Transcriptome analysis of neural progenitor cells derived from Lowe syndrome induced pluripotent stem cells: identification of candidate genes for the neurodevelopmental and eye manifestations.
ABSTRACT: BACKGROUND:Lowe syndrome (LS) is caused by loss-of-function mutations in the X-linked gene OCRL, which codes for an inositol polyphosphate 5-phosphatase that plays a key role in endosome recycling, clathrin-coated pit formation, and actin polymerization. It is characterized by congenital cataracts, intellectual and developmental disability, and renal proximal tubular dysfunction. Patients are also at high risk for developing glaucoma and seizures. We recently developed induced pluripotent stem cell (iPSC) lines from three patients with LS who have hypomorphic variants affecting the 3' end of the gene, and their neurotypical brothers to serve as controls. METHODS:In this study, we used RNA sequencing (RNA-seq) to obtain transcriptome profiles in LS and control neural progenitor cells (NPCs). RESULTS:In a comparison of the patient and control NPCs (n = 3), we found 16 differentially expressed genes (DEGs) at the multiple test adjusted p value (padj)
Project description:Lowe Syndrome (LS) is caused by loss-of-function mutations in the X-linked gene OCRL that codes for a 901 amino acid protein, inositol polyphosphate 5-phosphatase, which plays a key role in endosome recycling, clathrin coated pit formation and actin polymerization. It is characterized by congenital cataracts, intellectual and developmental disability, and renal proximal tubular dysfunction. Patients are also at high risk for developing glaucoma and seizures. We recently developed induced pluripotent stem cells (iPSCs) from three patients with LS who have hypomorphic variants affected the 3’ end of the gene, and their neurotypical brothers to serve as controls. In this study, we used RNA sequencing (RNA-seq) to obtain a transcriptome profile in neural progenitor cells (NPCs). Thirty-six differentially expressed genes (DEGs) were found in patient/control comparisons that were shared across all three sets of sibling pairs. Interestingly, two-thirds of these genes have previously been found to be involved in neuropsychiatric and neurodevelopmental disorders. Furthermore, 14 of the DEGs are strong candidate genes for a variety of eye disorders, including glaucoma development. The most notable example is EFEMP1, which was significantly increased in LS NPCs. These DEGs found in NPCs could reflect similar alterations occurring in eye tissues or their progenitor cells from LS. Overall, the RNA-seq findings present several molecular pathways that could explain the underlying basis for the neurodevelopmental and eye problems seen in boys with LS. Overall design: Three Lowe Syndrome patients and 3 sibling controls were analyzed in duplicate, using the Illumina HiSeq 2500 system.
Project description:Background:Lowe syndrome (LS) is a rare genetic disorder caused by loss of function mutations in the X-linked gene, OCRL, which codes for inositol polyphosphate 5-phosphatase. LS is characterized by the triad of congenital cataracts, neurodevelopmental impairment (primarily intellectual and developmental disabilities [IDD]), and renal proximal tubular dysfunction. Studies carried out over the years have shown that hypomorphic mutations in OCRL adversely affect endosome recycling and actin polymerization in kidney cells and patient-derived fibroblasts. The renal problem has been traced to an impaired recycling of megalin, a multi-ligand receptor that plays a key role in the reuptake of lipoproteins, amino acids, vitamin-binding proteins, and hormones. However, the neurodevelopmental aspects of the disorder have been difficult to study because the mouse knockout (KO) model does not display LS-related phenotypes. Fortunately, the discovery of induced pluripotent stem (iPS) cells has provided an opportunity to grow patient-specific neurons, which can be used to model neurodevelopmental disorders in vitro, as demonstrated in the many studies that have been published in the past few years in autism spectrum disorders (ASD), schizophrenia (SZ), bipolar disorder (BD), and IDD. Methods:We now report the first findings in neurons and neural progenitor cells (NPCs) generated from iPS cells derived from patients with LS and their typically developing male siblings, as well as an isogenic line in which the OCRL gene has been incapacitated by a null mutation generated using CRISPR-Cas9 gene editing. Results:We show that neuronal cells derived from patient-specific iPS cells containing hypomorphic variants are deficient in their capacity to produce F-filamentous actin (F-actin) fibers. Abnormalities were also found in the expression of WAVE-1, a component of the WAVE regulatory complex (WRC) that regulates actin polymerization. Curiously, neuronal cells carrying the engineered OCRL null mutation, in which OCRL protein is not expressed, did not show similar defects in F-actin and WAVE-1 expression. This is similar to the apparent lack of a phenotype in the mouse Ocrl KO model, and suggests that in the complete absence of OCRL protein, as opposed to producing a dysfunctional protein, as seen with the hypomorphic variants, there is partial compensation for the F-actin/WAVE-1 regulating function of OCRL. Conclusions:Alterations in F-actin polymerization and WRC have been found in a number of genetic subgroups of IDD and ASD. Thus, LS, a very rare genetic condition, is linked to a more expansive family of genes responsible for neurodevelopmental disorders that have shared pathogenic features.
Project description:Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-?, TGF-?, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.
Project description:Mutations in the OCRL1 gene result in the oculocerebrorenal syndrome of Lowe, with symptoms including congenital bilateral cataracts, glaucoma, renal failure, and neurological impairments. OCRL1 encodes an inositol polyphosphate 5-phosphatase which preferentially dephosphorylates phosphatidylinositide 4,5 bisphosphate (PI(4,5)P2). We have identified two novel mutations in two unrelated Lowe syndrome patients with congenital glaucoma. Novel deletion mutations are detected at c.739-742delAAAG in Lowe patient 1 and c.1595-1631del in Lowe patient 2. End stage glaucoma in patient 2 resulted in the enucleation of the eye, which on histology demonstrated corneal keloid, fibrous infiltration of the angle, ectropion uvea, retinal gliosis, and retinal ganglion cell loss. We measured OCRL protein levels in patient keratinocytes and found that Lowe 1 patient cells had significantly reduced OCRL protein as compared to the control keratinocytes. Genotype-phenotype correlation of OCRL1 mutations associated with congenital glaucoma revealed clustering of missense and deletion mutations in the 5-phosphatase domain and the RhoGAP-like domain. In conclusion, we report novel OCRL1 mutations in Lowe syndrome patients and the corresponding histopathologic analysis of one patient's ocular pathology.
Project description:Lowe syndrome (LS) is an X-linked disorder affecting the eyes, nervous system and kidneys, typically caused by missense or nonsense/frameshift OCRL mutations. We report a 6-month-old male clinically suspected to have LS, but without the Fanconi-type renal dysfunction. Using a targeted-exome sequencing-first approach, LS was diagnosed by the identification of a deletion involving 1.7 Mb at Xq25-q26.1, encompassing the entire OCRL gene and neighboring loci.
Project description:Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.
Project description:Mutations of the inositol 5-phosphatase OCRL cause Lowe syndrome (LS), characterized by congenital cataract, low IQ, and defective kidney proximal tubule resorption. A key subset of LS mutants abolishes OCRL's interactions with endocytic adaptors containing F&H peptide motifs. Converging unbiased methods examining human peptides and the unicellular phagocytic organism Dictyostelium discoideum reveal that, like OCRL, the Dictyostelium OCRL orthologue Dd5P4 binds two proteins closely related to the F&H proteins APPL1 and Ses1/2 (also referred to as IPIP27A/B). In addition, a novel conserved F&H interactor was identified, GxcU (in Dictyostelium) and the Cdc42-GEF FGD1-related F-actin binding protein (Frabin) (in human cells). Examining these proteins in D. discoideum, we find that, like OCRL, Dd5P4 acts at well-conserved and physically distinct endocytic stations. Dd5P4 functions in coordination with F&H proteins to control membrane deformation at multiple stages of endocytosis and suppresses GxcU-mediated activity during fluid-phase micropinocytosis. We also reveal that OCRL/Dd5P4 acts at the contractile vacuole, an exocytic osmoregulatory organelle. We propose F&H peptide-containing proteins may be key modifiers of LS phenotypes.
Project description:Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.
Project description:Glaucoma results in damage to the optic nerve and vision loss. The aim of this study was to screen more accurate biomarkers and targets for glaucoma. The datasets E-GEOD-7144 and E-MEXP-3427 were screened for differently expressed genes (DEGs) by significance analysis of microarrays. Functional and pathway enrichment analysis were processed. Pathway relationship networks and gene co-expression networks were constructed. DEGs of disease and treatment with the same symbols were of interest. RT-qPCR was processed to verify the expression of key DEGs. A total of 1,019 DEGs of glaucoma were identified and 93 DEGs in transforming growth factor-β1 (TGF-β1) and TGF-β1-2 treatment cases compared with the normal control group. Pathway relationship network of glaucoma was constructed with 25 nodes. The pathway relationship network of TGF-β1 and -2 treatment groups was constructed with 11 nodes. Glaucoma-related DEGs in GO terms and pathways were inserted and 180 common DEGs were obtained. Then, gene co-expression network of glaucoma-related DEGs was constructed with 91 nodes. Furthermore, DEGs of TGF-β1 and -2 treated glaucoma in GO terms and pathways were inserted, and 29 common DEGs were identified. Based on these DEGs, gene co-expression network was constructed with 12 nodes and 16 edges. Finally, a total of 6 important DEGs of disease and treatment were inserted and obtained. They were HGF, AKR1B10, AKR1C3, PPAP2B, INHBA and BCAT1. The expression of HGF, AKR1B10 and AKR1C3 was decreased in glaucoma samples and treatment samples. In conclusion, HGF, AKR1B10 and AKR1C3 may be key genes for glaucoma diagnosis and treatment.
Project description:The aim of the present study was to screen the differentially expressed genes (DEGs) associated with glaucoma and investigate the changing patterns of the expression of these genes. The GSE2378 gene microarray data of glaucoma was downloaded from the Gene Expression Omnibus database, which included seven normal samples and eight glaucoma astrocyte samples. Taking into account the corresponding associations between the probe ID and gene symbols, the DEGs were identified prior to and subsequent to the summation of probe level values using the Limma package in R language, followed by Gene Ontology (GO) and pathway enrichment analyses. Interaction networks of the DEGs were constructed using the Biomolecular Interaction Network Database, and cluster analysis of the genes in the networks was performed using ClusterONE. Subsequent to the summation of probe value, a total of 223 genes were identified as DEGs between the normal and glaucoma samples, including 74 downregulated and 149 upregulated genes. In addition, the DEGs were found to be associated with several functions, including response to wounding, extracellular region part and calcium ion binding. The most significantly enriched pathways were complement and coagulation cascades, arrhythmogenic right ventricular cardiomyopathy and extracellular matrix (ECM)?receptor interaction. Furthermore, interaction networks were constructed of the DEGs prior to and subsequent to the summation of probe values, and HNF4A and CEBPD were identified as hub genes. Additionally, 37 and 31 GO terms were identified to be enriched in the two DEGs of the networks prior to and subsequent to summation, respectively. The results indicated the identified genes associated with ECM as important, and the CEBPD gene was considered to be a critical gene in glaucoma. The findings of the present study offer a potential reference value in further investigations of glaucoma at the gene level.