Search

Half of patients with a ciliopathy syndrome remain unsolved after initial analysis of whole exome sequencing (WES) data, highlighting the need for improved variant filtering and annotation. By candidate gene curation of WES data, combined with homozygosity mapping, we detected a homozygous predicted synonymous allele in NPHP3 in two children with hepatorenal fibrocystic disease from a consanguineous family. Analyses on patient-derived RNA shows activation of a cryptic mid-exon splice donor leading to frameshift. Remarkably, the same rare variant was detected in four additional families with hepatorenal disease from UK, US, and Saudi patient cohorts and in addition, another synonymous NPHP3 variant was identified in an unsolved case from the Genomics England 100,000 Genomes data set. We conclude that synonymous NPHP3 variants, not reported before and discarded by pathogenicity pipelines, solved several families with a ciliopathy syndrome. These findings prompt careful reassessment of synonymous variants, especially if they are rare and located in candidate genes.

Background Mutations in ciliary genes cause a spectrum of both overlapping and distinct clinical syndromes (ciliopathies). CEP120 and CC2D2A are paradigmatic examples for this genetic heterogeneity and pleiotropy as mutations in both cause Joubert syndrome but are also associated with skeletal ciliopathies and Meckel syndrome, respectively. The molecular basis for this phenotypical variability is not understood but basal exon skipping likely contributes to tolerance for deleterious mutations via tissue‐specific preservation of the amount of expressed functional protein. Methods We systematically reviewed and annotated genetic variants and clinical presentations reported in CEP120‐ and CC2D2A‐associated disease and we combined in silico and ex vivo approaches to study tissue‐specific transcripts and identify molecular targets for exon skipping. Results We confirmed more severe clinical presentations associated with truncating CC2D2A mutations. We identified and confirmed basal exon skipping in the kidney, with possible relevance for organ‐specific disease manifestations. Finally, we proposed a multimodal approach to classify exons amenable to exon skipping. By mapping reported variants, 14 truncating mutations in 7 CC2D2A exons were identified as potentially rescuable by targeted exon skipping, an approach that is already in clinical use for other inherited human diseases. Conclusion Genotype‐phenotype correlations for CC2D2A support the deleteriousness of null alleles and CC2D2A, but not CEP120, offers potential for therapeutic exon skipping approaches., Mutations in CEP120 and CC2D2A cause Joubert syndrome but are also associated with skeletal ciliopathies and Meckel syndrome, respectively. Here we annotate genetic variants described in CEP120‐ and CC2D2A‐associated disease and confirm more severe clinical presentations with biallelic truncating CC2D2A mutations. Combining in silico and ex vivo studies, we identify alternative basal exon skipping in the kidney, with possible relevance for organ‐specific disease manifestations and propose a multimodal approach to classify exons amenable to exon skipping.

Joubert syndrome (JBTS) is an incurable multisystem ciliopathy syndrome. The most commonly mutated gene in JBTS patients with a cerebello-retinal-renal phenotype is CEP290 (alias JBTS5). The encoded CEP290 protein localises to the proximal end of the primary cilium, in the transition zone, where it controls ciliary protein composition and signalling. We examined primary cilium structure and composition in fibroblast cells derived from homozygous and compound heterozygous JBTS5 patients with nonsense mutations in CEP290 and show that elongation of cilia, impaired ciliogenesis and ciliary composition defects are typical features in JBTS5 cells. Targeted skipping of the mutated exon c.5668 G > T using antisense oligonucleotide (ASO) therapy leads to restoration of CEP290 protein expression and functions at the transition zone in homozygous and compound heterozygous JBTS5 cells, allowing a rescue of both cilia morphology and ciliary composition. This study, by demonstrating that targeted exon skipping is able to rescue ciliary protein composition defects, provides functional evidence for the efficacy of this approach in the treatment of JBTS.

Significance The treatment of genetic kidney disease is challenging, as this requires both the correction of the underlying gene defect and the delivery of the treatment. Here we show that by using antisense oligonucleotides, we can induce exon skipping of a mutated exon in CEP290, within renal epithelial cells derived from a patient with a ciliopathy syndrome called Joubert syndrome. This treatment rescues the truncated CEP290 protein to a near full-length protein and restores the ciliary phenotype. In a Cep290 murine model of Joubert syndrome, exon skipping is achievable with systemic treatment of an antisense oligonucleotide, which rescues both the ciliary and kidney disease phenotypes. This work paves the way toward personalized genetic therapies in patients with inherited kidney diseases., Genetic treatments of renal ciliopathies leading to cystic kidney disease would provide a real advance in current therapies. Mutations in CEP290 underlie a ciliopathy called Joubert syndrome (JBTS). Human disease phenotypes include cerebral, retinal, and renal disease, which typically progresses to end stage renal failure (ESRF) within the first two decades of life. While currently incurable, there is often a period of years between diagnosis and ESRF that provides a potential window for therapeutic intervention. By studying patient biopsies, patient-derived kidney cells, and a mouse model, we identify abnormal elongation of primary cilia as a key pathophysiological feature of CEP290-associated JBTS and show that antisense oligonucleotide (ASO)-induced splicing of the mutated exon (41, G1890*) restores protein expression in patient cells. We demonstrate that ASO-induced splicing leading to exon skipping is tolerated, resulting in correct localization of CEP290 protein to the ciliary transition zone, and restoration of normal cilia length in patient kidney cells. Using a gene trap Cep290 mouse model of JBTS, we show that systemic ASO treatment can reduce the cystic burden of diseased kidneys in vivo. These findings indicate that ASO treatment may represent a promising therapeutic approach for kidney disease in CEP290-associated ciliopathy syndromes.

BackgroundJoubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genesARL3andCEP120.MethodsWe sought to explore the developmental expression patterns ofARL3andCEP120in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characteriseARL3andCEP120expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource.ResultsBothARL3andCEP120are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes.ConclusionsThis study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology, however the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5’ and 3’ end, with >99% having one of two seed sequences (5 ‘ bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3 ‘UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published datasets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

The majority of multi-exon genes undergo alternative splicing to produce different mRNA transcripts and this may occur in a tissue-specific manner. Assessment of mRNA transcripts isolated from blood samples may sometimes be unhelpful in determining the affect on function of putative splice-site variants affecting kidney-specific mRNA transcripts. Here we present data demonstrating the power of using human urine-derived renal epithelial cells (hUREC) as a source of kidney RNA. We report clinical and molecular genetic data from three affected cases from two families all with end-stage renal disease by 15 years of age. In both families, heterozygous variants which are predicted to effect function in NPHP3 were found on one allele, in combination with a synonymous SNV (c.2154C>T; p.Phe718=), 18 base pairs from the exon–intron boundary within exon 15 of NPHP3. The only mRNA transcript amplified from wild-type whole blood showed complete splicing out of exon 15. Urine samples obtained from control subjects and the father of family 2, who carried the synonymous SNV variant, were therefore used to culture hUREC and allowed us to obtain kidney-specific mRNA. Control kidney mRNA showed retention of exon 15, while the mRNA from the patient’s father confirmed evidence of a heterozygous alternate splicing of exon 15 of NPHP3. Analysis of RNA derived from hUREC allows for a comparison of kidney-specific and whole-blood RNA transcripts and for assessment of the effect on function of putative splice variants leading to end-stage kidney disease.

Joubert syndrome (JBTS) is the archetypal ciliopathy caused by mutation of genes encoding ciliary proteins leading to multi-system phenotypes, including a cerebello-retinal-renal syndrome. JBTS is genetically heterogeneous, however mutations in CEP290 are a common underlying cause. The renal manifestation of JBTS is a juvenile-onset cystic kidney disease, known as nephronophthisis, typically progressing to end-stage renal failure within the first two decades of life, thus providing a potential window for therapeutic intervention. In order to increase understanding of JBTS and its associated kidney disease and to explore potential treatments, we conducted a comprehensive analysis of primary renal epithelial cells directly isolated from patient urine (human urine-derived renal epithelial cells, hURECs). We demonstrate that hURECs from a JBTS patient with renal disease have elongated and disorganized primary cilia and that this ciliary phenotype is specifically associated with an absence of CEP290 protein. Treatment with the Sonic hedgehog (Shh) pathway agonist purmorphamine or cyclin-dependent kinase inhibition (using roscovitine and siRNA directed towards cyclin-dependent kinase 5) ameliorated the cilia phenotype. In addition, purmorphamine treatment was shown to reduce cyclin-dependent kinase 5 in patient cells, suggesting a convergence of these signalling pathways. To our knowledge, this is the most extensive analysis of primary renal epithelial cells from JBTS patients to date. It demonstrates the feasibility and power of this approach to directly assess the consequences of patient-specific mutations in a physiologically relevant context and a previously unrecognized convergence of Shh agonism and cyclin-dependent kinase inhibition as potential therapeutic targets.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997–109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of inherited genetic disorders that share a defect in the formation, maintenance or functioning of the primary cilium complex, causing progressive cystic kidney disease and other clinical manifestations. Mutations in centrosomal protein 164 kDa (CEP164), also known as NPHP15, have been identified as a cause of NPHP-RC. Here we have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) to perform immunohistochemistry studies on human embryonic and foetal tissues to determine the expression patterns of CEP164 during development. Notably expression is widespread, yet defined, in multiple organs including the kidney, retina and cerebellum. Murine studies demonstrated an almost identical Cep164 expression pattern. Taken together, these data support a conserved role for CEP164 throughout the development of numerous organs, which, we suggest, accounts for the multi-system disease phenotype of CEP164-mediated NPHP-RC.

Juvenile ciliopathy syndromes that are associated with renal cysts and premature renal failure are commonly the result of mutations in the gene encoding centrosomal protein CEP290. In addition to centrosomes and the transition zone at the base of the primary cilium, CEP290 also localizes to the nucleus; however, the nuclear function of CEP290 is unknown. Here, we demonstrate that reduction of cellular CEP290 in primary human and mouse kidney cells as well as in zebrafish embryos leads to enhanced DNA damage signaling and accumulation of DNA breaks ex vivo and in vivo. Compared with those from WT mice, primary kidney cells from Cep290-deficient mice exhibited supernumerary centrioles, decreased replication fork velocity, fork asymmetry, and increased levels of cyclin-dependent kinases (CDKs). Treatment of Cep290-deficient cells with CDK inhibitors rescued DNA damage and centriole number. Moreover, the loss of primary cilia that results from CEP290 dysfunction was rescued in 3D cell culture spheroids of primary murine kidney cells after exposure to CDK inhibitors. Together, our results provide a link between CEP290 and DNA replication stress and suggest CDK inhibition as a potential treatment strategy for a wide range of ciliopathy syndromes.

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and highly expressed in a wide variety of other malignancies. WT1 is a transcription factor that is likely to have additional, post-transcriptional, regulatory roles, although the molecular mechanisms by which WT1 acts remain poorly understood. We have combined genetic and biochemical approaches to show, that endogenous WT1 binds to heterogeneous nuclear ribonuclear protein U (hnRNP-U), that this interaction does not require any other proteins or nucleic acids, involves the zinc-fingers of WT1 and the middle domain of hnRNP-U, and that hnRNP-U can modulate WT1 transcriptional activation of a bona fide WT1 target gene. These findings increase our knowledge of how WT1 exerts its transcriptional regulatory role and suggests that hnRNP-U may be a candidate Wilms' tumour gene at 1q44.

The Wilms tumor-suppressor gene, WT1 , plays a key role in urogenital development, and WT1 dysfunction is implicated in both neoplastic (Wilms tumor, mesothelioma, leukemias, and breast cancer) and nonneoplastic (glomerulosclerosis) disease. The analysis of diseases linked specifically with WT1 mutations, such as Denys–Drash syndrome (DDS), can provide valuable insight concerning the role of WT1 in development and disease. DDS is a rare childhood disease characterized by a nephropathy involving mesangial sclerosis, XY pseudohermaphroditism, and/or Wilms tumor (WT). DDS patients are constitutionally heterozygous for exonic point mutations in WT1 , which include mutations predicted to truncate the protein within the C-terminal zinc finger (ZF) region. We report that heterozygosity for a targeted murine Wt1 allele, Wt1 tmT396 , which truncates ZF3 at codon 396, induces mesangial sclerosis characteristic of DDS in adult heterozygous and chimeric mice. Male genital defects also were evident and there was a single case of Wilms tumor in which the transcript of the nontargeted allele showed an exon 9 skipping event, implying a causal link between Wt1 dysfunction and Wilms tumorigenesis in mice. However, the mutant WT1 tmT396 protein accounted for only 5% of WT1 in both heterozygous embryonic stem cells and the WT. This has implications regarding the mechanism by which the mutant allele exerts its effect.

The Wilms’ tumour suppressor gene, WT1, is mutated/deleted in approximately 15% of Wilms’ tumours, highly expressed in the majority of other cancers and is essential for normal embryonic development. The gene encodes multiple isoforms of a zinc-finger protein with diverse cellular functions, in particular participating in both transcriptional and post-transcriptional gene regulation. Physical interactions of other cellular proteins with WT1 are known to modulate its function. However, despite the isolation of several WT1-binding proteins, the mechanisms involved in regulating WT1 activities are not clearly understood. In this study we report the identification of the Krüppel-like zinc finger protein, ZNF224, as a novel human WT1-associating protein and demonstrate that ZNF224 and its isoform ZNF255 show a specific pattern of interaction with the WT1 splicing variants WT1(-KTS) and WT1(+KTS). These interactions occur in different sub-cellular compartments and are devoted to control different cellular pathways. The nuclear interaction between ZNF224 and WT1(-KTS) results in an increase in trascriptional activation mediated by WT1, implying that ZNF224 acts as a co-regulator of WT1, whereas, on the contrary, the results obtained for ZNF255 suggest a role for this protein in RNA processing together with WT1. Moreover, our data give the first functional information about the involvement of ZNF255 in a specific molecular pathway, RNA maturation and processing.

Mutations in the WT1 gene are found in a subset of Wilms' tumours and in certain other disorders such as Denys-Drash syndrome. The WT1 gene product is a zinc finger transcription factor for which many target genes have been suggested. Here we utilise gene targeting to generate cells containing only truncated forms of WT1, in which the DNA-binding region is disrupted. Examination of gene expression in these cells using cDNA macroarrays suggests two novel WT1 transcriptional targets, cyclin G1 (Ccng1), and insulin-like growth factor binding protein 4 (Igfbp4).

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.