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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.

Background The triad of a presacral mass, sacral agenesis and an anorectal anomaly constitutes the rare Currarino syndrome (CS), which is caused by dorsal–ventral patterning defects during embryonic development. The major causative CS gene is MNX1 , encoding a homeobox protein. Main body In the majority of patients, CS occurs as an autosomal dominant trait; however, a female predominance observed, implies that CS may underlie an additional mode(s) of inheritance. Often, the diagnosis of CS is established solely by clinical findings, impacting a detailed analysis of the disease. Our combined data, evaluating more than 60 studies reporting patients with CS-associated mutations, revealed a slightly higher incidence rate in females with a female-to-male ratio of 1.39:1. Overall, MNX1 mutation analysis was successful in only 57.4% of all CS patients investigated, with no mutation detected in 7.7% of the familial and 68% of the sporadic patients. Our studies failed to detect the presence of an expressed MNX1 isoform that might explain at least some of these mutation-negative cases. Conclusion Aside from MNX1 , other genes or regulatory regions may contribute to CS and we discuss several cytogenetic studies and whole-exome sequencing data that have implicated further loci/genes in its etiology.

Pulmonary hypertension (PH) contributes to high mortality in congenital diaphragmatic hernia (CDH). A better understanding of the regulatory mechanisms underlying the pathology in CDH might allow the identification of prognostic biomarkers and potential therapeutic targets. We report the results from an expression profiling of circulating microRNAs (miRNAs) in direct post-pulmonary blood flow of 18 CDH newborns. Seven miRNAs differentially expressed in children that either died or developed chronic lung disease (CLD) up to 28 days after birth, compared to those who survived without developing CLD during this period, were identified. Target gene and pathway analyses indicate that these miRNAs functions include regulation of the cell cycle, inflammation and morphogenesis, by targeting molecules responsive to growth factors, cytokines and cellular stressors. Furthermore, we identified hub molecules by constructing a protein-protein interaction network of shared targets, and ranked the relative importance of the identified miRNAs. Our results suggest that dysregulations in miRNAs let-7b-5p, -7c-5p, miR-1307-3p, -185-3p, -8084, -331-3p and -210-3p may be detrimental for the development and function of the lungs and pulmonary vasculature, compromise cardiac function and contribute to the development of CLD in CDH. Further investigation of the biomarker and therapeutic potential of these circulating miRNAs is encouraged.

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.

Besides visceral heterotaxia, Pkd1l1 null mouse embryos exhibit general edema and perinatal lethality. In humans, congenital chylothorax (CCT) is a frequent cause of fetal hydrops. In 2021, Correa and colleagues reported ultrarare compound heterozygous variants in PKD1L1 exhibiting in two consecutive fetuses with severe hydrops, implicating a direct role of PKD1L1 in fetal hydrops formation. Here, we performed an exome survey and identified ultrarare compound heterozygous variants in PKD1L1 in two of the five case–parent trios with CCT. In one family, the affected carried the ultrarare missense variants c.1543G>A(p.Gly515Arg) and c.3845T>A(p.Val1282Glu). In the other family, the affected carried the ultrarare loss-of-function variant (LoF) c.863delA(p.Asn288Thrfs*3) and the ultrarare missense variant c.6549G>T(p.Gln2183His). Investigation of the variants’ impact on PKD1L1 protein localization suggests the missense variants cause protein dysfunction and the LoF variant causes protein mislocalization. Further analysis of Pkd1l1 mutant mouse embryos revealed about 20% of Pkd1l1−/− embryos display general edema and pleural effusion at 14.5 dpc. Immunofluorescence staining at 14.5 dpc in Pkd1l1−/− embryos displayed both normal and massively altered lymphatic vessel morphologies. Together, our studies suggest the implication of PKD1L1 in congenital lymphatic anomalies, including CCTs.

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.MethodsHere, 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.ResultsIn 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.ConclusionThe 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.

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.

BackgroundPrevious studies in mouse, Xenopus and zebrafish embryos show strong tfap2e expression in progenitor cells of neuronal and neural crest tissues suggesting its involvement in neural crest specification. However, the role of human transcription factor activator protein 2 (TFAP2E) in human embryonic central nervous system (CNS), orofacial and maxillofacial development is unknown.MethodsThrough a collaborative work, exome survey was performed in families with congenital CNS, orofacial and maxillofacial anomalies. Exome variant prioritisation prompted TFAP2E gene for functional analysis in zebrafish embryos. Embryonic morphology and development were assessed after antisense morpholino (MO) knockdown (KD), CRISPR/Cas9 knockout and overexpression of tfap2e in fluorescent zebrafish reporter lines using in vivo microscopy. Computational structural protein modelling of the identified human variants was performed.ResultsIn total, exome survey identified novel or ultra-rare heterozygous missense variants in TFAP2E in seven individuals from five independent families with predominantly CNS, orofacial and maxillofacial anomalies. One variant was found de novo and another variant segregated in an affected multiplex family. Protein modelling of the identified variants indicated potential distortion of TFAP2E in the transactivation or dimerisation domain. MO KD and CRISPR/Cas9 knockout of tfap2e in zebrafish revealed hydrocephalus and a significant reduction of brain volume, consistent with a microencephaly phenotype. Furthermore, mRNA overexpression of TFAP2E indicates dosage-sensitive phenotype expression. In addition, zebrafish showed orofacial and maxillofacial anomalies following tfap2e KD, recapitulating the human phenotype.ConclusionOur human genetic data and analysis of Tfap2e manipulation in zebrafish indicate a potential role of TFAP2E in human CNS, orofacial and maxillofacial anomalies.

Anorectal malformations (ARM) represent a rare birth defect of the hindgut that occur in approximately 1 in 3000 live births. Around 60% of ARM occur with associated anomalies including defined genetic syndromes and associations with chromosomal aberrations. The etiology of ARM is heterogeneous, with the individual environmental or genetic risk factors remaining unknown for the majority of cases. The occurrence of familial ARM and previous epidemiologic analysis suggest autosomal dominant inheritance in a substantial subset of ARM patients. The implicated mortality and reduced fecundity in patients with ARM would lead to allele loss. However, mutational de novo events among the affected individuals could compensate for the evolutionary pressure. With the implementation of exome sequencing, array-based molecular karyotyping and family-based rare variant analyses, the technologies are available to identify the respective factors. This review discusses the identification of disease-causing variants among individuals with ARM. It highlights the role of mutational de novo events.

VACTERL association and Scimitar syndrome are rare congenital diseases. In this study, we report on a neonate with prenatal suspicion of VACTERL association and small left-sided cardiac structures, which, only on postnatal angiography, could be revealed to be part of a Scimitar syndrome. As this is the second reported case of VACTERL association and Scimitar syndrome, the presence of Scimitar syndrome should be considered in the prenatal and postnatal evaluation of VACTERL association.