Natural History and Clinicopathological Associations of TRPC6-Associated Podocytopathy.

BACKGROUND: Understanding the genetic basis of human diseases has become integral to drug development and precision medicine. Recent advancements have enabled the identification of molecular pathways driving diseases, leading to targeted treatment strategies. The increasing investment in rare diseases by the biotech industry underscores the importance of genetic evidence in drug discovery and approval processes. Here we studied a monogenic Mendelian kidney disease, TRPC6-associated podocytopathy (TRPC6-AP), to present its natural history, genetic spectrum, and clinicopathological associations in a large cohort of patients with causal variants in TRPC6, in order to help define the specific features of disease and further facilitate drug development and clinical trials design. METHODS: the study involved 64 individuals from 39 families with TRPC6 causal missense variants. Clinical data, including age of onset, laboratory results, response to treatment, kidney biopsy findings, and genetic information, were collected from multiple centers nationally and internationally. Exome or targeted sequencing was performed and variant classification was based on strict criteria. Structural and functional analyses of TRPC6 variants were conducted to understand their impact on protein function. In depth re-analysis of light and electron microscopy specimens for 9 available kidney biopsies was conducted to identify pathological features and correlates of TRPC6-AP. RESULTS: Large-scale sequencing data did not support causality for TRPC6 protein-truncating variants. We identified 21 unique TRPC6 missense variants, clustering in three distinct regions of the protein, and with different effects on TRPC6 3D protein structure. Kidney biopsy analysis revealed FSGS patterns of injury in most cases, along with distinctive podocyte features including diffuse foot process effacement and swollen cell bodies. The majority of patients presented in adolescence or early adulthood but with ample variation (average 22, SD ± 14 years), with frequent progression to kidney failure but with variability in time between presentation and ESKD. CONCLUSIONS: This study provides insights into the genetic spectrum, clinicopathological associations, and natural history of TRPC6-AP.

Risk of meningomyelocele mediated by the common 22q11.2 deletion.

Meningomyelocele is one of the most severe forms of neural tube defects (NTDs) and the most frequent structural birth defect of the central nervous system. We assembled the Spina Bifida Sequencing Consortium to identify causes. Exome and genome sequencing of 715 parent-offspring trios identified six patients with chromosomal 22q11.2 deletions, suggesting a 23-fold increased risk compared with the general population. Furthermore, analysis of a separate 22q11.2 deletion cohort suggested a 12- to 15-fold increased NTD risk of meningomyelocele. The loss of Crkl, one of several neural tube-expressed genes within the minimal deletion interval, was sufficient to replicate NTDs in mice, where both penetrance and expressivity were exacerbated by maternal folate deficiency. Thus, the common 22q11.2 deletion confers substantial meningomyelocele risk, which is partially alleviated by folate supplementation.

Strong protective effect of the APOL1 p.N264K variant against G2-associated focal segmental glomerulosclerosis and kidney disease.

African Americans have a significantly higher risk of developing chronic kidney disease, especially focal segmental glomerulosclerosis -, than European Americans. Two coding variants (G1 and G2) in the APOL1 gene play a major role in this disparity. While 13% of African Americans carry the high-risk recessive genotypes, only a fraction of these individuals develops FSGS or kidney failure, indicating the involvement of additional disease modifiers. Here, we show that the presence of the APOL1 p.N264K missense variant, when co-inherited with the G2 APOL1 risk allele, substantially reduces the penetrance of the G1G2 and G2G2 high-risk genotypes by rendering these genotypes low-risk. These results align with prior functional evidence showing that the p.N264K variant reduces the toxicity of the APOL1 high-risk alleles. These findings have important implications for our understanding of the mechanisms of APOL1-associated nephropathy, as well as for the clinical management of individuals with high-risk genotypes that include the G2 allele.

Mouse and human studies support DSTYK loss of function as a low-penetrance and variable expressivity risk factor for congenital urinary tract anomalies.

PURPOSE: Previous work identified rare variants in DSTYK associated with human congenital anomalies of the kidney and urinary tract (CAKUT). Here, we present a series of mouse and human studies to clarify the association, penetrance, and expressivity of DSTYK variants. METHODS: We phenotypically characterized Dstyk knockout mice of 3 separate inbred backgrounds and re-analyzed the original family segregating the DSTYK c.654+1G>A splice-site variant (referred to as "SSV" below). DSTYK loss of function (LOF) and SSVs were annotated in individuals with CAKUT, epilepsy, or amyotrophic lateral sclerosis vs controls. A phenome-wide association study analysis was also performed using United Kingdom Biobank (UKBB) data. RESULTS: Results demonstrate ∼20% to 25% penetrance of obstructive uropathy, at least, in C57BL/6J and FVB/NJ Dstyk-/- mice. Phenotypic penetrance increased to ∼40% in C3H/HeJ mutants, with mild-to-moderate severity. Re-analysis of the original family segregating the rare SSV showed low penetrance (43.8%) and no alternative genetic causes for CAKUT. LOF DSTYK variants burden showed significant excess for CAKUT and epilepsy vs controls and an exploratory phenome-wide association study supported association with neurological disorders. CONCLUSION: These data support causality for DSTYK LOF variants and highlights the need for large-scale sequencing studies (here >200,000 cases) to accurately assess causality for genes and variants to lowly penetrant traits with common population prevalence.

Strong protective effect of the APOL1 p.N264K variant against G2-associated focal segmental glomerulosclerosis and kidney disease.

Black Americans have a significantly higher risk of developing chronic kidney disease (CKD), especially focal segmental glomerulosclerosis (FSGS), than European Americans. Two coding variants (G1 and G2) in the APOL1 gene play a major role in this disparity. While 13% of Black Americans carry the high-risk recessive genotypes, only a fraction of these individuals develops FSGS or kidney failure, indicating the involvement of additional disease modifiers. Here, we show that the presence of the APOL1 p.N264K missense variant, when co-inherited with the G2 APOL1 risk allele, substantially reduces the penetrance of the G1G2 and G2G2 high-risk genotypes by rendering these genotypes low-risk. These results align with prior functional evidence showing that the p.N264K variant reduces the toxicity of the APOL1 high-risk alleles. These findings have important implications for our understanding of the mechanisms of APOL1 -associated nephropathy, as well as for the clinical management of individuals with high-risk genotypes that include the G2 allele.

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.

Author Correction: The copy number variation landscape of congenital anomalies of the kidney and urinary tract.

In the version of this article initially published, affiliation 38 incorrectly read "ICNU-Nephrology and Urology Department, Barcelona, Spain"; "Renal Division, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain" is the correct affiliation. The error has been corrected in the HTML and PDF versions of the article.

The copy number variation landscape of congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) are a major cause of pediatric kidney failure. We performed a genome-wide analysis of copy number variants (CNVs) in 2,824 cases and 21,498 controls. Affected individuals carried a significant burden of rare exonic (that is, affecting coding regions) CNVs and were enriched for known genomic disorders (GD). Kidney anomaly (KA) cases were most enriched for exonic CNVs, encompassing GD-CNVs and novel deletions; obstructive uropathy (OU) had a lower CNV burden and an intermediate prevalence of GD-CNVs; and vesicoureteral reflux (VUR) had the fewest GD-CNVs but was enriched for novel exonic CNVs, particularly duplications. Six loci (1q21, 4p16.1-p16.3, 16p11.2, 16p13.11, 17q12 and 22q11.2) accounted for 65% of patients with GD-CNVs. Deletions at 17q12, 4p16.1-p16.3 and 22q11.2 were specific for KA; the 16p11.2 locus showed extensive pleiotropy. Using a multidisciplinary approach, we identified TBX6 as a driver for the CAKUT subphenotypes in the 16p11.2 microdeletion syndrome.

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10-5 for novel LOF, increased to p = 4.1 × 10-6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10-7). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD.

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome.

BACKGROUND: The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS: We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS: We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10-14). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS: We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.).

A susceptibility gene for kidney disease in an obese mouse model of type II diabetes maps to chromosome 8.

Most mouse models of diabetes do not fully reproduce features of human diabetic nephropathy, limiting their utility in inferring mechanisms of human disease. Here we performed detailed phenotypic and genetic characterization of leptin-receptor (Lepr) deficient mice on the FVB/NJ background (FVB(db/db)), an obese model of type II diabetes, to determine their suitability to model human diabetic nephropathy. These mice have sustained hyperglycemia, significant albuminuria and characteristic diabetic renal findings including mesangial sclerosis and nodular glomerulosclerosis after 6 months of age. In contrast, equally obese, hyperglycemic Lepr/Sur1 deficient C57BL/6J (Sur1 has defective insulin secretion) mice have minimal evidence of nephropathy. A genome-wide scan in 165 Lepr deficient backcross progeny derived from FVB/NJ and C57BL/6J identified a major locus influencing nephropathy and albuminuria on chromosome 8B1-C5 (Dbnph1 locus, peak lod score 5.0). This locus was distinct from those contrasting susceptibility to beta cell hypertrophy and HIV-nephropathy between the same parental strains, indicating specificity to diabetic kidney disease. Genome-wide expression profiling showed that high and low risk Dbnph1 genotypes were associated with significant enrichment for oxidative phosphorylation and lipid clearance, respectively; molecular pathways shared with human diabetic nephropathy. Hence, we found that the FVB(db/db) mouse recapitulates many clinical, histopathological and molecular features of human diabetic nephropathy. Identifying underlying susceptibility gene(s) and downstream dysregulated pathways in these mice may provide insight into the disease pathogenesis in humans.

Susceptibility loci for murine HIV-associated nephropathy encode trans-regulators of podocyte gene expression.

Multiple studies have linked podocyte gene variants to diverse sporadic nephropathies, including HIV-1-associated nephropathy (HIVAN). We previously used linkage analysis to identify a major HIVAN susceptibility locus in mouse, HIVAN1. We performed expression quantitative trait locus (eQTL) analysis of podocyte genes in HIV-1 transgenic mice to gain further insight into genetic susceptibility to HIVAN. In 2 independent crosses, we found that transcript levels of the podocyte gene nephrosis 2 homolog (Nphs2), were heritable and controlled by an ancestral cis-eQTL that conferred a 3-fold variation in expression and produced reactive changes in other podocyte genes. In addition, Nphs2 expression was controlled by 2 trans-eQTLs that localized to the nephropathy susceptibility intervals HIVAN1 and HIVAN2. Transregulation of podocyte genes was observed in the absence of HIV-1 or glomerulosclerosis, indicating that nephropathy susceptibility alleles induce latent perturbations in the podocyte expression network. Presence of the HIV-1 transgene interfered with transregulation, demonstrating effects of gene-environment interactions on disease. These data demonstrate that transcript levels of Nphs2 and related podocyte-expressed genes are networked and suggest that the genetic lesions introduced by HIVAN susceptibility alleles perturb this regulatory pathway and transcriptional responses to HIV-1, increasing susceptibility to nephropathy.

Accelerated development of collapsing glomerulopathy in mice congenic for the HIVAN1 locus.

HIV-1 transgenic mice on the FVB/NJ background (TgFVB) are a well validated model of HIV-associated nephropathy (HIVAN). A mapping study between TgFVB and CAST/EiJ (CAST) strains showed this trait to be influenced by a major susceptibility locus on chromosome 3A1-A3 (HIVAN1), with CAST alleles associated with increased risk of disease. We introgressed a 50 Mb interval, encompassing this HIVAN1 locus, from CAST into the TgFVB genome (TgFVB-HIVAN1(CAST) congenic mice). Compared to the TgFVB strain, these congenic mice developed an earlier onset of proteinuria, a rapid progression to kidney failure, and increased mortality. A prospective study of these congenic mice also showed that they had a significantly greater histologic and biochemical evidence of glomerulopathy with one-third of mice developing global glomerulosclerosis by 6 weeks of age. An F2 cross between TgFVB and the congenic mice identified a significant linkage (LOD=3.7) to a 10 cM interval within the HIVAN1 region between D3Mit167 and D3Mit67 resulting in a 60% reduction of the original interval. These data independently confirm that a gene on chromosome 3A1-A3 increases susceptibility to HIVAN, resulting in early onset and rapid progression of kidney disease. These mice represent a new model to study the development and progression of collapsing glomerulopathy.

Genetic susceptibility, HIV infection, and the kidney.

In recent years, the sequencing of mammalian and microbial genomes has provided the opportunity to study how genetic variation in the host and pathogen influence the course of infectious disease. In the case of HIV-1 infection, such studies have led to identification of key viral proteins that determine pathogenicity, immune evasion, or drug resistance. In addition, candidate gene association studies have uncovered a large number of host genetic variants that influence the outcome of infection and some organ-specific complications. HIV-associated nephropathy (HIVAN) is a pathologically distinct complication of HIV infection. Interindividual variability in incidence, skewed ethnic distribution, and familial aggregation of HIVAN with other forms of ESRD have suggested genetic susceptibility as a major contributing factor. This article reviews the host genetic factors that influence the course of HIV-1 infection and discusses murine models that have increased the understanding of HIVAN pathogenesis and demonstrated the role of genetic background on determination of disease.

Associative Pavlovian conditioning leads to an increase in spinophilin-immunoreactive dendritic spines in the lateral amygdala.

Changes in dendritic spine number and shape are believed to reflect structural plasticity consequent to learning. Previous studies have strongly suggested that the dorsal subnucleus of the lateral amygdala is an important site of physiological plasticity in Pavlovian fear conditioning. In the present study, we examined the effect of auditory fear conditioning on dendritic spine numbers in the dorsal subnucleus of the lateral amygdala using an immunolabelling procedure to visualize the spine-associated protein spinophilin. Associatively conditioned rats that received paired tone and shock presentations had 35% more total spinophilin-immunoreactive spines than animals that had unpaired stimulation, consistent with the idea that changes in the number of dendritic spines occur during learning and account in part for memory.