Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Knockout mouse models as a resource for the study of rare diseases.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.

AOX delays the onset of the lethal phenotype in a mouse model of Uqcrh (complex III) disease.

The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.

Implication of FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

Post-synaptic scaffold protein TANC2 in psychiatric and somatic disease risk.

Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2)-disrupting variants were disease causing in NDD patients. The post-synaptic scaffold protein TANC2 is essential for dendrite formation in synaptic plasticity and plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2-disrupted function model in which mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain, such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility, underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods.

EUCOMM--the European conditional mouse mutagenesis program.

Functional analysis of the mammalian genome is an enormous challenge for biomedical scientists. To facilitate this endeavour, the European Conditional Mouse Mutagenesis Program (EUCOMM) aims at generating up to 12 000 mutations by gene trapping and up to 8000 mutations by gene targeting in mouse embryonic stem (ES) cells. These mutations can be rendered into conditional alleles, allowing Cre recombinase-mediated disruption of gene function in a time- and tissue-specific manner. Furthermore, the EUCOMM program will generate up to 320 mouse lines from the EUCOMM resource and up to 20 new Cre driver mouse lines. The EUCOMM resource of vectors, mutant ES cell lines and mutant mice will be openly available to the scientific community. EUCOMM will be one of the cornerstones of an international effort to create a global mouse mutant resource.

High-throughput trapping of secretory pathway genes in mouse embryonic stem cells.

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.

Genomewide production of multipurpose alleles for the functional analysis of the mouse genome.

A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.

L-type Ca2+ channels of the embryonic mouse heart.

In the heart, where Ca(2+) influx across the sarcolemma is essential for contraction, L-type Ca(2+) channels represent the major entry pathway of Ca(2+). Mice with a homozygous deletion of the L-type Ca(v)1.2 Ca(2+) channel gene die before day 14.5 p.c. Electrophysiological and pharmacological investigations on Ca(v)1.2-/- cardiomyocytes demonstrated that contractions depended on the influx of Ca(2+) through an L-type-like Ca(2+) channel. We analyzed now the expression pattern of various L-type Ca(2+) channels. Amplification of the alternative exons 1a and 1b revealed that embryonic cardiac cells express both Ca(v)1.2a and Ca(v)1.2b subunits. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications indicated the expression of Ca(v)1.1 and Ca(v)1.3 in about a 1:10 ratio in Ca(v)1.2-/- embryos. Two different amino termini of the Ca(v)1.3 cDNA were found in the embryonic heart, which both gave rise to functional channels. Ca(v)1.3(1a) and Ca(v)1.3(1b) channels have similar current kinetics and voltage-dependencies as described for Ca(v)1.3(8A) channels [J. Biol. Chem. 276 (2001) 22100], but the properties of Ca(v)1.3(1a) or Ca(v)1.3(1b) channels are different from that of the L-type-like current in Ca(v)1.2-/- cardiomyocytes. The I(Ba) of Ca(v)1.3(1a) was blocked by the dihydropyridine nisoldipine with an IC(50) value of 0.13 microM at a holding potential of -80 mV. In embryonic Ca(v)1.2+/+ cardiomyocytes, I(Ba) was blocked by nisoldipine with an IC(50) value of 0.1 microM. Although the expressed Ca(v)1.3 channel has a similar affinity for nisoldipine as Ca(v)1.2+/+ cardiomyocytes, the L-type-like Ca(2+) channel found in Ca(v)1.2+/+ and -/- cardiomyocytes is not identical with the new Ca(v)1.3 splice variants.

[Potential screening model for tissue specific calcium channel blockers]

Cardiovascular diseases are still the major cause of death in industrialized countries. The available therapies need improvement and specially tissue specific drugs. We have tested the possibility to establish cell lines expressing tissue specific cardiac muscular or smooth muscle L-type calcium channels. The cardiac and the smooth muscle L-type calcium channel differ in the IS6 segment of the alphaIC-gene. The established cell lines expressing the alphaIC-a or the alphaIC-b splice variants of the alphaIC-gene can be used for the screening of tissue specific calcium channel blockers.