dominant CAKUT genes. Our report represents one of the most in-depth diagnostic studies of monogenic causes of isolated CAKUT in children.

Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association.

Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole-exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor-associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat-shock protein 90-related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle's loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or VACTERL association with CAKUT.

Heterozygous FGF8 mutations in patients presenting cryptorchidism and multiple VATER/VACTERL features without limb anomalies.

BACKGROUND: The acronym VATER/VACTERL association describes the combination of at least three of the following cardinal features: vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. Although fibroblast growth factor-8 (FGF8) mutations have mainly found in patients with Kallmann syndrome, mice with a hypomorphic Fgf8 allele or complete gene invalidation display, aside from gonadotropin-releasing hormone deficiency, parts or even the entire spectrum of human VATER/VACTERL association. METHODS: We performed FGF8 gene analysis in 49 patients with VATER/VACTERL association and 27 patients presenting with a VATER/VACTERL-like phenotype (two cardinal features). RESULTS: We identified two heterozygous FGF8 mutations in patients displaying either VATER/VACTERL association (p.Gly29_Arg34dup) or a VATER/VACTERL-like phenotype (p.Pro26Leu) without limb anomalies. Whereas the duplication mutation has not been reported before, p.Pro26Leu was once observed in a Kallmann syndrome patient. Both our patients had additional bilateral cryptorchidism, a key phenotypic feature in males with FGF8 associated Kallmann syndrome. Each mutation was paternally inherited. Besides delayed puberty in both and additional unilateral cryptorchidism in one of the fathers, they were otherwise healthy. Serum hormone levels downstream the gonadotropin-releasing hormone in both patients and their fathers were within normal range. CONCLUSION: Our results suggest FGF8 mutations to contribute to the formation of the VATER/VACTERL association. Further studies are needed to support this observation.

Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans.

Congenital anomalies of the kidney and urinary tract (CAKUT) cover a wide range of structural malformations that result from defects in the morphogenesis of the kidney and/or urinary tract. These anomalies account for about 40-50 % of children with chronic kidney disease worldwide. Knowledge from genetically modified mouse models suggests that single gene mutations in renal developmental genes may lead to CAKUT in humans. However, until recently, only a handful of CAKUT-causing genes were reported, most of them in familial syndromic cases. Recent findings suggest that CAKUT may arise from mutations in a multitude of different single gene causes. We focus here on single-gene causes of CAKUT and their developmental origin. Currently, more than 20 monogenic CAKUT-causing genes have been identified. High-throughput sequencing techniques make it likely that additional CAKUT-causing genes will be identified in the near future.

Bi-allelic variants in CELSR3 are implicated in central nervous system and urinary tract anomalies.

CELSR3 codes for a planar cell polarity protein. We describe twelve affected individuals from eleven independent families with bi-allelic variants in CELSR3. Affected individuals presented with an overlapping phenotypic spectrum comprising central nervous system (CNS) anomalies (7/12), combined CNS anomalies and congenital anomalies of the kidneys and urinary tract (CAKUT) (3/12) and CAKUT only (2/12). Computational simulation of the 3D protein structure suggests the position of the identified variants to be implicated in penetrance and phenotype expression. CELSR3 immunolocalization in human embryonic urinary tract and transient suppression and rescue experiments of Celsr3 in fluorescent zebrafish reporter lines further support an embryonic role of CELSR3 in CNS and urinary tract formation.

De novo 13q deletions in two patients with mild anorectal malformations as part of VATER/VACTERL and VATER/VACTERL-like association and analysis of EFNB2 in patients with anorectal malformations.

Anorectal malformations (ARMs) comprise a broad spectrum of conditions ranging from mild anal anomalies to complex cloacal malformations. In 40-50% of cases, ARM occurs within the context of defined genetic syndromes or complex multiple congenital anomalies, such as VATER/VACTERL (vertebral defects [V], ARMs [A], cardiac defects [C], tracheoesophageal fistula with or without esophageal atresia [TE], renal malformations [R], and limb defects [L]) association. Here, we report the identification of deletions at chromosome 13q using single nucleotide polymorphism-based array analysis in two patients with mild ARM as part of VATER/VACTERL and VATER/VACTERL-like associations. Both deletions overlap the previously defined critical region for ARM. Heterozygous Efnb2 murine knockout models presenting with mild ARM suggest EFNB2 as an excellent candidate gene in this region. Our patients showed a mild ARM phenotype, closely resembling that of the mouse. We performed a comprehensive mutation analysis of the EFNB2 gene in 331 patients with isolated ARM, or ARM as part of VATER/VACTERL or VATER/VACTERL-like associations. However, we did not identify any disease-causing mutations. Given the convincing argument for EFNB2 as a candidate gene for ARM, analyses of larger samples and screening of functionally relevant non-coding regions of EFNB2 are warranted. In conclusion, our report underlines the association of chromosome 13q deletions with ARM, suggesting that routine molecular diagnostic workup should include the search for these deletions. Despite the negative results of our mutation screening, we still consider EFNB2 an excellent candidate gene for contributing to the development of ARM in humans.

Modelling human lower urinary tract malformations in zebrafish.

Advances in molecular biology are improving our understanding of the genetic causes underlying human congenital lower urinary tract (i.e., bladder and urethral) malformations. This has recently led to the identification of the first disease-causing variants in the gene BNC2 for isolated lower urinary tract anatomical obstruction (LUTO), and of WNT3 and SLC20A1 as genes implicated in the pathogenesis of the group of conditions called bladder-exstrophy-epispadias complex (BEEC). Implicating candidate genes from human genetic data requires evidence of their influence on lower urinary tract development and evidence of the found genetic variants' pathogenicity. The zebrafish (Danio rerio) has many advantages for use as a vertebrate model organism for the lower urinary tract. Rapid reproduction with numerous offspring, comparable anatomical kidney and lower urinary tract homology, and easy genetic manipulability by Morpholino®-based knockdown or CRISPR/Cas editing are among its advantages. In addition, established marker staining for well-known molecules involved in urinary tract development using whole-mount in situ hybridization (WISH) and the usage of transgenic lines expressing fluorescent protein under a tissue-specific promoter allow easy visualization of phenotypic abnormalities of genetically modified zebrafish. Assays to examine the functionality of the excretory organs can also be modeled in vivo with the zebrafish. The approach of using these multiple techniques in zebrafish not only enables rapid and efficient investigation of candidate genes for lower urinary tract malformations derived from human data, but also cautiously allows transferability of causality from a non-mammalian vertebrate to humans.

Exome Survey and Candidate Gene Re-Sequencing Identifies Novel Exstrophy Candidate Genes and Implicates LZTR1 in Disease Formation.

BACKGROUND: The bladder exstrophy-epispadias complex (BEEC) is a spectrum of congenital abnormalities that involves the abdominal wall, the bony pelvis, the urinary tract, the external genitalia, and, in severe cases, the gastrointestinal tract as well. METHODS: Herein, we performed an exome analysis of case-parent trios with cloacal exstrophy (CE), the most severe form of the BEEC. Furthermore, we surveyed the exome of a sib-pair presenting with classic bladder exstrophy (CBE) and epispadias (E) only. Moreover, we performed large-scale re-sequencing of CBE individuals for novel candidate genes that were derived from the current exome analysis, as well as for previously reported candidate genes within the CBE phenocritical region, 22q11.2. RESULTS: The exome survey in the CE case-parent trios identified two candidate genes harboring de novo variants (NR1H2, GKAP1), four candidate genes with autosomal-recessive biallelic variants (AKR1B10, CLSTN3, NDST4, PLEKHB1) and one candidate gene with suggestive uniparental disomy (SVEP1). However, re-sequencing did not identify any additional variant carriers in these candidate genes. Analysis of the affected sib-pair revealed no candidate gene. Re-sequencing of the genes within the 22q11.2 CBE phenocritical region identified two highly conserved frameshift variants that led to early termination in two independent CBE males, in LZTR1 (c.978_985del, p.Ser327fster6) and in SLC7A4 (c.1087delC, p.Arg363fster68). CONCLUSIONS: According to previous studies, our study further implicates LZTR1 in CBE formation. Exome analysis-derived candidate genes from CE individuals may not represent a frequent indicator for other BEEC phenotypes and warrant molecular analysis before their involvement in disease formation can be assumed.

Genome-wide identification of disease-causing copy number variations in 450 individuals with anorectal malformations.

Anorectal malformations (ARM) represent a spectrum of rare malformations originating from a perturbated development of the embryonic hindgut. Approximately 60% occur as a part of a defined genetic syndrome or within the spectrum of additional congenital anomalies. Rare copy number variations (CNVs) have been associated with both syndromic and non-syndromic forms. The present study represents the largest study to date to explore the contribution of CNVs to the expression of ARMs. SNP-array-based molecular karyotyping was applied in 450 individuals with ARM and 4392 healthy controls. CNVs were identified from raw intensity data using PennCNV. Overlapping CNVs between cases and controls were discarded. Remaining CNVs were filtered using a stringent filter algorithm of nine filter steps. Prioritized CNVs were confirmed using qPCR. Filtering prioritized and qPCR confirmed four microscopic chromosomal anomalies and nine submicroscopic CNVs comprising seven microdeletions (del2p13.2, del4p16.2, del7q31.33, del9p24.1, del16q12.1, del18q32, del22q11.21) and two microduplications (dup2p13.2, dup17q12) in 14 individuals (12 singletons and one affected sib-pair). Within these CNVs, based on their embryonic expression data and function, we suggest FOXK2, LPP, and SALL3 as putative candidate genes. Overall, our CNV analysis identified putative microscopic and submicroscopic chromosomal rearrangements in 3% of cases. Functional characterization and re-sequencing of suggested candidate genes is warranted.