Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes-Brocks syndrome 2.

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient's features overlapped Townes-Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1-/- murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

Inflammation-like changes in the urothelium of Lifr-deficient mice and LIFR-haploinsufficient humans with urinary tract anomalies.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause of end-stage kidney disease in children. While the genetic aberrations underlying CAKUT pathogenesis are increasingly being elucidated, their consequences on a cellular and molecular level commonly remain unclear. Recently, we reported rare heterozygous deleterious LIFR variants in 3.3% of CAKUT patients, including a novel de novo frameshift variant, identified by whole-exome sequencing, in a patient with severe bilateral CAKUT. We also demonstrated CAKUT phenotypes in Lifr-/- and Lifr+/- mice, including a narrowed ureteric lumen due to muscular hypertrophy and a thickened urothelium. Here, we show that both in the ureter and bladder of Lifr-/- and Lifr+/- embryos, differentiation of the three urothelial cell types (basal, intermediate and superficial cells) occurs normally but that the turnover of superficial cells is elevated due to increased proliferation, enhanced differentiation from their progenitor cells (intermediate cells) and, importantly, shedding into the ureteric lumen. Microarray-based analysis of genome-wide transcriptional changes in Lifr-/- versus Lifr+/+ ureters identified gene networks associated with an antimicrobial inflammatory response. Finally, in a reverse phenotyping effort, significantly more superficial cells were detected in the urine of CAKUT patients with versus without LIFR variants indicating conserved LIFR-dependent urinary tract changes in the murine and human context. Our data suggest that LIFR signaling is required in the epithelium of the urinary tract to suppress an antimicrobial response under homeostatic conditions and that genetically induced inflammation-like changes underlie CAKUT pathogenesis in Lifr deficiency and LIFR haploinsufficiency.

FOCAD loss impacts microtubule assembly, G2/M progression and patient survival in astrocytic gliomas.

In search of novel genes associated with glioma pathogenesis, we have previously shown frequent deletions of the KIAA1797/FOCAD gene in malignant gliomas, and a tumor suppressor function of the encoded focadhesin impacting proliferation and migration of glioma cells in vitro and in vivo. Here, we examined an association of reduced FOCAD gene copy number with overall survival of patients with astrocytic gliomas, and addressed the molecular mechanisms that govern the suppressive effect of focadhesin on glioma growth. FOCAD loss was associated with inferior outcome in patients with isocitrate dehydrogenase 1 or 2 (IDH)-mutant astrocytic gliomas of WHO grades II-IV. Multivariate analysis considering age at diagnosis as well as IDH mutation, MGMT promoter methylation, and CDKN2A/B homozygous deletion status confirmed reduced FOCAD gene copy number as a prognostic factor for overall survival. Using a yeast two-hybrid screen and pull-down assays, tubulin beta-6 and other tubulin family members were identified as novel focadhesin-interacting partners. Tubulins and focadhesin co-localized to centrosomes where focadhesin was enriched in proximity to centrioles. Focadhesin was recruited to microtubules via its interaction partner SLAIN motif family member 2 and reduced microtubule assembly rates, possibly explaining the focadhesin-dependent decrease in cell migration. During the cell cycle, focadhesin levels peaked in G2/M phase and influenced time-dependent G2/M progression potentially via polo like kinase 1 phosphorylation, providing a possible explanation for focadhesin-dependent cell growth reduction. We conclude that FOCAD loss may promote biological aggressiveness and worsen clinical outcome of diffuse astrocytic gliomas by enhancing microtubule assembly and accelerating G2/M phase progression.

Diagnostic Yield and Benefits of Whole Exome Sequencing in CAKUT Patients Diagnosed in the First Thousand Days of Life.

Introduction: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause of chronic kidney disease (CKD) and the need for kidney replacement therapy (KRT) in children. Although more than 60 genes are known to cause CAKUT if mutated, genetic etiology is detected, on average, in only 16% of unselected CAKUT cases, making genetic testing unproductive. Methods: Whole exome sequencing (WES) was performed in 100 patients with CAKUT diagnosed in the first 1000 days of life with CKD stages 1 to 5D/T. Variants in 58 established CAKUT-associated genes were extracted, classified according to the American College of Medical Genetics and Genomics guidelines, and their translational value was assessed. Results: In 25% of these mostly sporadic patients with CAKUT, a rare likely pathogenic or pathogenic variant was identified in 1 or 2 of 15 CAKUT-associated genes, including GATA3, HNF1B, LIFR, PAX2, SALL1, and TBC1D1. Of the 27 variants detected, 52% were loss-of-function and 18.5% de novo variants. The diagnostic yield was significantly higher in patients requiring KRT before 3 years of age (43%, odds ratio 2.95) and in patients with extrarenal features (41%, odds ratio 3.5) compared with patients lacking these criteria. Considering that all affected genes were previously associated with extrarenal complications, including treatable conditions, such as diabetes, hyperuricemia, hypomagnesemia, and hypoparathyroidism, the genetic diagnosis allowed preventive measures and/or early treatment in 25% of patients. Conclusion: WES offers significant advantages for the diagnosis and management of patients with CAKUT diagnosed before 3 years of age, especially in patients who require KRT or have extrarenal anomalies.

A SUMO4 initiator codon variant in amyotrophic lateral sclerosis reduces SUMO4 expression and alters stress granule dynamics.

BACKGROUND: Recent evidence points toward a role of the small ubiquitin-like modifier (SUMO) system, including SUMO4, in protecting from stress insults and neurodegeneration, such as the progressive motor neuron disease amyotrophic lateral sclerosis (ALS), e.g., by regulating stress granule (SG) dynamics. Here, we investigated whether SUMO4 variants play a role in ALS pathogenesis. METHODS: Whole-exome or targeted SUMO4 sequencing was done in 222 unrelated European ALS patients. The consequences of the identified initiator codon variant were analyzed at the mRNA, protein and cellular level. SUMO4 expression was quantified in human tissues. All patients were subjected to clinical, electrophysiological, and neuroradiological characterization. RESULTS: A rare heterozygous SUMO4 variant, i.e., SUMO4:c.2T>C p.Met1?, was detected in four of 222 (1.8%) ALS patients, significantly more frequently than in two control cohorts (0.3% each). SUMO4 mRNA and protein expression was diminished in whole blood or fibroblasts of a SUMO4 variant carrier versus controls. Pertinent stress factors, i.e., head trauma or cancer (treated by radiochemotherapy), were significantly more frequent in SUMO4 variant carrier versus non-carrier ALS patients. The mean number of SGs per cell was significantly higher in fibroblasts of a SUMO4 variant carrier compared to controls at baseline, upon oxidative stress, and after recovery, and SUMOylation of ALS-associated valosin-containing protein by SUMO4 was decreased. SUMO4 mRNA expression was highest in brain of all human tissues analyzed. CONCLUSIONS: Our results are consistent with SUMO4 haploinsufficiency as a contributor to ALS pathogenesis impacting SG dynamics and possibly acting in conjunction with environmental oxidative stress-related factors.

SPG7 mutations in amyotrophic lateral sclerosis: a genetic link to hereditary spastic paraplegia.

Amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP) are motor neuron diseases sharing clinical, pathological, and genetic similarities. While biallelic SPG7 mutations are known to cause recessively inherited HSP, heterozygous SPG7 mutations have repeatedly been identified in HSP and recently also in ALS cases. However, the frequency and clinical impact of rare SPG7 variants have not been studied in a larger ALS cohort. Here, whole-exome (WES) or targeted SPG7 sequencing was done in a cohort of 214 European ALS patients. The consequences of a splice site variant were analyzed on the mRNA level. The resulting protein alterations were visualized in a crystal structure model. All patients were subjected to clinical, electrophysiological, and neuroradiological characterization. In 9 of 214 (4.2%) ALS cases, we identified five different rare heterozygous SPG7 variants, all of which were previously reported in patients with HSP or ALS. All detected SPG7 variants affect the AAA+ domain of the encoded mitochondrial metalloprotease paraplegin and impair its stability or function according to predictions from mRNA analysis or crystal structure modeling. ALS patients with SPG7 mutations more frequently presented with cerebellar symptoms, flail arm or leg syndrome compared to those without SPG7 mutations, and showed a partial clinical overlap with HSP. Brain MRI findings in SPG7 mutation carriers included cerebellar atrophy and patterns suggestive of frontotemporal dementia. Collectively, our findings suggest that SPG7 acts as a genetic risk factor for ALS. ALS patients carrying SPG7 mutations present with distinct features overlapping with HSP, particularly regarding cerebellar findings.

Rare heterozygous GDF6 variants in patients with renal anomalies.

Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis.