Reverse phenotyping facilitates disease allele calling in exome sequencing of patients with CAKUT.

PURPOSE: Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the leading cause of chronic kidney disease in children. In total, 174 monogenic causes of isolated or syndromic CAKUT are known. However, syndromic features may be overlooked when the initial clinical diagnosis of CAKUT is made. We hypothesized that the yield of a molecular genetic diagnosis by exome sequencing (ES) can be increased by applying reverse phenotyping, by re-examining the case for signs/symptoms of the suspected clinical syndrome that results from the genetic variant detected by ES. METHODS: We conducted ES in an international cohort of 731 unrelated families with CAKUT. We evaluated ES data for variants in 174 genes, in which variants are known to cause isolated or syndromic CAKUT. In cases in which ES suggested a previously unreported syndromic phenotype, we conducted reverse phenotyping. RESULTS: In 83 of 731 (11.4%) families, we detected a likely CAKUT-causing genetic variant consistent with an isolated or syndromic CAKUT phenotype. In 19 of these 83 families (22.9%), reverse phenotyping yielded syndromic clinical findings, thereby strengthening the genotype-phenotype correlation. CONCLUSION: We conclude that employing reverse phenotyping in the evaluation of syndromic CAKUT genes by ES provides an important tool to facilitate molecular genetic diagnostics in CAKUT.

Ttc30a affects tubulin modifications in a model for ciliary chondrodysplasia with polycystic kidney disease.

Skeletal ciliopathies (e.g., Jeune syndrome, short rib polydactyly syndrome, and Sensenbrenner syndrome) are frequently associated with nephronophthisis-like cystic kidney disease and other organ manifestations. Despite recent progress in genetic mapping of causative loci, a common molecular mechanism of cartilage defects and cystic kidneys has remained elusive. Targeting two ciliary chondrodysplasia loci (ift80 and ift172) by CRISPR/Cas9 mutagenesis, we established models for skeletal ciliopathies in Xenopus tropicalis Froglets exhibited severe limb deformities, polydactyly, and cystic kidneys, closely matching the phenotype of affected patients. A data mining-based in silico screen found ttc30a to be related to known skeletal ciliopathy genes. CRISPR/Cas9 targeting replicated limb malformations and renal cysts identical to the models of established disease genes. Loss of Ttc30a impaired embryonic renal excretion and ciliogenesis because of altered posttranslational tubulin acetylation, glycylation, and defective axoneme compartmentalization. Ttc30a/b transcripts are enriched in chondrocytes and osteocytes of single-cell RNA-sequenced embryonic mouse limbs. We identify TTC30A/B as an essential node in the network of ciliary chondrodysplasia and nephronophthisis-like disease proteins and suggest that tubulin modifications and cilia segmentation contribute to skeletal and renal ciliopathy manifestations of ciliopathies in a cell type-specific manner. These findings have implications for potential therapeutic strategies.

Recessive NOS1AP variants impair actin remodeling and cause glomerulopathy in humans and mice.

Nephrotic syndrome (NS) is a leading cause of chronic kidney disease. We found recessive NOS1AP variants in two families with early-onset NS by exome sequencing. Overexpression of wild-type (WT) NOS1AP, but not cDNA constructs bearing patient variants, increased active CDC42 and promoted filopodia and podosome formation. Pharmacologic inhibition of CDC42 or its effectors, formin proteins, reduced NOS1AP-induced filopodia formation. NOS1AP knockdown reduced podocyte migration rate (PMR), which was rescued by overexpression of WT Nos1ap but not by constructs bearing patient variants. PMR in NOS1AP knockdown podocytes was also rescued by constitutively active CDC42Q61L or the formin DIAPH3 Modeling a NOS1AP patient variant in knock-in human kidney organoids revealed malformed glomeruli with increased apoptosis. Nos1apEx3-/Ex3- mice recapitulated the human phenotype, exhibiting proteinuria, foot process effacement, and glomerulosclerosis. These findings demonstrate that recessive NOS1AP variants impair CDC42/DIAPH-dependent actin remodeling, cause aberrant organoid glomerulogenesis, and lead to a glomerulopathy in humans and mice.

Generation of Monogenic Candidate Genes for Human Nephrotic Syndrome Using 3 Independent Approaches.

INTRODUCTION: Steroid-resistant nephrotic syndrome (SRNS) is the second most common cause of chronic kidney disease during childhood. Identification of 63 monogenic human genes has delineated 12 distinct pathogenic pathways. METHODS: Here, we generated 2 independent sets of nephrotic syndrome (NS) candidate genes to augment the discovery of additional monogenic causes based on whole-exome sequencing (WES) data from 1382 families with NS. RESULTS: We first identified 63 known monogenic causes of NS in mice from public databases and scientific publications, and 12 of these genes overlapped with the 63 known human monogenic SRNS genes. Second, we used a set of 64 genes that are regulated by the transcription factor Wilms tumor 1 (WT1), which causes SRNS if mutated. Thirteen of these WT1-regulated genes overlapped with human or murine NS genes. Finally, we overlapped these lists of murine and WT1 candidate genes with our list of 120 candidate genes generated from WES in 1382 NS families, to identify novel candidate genes for monogenic human SRNS. Using this approach, we identified 7 overlapping genes, of which 3 genes were shared by all datasets, including SYNPO. We show that loss-of-function of SYNPO leads to decreased CDC42 activity and reduced podocyte migration rate, both of which are rescued by overexpression of wild-type complementary DNA (cDNA), but not by cDNA representing the patient mutation. CONCLUSION: Thus, we identified 3 novel candidate genes for human SRNS using 3 independent, nonoverlapping hypotheses, and generated functional evidence for SYNPO as a novel potential monogenic cause of NS.

Recessive Mutations in SYNPO2 as a Candidate of Monogenic Nephrotic Syndrome.

INTRODUCTION: Most of the approximately 60 genes that if mutated cause steroid-resistant nephrotic syndrome (SRNS) are highly expressed in the glomerular podocyte, rendering SRNS a "podocytopathy." METHODS: We performed whole-exome sequencing (WES) in 1200 nephrotic syndrome (NS) patients. RESULTS: We discovered homozygous truncating and homozygous missense mutation in SYNPO2 (synaptopodin-2) (p.Lys1124∗ and p.Ala1134Thr) in 2 patients with childhood-onset NS. We found SYNPO2 expression in both podocytes and mesangial cells; however, notably, immunofluorescence staining of adult human and rat kidney cryosections indicated that SYNPO2 is localized mainly in mesangial cells. Subcellular localization studies reveal that in these cells SYNPO2 partially co-localizes with α-actinin and filamin A-containing F-actin filaments. Upon transfection in mesangial cells or podocytes, EGFP-SYNPO2 co-localized with α-actinin-4, which gene is mutated in autosomal dominant SRNS in humans. SYNPO2 overexpression increases mesangial cell migration rate (MMR), whereas shRNA knockdown reduces MMR. Decreased MMR was rescued by transfection of wild-type mouse Synpo2 cDNA but only partially by cDNA representing mutations from the NS patients. The increased mesangial cell migration rate (MMR) by SYNPO2 overexpression was inhibited by ARP complex inhibitor CK666. SYNPO2 shRNA knockdown in podocytes decreased active Rac1, which was rescued by transfection of wild-type SYNPO2 cDNA but not by cDNA representing any of the 2 mutant variants. CONCLUSION: We show that SYNPO2 variants may lead to Rac1-ARP3 dysregulation, and may play a role in the pathogenesis of nephrotic syndrome.

De novo TRIM8 variants impair its protein localization to nuclear bodies and cause developmental delay, epilepsy, and focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

Mutations in transcription factor CP2-like 1 may cause a novel syndrome with distal renal tubulopathy in humans.

BACKGROUND: An underlying monogenic cause of early-onset chronic kidney disease (CKD) can be detected in ∼20% of individuals. For many etiologies of CKD manifesting before 25 years of age, >200 monogenic causative genes have been identified to date, leading to the elucidation of mechanisms of renal pathogenesis. METHODS: In 51 families with echogenic kidneys and CKD, we performed whole-exome sequencing to identify novel monogenic causes of CKD. RESULTS: We discovered a homozygous truncating mutation in the transcription factor gene transcription factor CP2-like 1 (TFCP2L1) in an Arabic patient of consanguineous descent. The patient developed CKD by the age of 2 months and had episodes of severe hypochloremic, hyponatremic and hypokalemic alkalosis, seizures, developmental delay and hypotonia together with cataracts. We found that TFCP2L1 was localized throughout kidney development particularly in the distal nephron. Interestingly, TFCP2L1 induced the growth and development of renal tubules from rat mesenchymal cells. Conversely, the deletion of TFCP2L1 in mice was previously shown to lead to reduced expression of renal cell markers including ion transporters and cell identity proteins expressed in different segments of the distal nephron. TFCP2L1 localized to the nucleus in HEK293T cells only upon coexpression with its paralog upstream-binding protein 1 (UBP1). A TFCP2L1 mutant complementary DNA (cDNA) clone that represented the patient's mutation failed to form homo- and heterodimers with UBP1, an essential step for its transcriptional activity. CONCLUSION: Here, we identified a loss-of-function TFCP2L1 mutation as a potential novel cause of CKD in childhood accompanied by a salt-losing tubulopathy.

DAAM2 Variants Cause Nephrotic Syndrome via Actin Dysregulation.

The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections. Further, the variants impair DAAM2-dependent actin remodeling processes: wild-type DAAM2 cDNA, but not cDNA representing missense variants found in individuals with NS, rescued reduced podocyte migration rate (PMR) and restored reduced filopodia formation in shRNA-induced DAAM2-knockdown podocytes. Filopodia restoration was also induced by the formin-activating molecule IMM-01. DAAM2 also co-localizes and co-immunoprecipitates with INF2, which is intriguing since variants in both formins cause NS. Using in vitro bulk and TIRF microscopy assays, we find that DAAM2 variants alter actin assembly activities of the formin. In a Xenopus daam2-CRISPR knockout model, we demonstrate actin dysregulation in vivo and glomerular maldevelopment that is rescued by WT-DAAM2 mRNA. We conclude that DAAM2 variants are a likely cause of monogenic human SRNS due to actin dysregulation in podocytes. Further, we provide evidence that DAAM2-associated SRNS may be amenable to treatment using actin regulating compounds.

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.

Erratum: Azathioprine hypersensitivity syndrome in anti-myeloperoxidase anti-neutrophil cytoplasmic antibody-associated vasculitis.

[This corrects the article DOI: 10.1093/ckj/sfy038.].

Azathioprine hypersensitivity syndrome in anti-myeloperoxidase anti-neutrophil cytoplasmic antibody-associated vasculitis.

Two patients with anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and rapid onset of high fever, tachycardia and systemic hypotension accompanied by elevated laboratory markers of infection were diagnosed with azathioprine hypersensitivity syndrome only after repeat exposure. Azathioprine hypersensitivity can closely mimic sepsis and/or vasculitis activity and should be considered in AAV, a condition with frequent use of this drug. We discuss the pitfalls in diagnosis and the possible pathophysiologic background.

Loss of Kynurenine 3-Mono-oxygenase Causes Proteinuria.

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes.