CRX Gene Research Articles

Enhanced Transcriptional Activation in Developing Mouse Photoreceptors.

Retinal development in the mouse continues past birth and provides a widely used model system in which photoreceptor formation can be observed and manipulated. This experimental paradigm provides opportunities for both gain-of-function and loss-of-function studies, which can be accomplished through in vivo or ex vivo plasmid delivery and electroporation. However, the cis-regulatory elements used to implement this approach have not been fully evaluated or optimized for the unique transcriptional environment of photoreceptors. Here we investigate whether the use of a photoreceptor cis-regulatory element from the Crx gene in combination with broadly active promoter elements can increase the targeting of developing photoreceptors in the mouse. This study characterizes the in vivo activity of this element for the first time, as well as explores its use as a tool for gain-of-function and loss-of-function experiments. We report that a cis-regulatory element from the Crx gene, in combination with broadly active promoter elements, increases the targeting of developing rod photoreceptors in the mouse. Additionally, the same element can be used to target developing cones at embryonic time points by ex vivo electroporation. Utility of this combined element includes greater reporter expression, as well as enhanced overexpression and loss-of-function phenotypes in photoreceptors. This study highlights the importance of identifying and testing relevant cis-regulatory elements when planning cell subtype-specific experiments. The use of specific hybrid elements will provide a more efficacious gene delivery system to study mammalian photoreceptor formation, which will benefit research with potential therapeutic relevance for blinding diseases.

Hydroxychloroquine retinopathy in a 23-year-old male.

To report a case of hydroxychloroquine (HCQ) retinopathy after long-term exposure in a 23-year-old male. Multimodal imaging including fundus photography, fundus autofluorescence (FAF), spectral domain optical coherence tomography (SD-OCT), and en face OCT were performed, in addition to functional testing with full-field electroretinography (ERG) and Humphrey visual field (HVF). A 23-year-old man with a history of juvenile systemic lupus erythematosus and HCQ treatment for 13 years at a dosage of 200 mg/d (cumulative dose: 949 grams) presented to the retinal clinic (DS). Although fundus photography and FAF were unremarkable, SD-OCT and en face OCT showed paracentral ellipsoid zone loss with thinning of the outer nuclear layer, OS greater than OD consistent with HCQ retinopathy. HVF 10-2 showed possible nasal loss OU. Genetic testing revealed heterozygous mutations in the CNGA1 and CRX genes but full-field ERG was unremarkable without evidence of a cone dystrophy. Family history was negative for genetic disease. This report highlights a case of HCQ retinopathy in a young patient who started treatment at the age of 10 years. There are currently no specific guidelines for the screening of pediatric patients, and studies evaluating the effect of HCQ on children are lacking. Whether the genetic carrier status rendered the patient more susceptible to the toxic effect of HCQ remains to be determined.

Genome Mining for Diazo-Synthesis-Related Genes in Streptomyces sp. CS057 Unveiled the Cryptic Biosynthetic Gene Cluster crx for the Novel 3,4-AHBA-Derived Compound Crexazone 2.

Natural products play a crucial role in drug development, addressing the escalating microbial resistance to antibiotics and the treatment of emerging diseases. Progress in genome sequencing techniques, coupled with the development of bioinformatics tools and the exploration of uncharted habitats, has highlighted the biosynthetic potential of actinomycetes. By in silico screening for diazo-related gene genomes from twelve Streptomyces strains isolated from Attini leaf-cutting ants, the new crx biosynthetic gene cluster (BGC) was identified in Streptomyces sp. CS057. This cluster, highly conserved in several Streptomyces strains, contains genes related to diazo group formation and genes for the biosynthesis of 3,4-AHBA. By overexpressing the LuxR-like regulatory gene crxR1, we were able to activate the crx cluster, which encodes the biosynthesis of three 3,4-AHBA-derived compounds that we named crexazones (CRXs). The chemical structure of crexazones (CRXs) was determined by LC-DAD-HRMS-based dereplication and NMR spectroscopic analyses and was found to correspond to two known compounds, 3-acetamido-4-hydroxybenzoic acid (CRX1) and the phenoxazinone texazone (CRX3), and a novel 3,4-AHBA-containing compound herein designated as CRX2. Experimental proof linking the crx BGC to their encoded compounds was achieved by generating mutants in selected crx genes.

Clinical, Genetic, and Histopathological Characteristics of CRX-associated Retinal Dystrophies

PURPOSETo describe phenotypic, genotypic, and histopathological features of inherited retinal dystrophies associated with the CRX gene (CRX-RDs). DESIGNRetrospective multicenter cohort study including histopathology. SUBJECTSThirty-nine patients from 31 families with pathogenic variants in the CRX gene. METHODSClinical data of 152 visits were collected from medical records with a median follow-up time of 9.1 years (IQR, 3.3-15.3 years; range, 0.0-48.8 years). Histopathologic examination of the eye of a 17-year-old patient with advanced early-onset CRX-RD was performed. MAIN OUTCOME MEASURESVisual acuity, retinal imaging, electroretinography (ERG), genotype-phenotype correlation, and histopathological examination were evaluated. RESULTSThe age at onset ranged from birth to the 8th decade of life. Median visual acuity was 1.00 logMAR (IQR, 0.69-1.48 logMAR; range, 0.06-3.00 logMAR) at a mean age of 52.0 ± 19.9 years (range, 4.6-81.9 years). Sufficient imaging was available for 36 out of 39 patients (92.3%), and all showed degeneration of at least the macula. Out of these 36 patients, 22 (61.1%) had only macular dystrophy. Another 10 patients (27.8%) had additional degeneration beyond the vascular arcades, and 4 patients (11.1%) panretinal degeneration. Two patients (5.1%) had Leber congenital amaurosis (LCA). In total, 21 different disease-associated heterozygous CRX variants were identified (10 missense, 8 frameshift, 2 deletion, 1 deletion-insertion variants). Missense variants in the CRX homeodomain and two variants deleting all functional domains, thus causing haploinsufficiency, generally tended to cause milder late-onset phenotypes.Histopathologic examination of the eye of a 17-year-old patient with advanced early-onset retinal dystrophy due to a heterozygous deletion of exons 3 and 4 of the CRX gene revealed loss of laminar integrity and widespread photoreceptor degeneration especially in the central retina, with extensive loss of photoreceptor nuclei and outer segments. CONCLUSIONThis study illustrates the large clinical and genetic heterogenic spectrum of CRX-RDs, ranging from LCA to mild late-onset maculopathy resembling occult macular dystrophy. Haploinsufficiency and missense variants tended to be associated with milder phenotypes. Patients showed degeneration predominantly affecting the central retina on imaging. The histopathological findings also mirror these clinical findings and features similar to previously reported animal models of CRX-RDs.

Heparin-Binding Epidermal-like Growth Factor (HB-EGF) Reduces Cell Death in an Organoid Model of Retinal Damage

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration.

Enhanced Plasmid-Based Transcriptional Activation in Developing Mouse Photoreceptors.

Rod photoreceptor formation in the postnatal mouse is a widely used model system for studying mammalian photoreceptor development. This experimental paradigm provides opportunities for both gain and loss-of-function studies which can be accomplished through in vivo plasmid delivery and electroporation. However, the cis-regulatory elements used to implement this approach have not been fully evaluated or optimized for the unique transcriptional environment of photoreceptors. Here we report that the use of a photoreceptor cis-regulatory element from the Crx gene in combination with broadly active promoter elements can increase the targeting of developing rod photoreceptors in the mouse. This can lead to greater reporter expression, as well as enhanced misexpression and loss-of-function phenotypes in these cells. This study also highlights the importance of identifying and testing relevant cis-regulatory elements when planning cell subtype specific experiments. The use of the specific hybrid elements in this study will provide a more efficacious gene delivery system to study mammalian photoreceptor formation.

MIR96 Has Good Potential to Differentiate Human Bone Marrow-Derived Mesenchymal Stem Cells into Photoreceptor-Like Cells.

MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.

Four different gene-related cone-rod dystrophy: clinical and genetic findings in six Chinese families with diverse modes of inheritance.

Purpose: To investigate pathogenic variants in six families with cone-rod dystrophy (CORD) presenting various inheritance patterns by using whole-exome sequencing (WES) and analyzing phenotypic features. Methods: A total of six families with CORD were enrolled in Ningxia Eye Hospital for this study. The probands and their family members received comprehensive ophthalmic examinations, and DNA was abstracted from patients and family members. Whole-exome sequencing was performed on probands to screen the causative variants, and all suspected pathogenic variants were determined via Sanger sequencing. Furthermore, co-segregation analysis was performed on available family members. The pathogenicity of novel variants was predicted using in silico analysis and evaluated according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Results: Of the six families, two families were assigned as X-linked recessive (XL), two families were assigned as autosomal recessive (AR), and two families were assigned as autosomal dominant (AD). Pathogenic variants were detected in CACNA1F in two X-linked recessive probands, among which family 1 had a hemizygous frameshift variant c.2201del (p.Val734Glyfs*17) and family 2 had a hemizygous missense variant c.245G>A (p.Arg82Gln). Both probands had high myopia, with fundus tessellation accompanied by abnormalities in the outer structure of the macular area. The homozygous splice variant c.2373 + 5G>T in PROM1 and the homozygous nonsense variant c.604C>T (p.Arg202Ter) in ADAM9 were detected in two autosomal recessive families of the probands. Both probands showed different degrees of atrophy in the macular area, and the lesions showed hypofluorescence changes in autofluorescence. The heterozygous variation in CRX c.682C>T (p.Gln228Ter) was detected in two autosomal dominant families. The onset age of the two probands was late, with better vision and severe macular atrophy. According to ACMG guidelines and the analysis of online in silico tools, all variations were labeled as potentially harmful or pathogenic. Conclusion: Pathogenic variants in CACNA1F, PROM1, ADAM9, and CRX genes were identified in six families affected by the diverse inheritance patterns of CORD. Furthermore, the potential impact of the nonsense-mediated decay (NMD) mechanism on the manifestation of CORD phenotypes was examined and addressed. Simultaneously, the spectrum of pathogenic variants and clinical phenotypes associated with the CORD gene was extended.

IDENTYFIKACJA MUTACJI WYBRANYCH GENÓW POWODUJ ˛ACYCH ZWYRODNIENIE SIATKÓWKI U KOTA DOMOWEGO (FELIS CATUS) – ANALIZA IN SILICO

Retinal degenerations are a series of genetically inherited diseases resulting in significant visual impairment and blindness. Among domestic cat breeds, there are degenerations of different courses associated with mutations in CEP290, CRX, AIPL1 and KIF3B genes. The aim of this study was to design diagnostic tests to identify the mutated alleles. The primers for PCR and restriction enzymes for PCR-RFLP were designed to detect mutations in genes. Mutation in the nucleotide sequence encoding AIPL1 protein causes a change in the protein structure, where a monomer is formed instead of a homodimer. Interactions of CEP290, CRX, AIPL1 and KIF3B proteins with other proteins that play a role in the proper functioning of the retina were observed. The occurring interactions between some of these proteins suggest a possible link between diseases caused by mutations of genes encoding these proteins. In other animal species, co-expression of the analyzed genes with other genes affecting retinal functions was noted.

Genotypic Profile and Clinical Characteristics of CRX-Associated Retinopathy in Koreans.

This study aimed to investigate the clinical characteristics of Korean patients with retinal dystrophy associated with pathogenic variants of cone rod homeobox-containing gene (CRX). We retrospectively enrolled Korean patients with CRX-associated retinal dystrophy (CRX-RD) who visited two tertiary referral hospitals. Pathogenic variants were identified using targeted panel sequencing or whole-exome sequencing. We analyzed clinical features and phenotypic spectra according to genotype. Eleven patients with CRX-RD were included in this study. Six patients with cone-rod dystrophy (CORD), two with macular dystrophy (MD), two with Leber congenital amaurosis (LCA), and one with retinitis pigmentosa (RP) were included. One patient (9.1%) had autosomal recessive inheritance, and the other ten patients (90.9%) had autosomal dominant inheritance. Six patients (54.5%) were male, and the mean age of symptom onset was 27.0 ± 17.9 years. At the first presentation, the mean age was 39.4 ± 20.6 years, and best-corrected visual acuity (BCVA) (logMAR) was 0.76 ± 0.90 in the better eye. Negative electroretinography (ERG) was observed in seven (63.6%) patients. Nine pathogenic variants were identified, including two novel variants, c.101-1G>A and c.898T>C:p.(*300Glnext*118). Taken together with the variants reported in prior studies, all variants within the homeodomain are missense variants, whereas most variants downstream of the homeodomain are truncating variants (88%). The clinical features of pathogenic variants within the homeodomain are either CORD or MD with bull's eye maculopathy, whereas variants downstream of the homeodomain cause more diverse phenotypes, with CORD and MD in 36%, LCA in 40%, and RP in 24%. This is the first case series in Korea to investigate the CRX-RD genotype-phenotype correlation. Pathogenic variants downstream of the homeodomain of the CRX gene are present as RP, LCA, and CORD, whereas pathogenic variants within the homeodomain are mainly present as CORD or MD with bull's eye maculopathy. This trend was similar to previous genotype-phenotype analyses of CRX-RD. Further molecular biologic research on this correlation is required.

Pathogenicity of Fusarium oxysporum and Fusarium proliferatum isolates from symptomless onions (Allium cepa) and onions with Fusarium basal rot

AbstractSeveral Fusarium species cause disease in onion (Allium cepa), resulting in significant yield losses. In this study, isolates of Fusarium oxysporum and Fusarium proliferatum from diseased and symptomless mature onion bulbs were tested for pathogenicity on onion seedlings and mature bulbs. For F. oxysporum isolates, the outcome of the test on mature bulbs correlated well with the symptom status of the original bulb, whereas for F. proliferatum, such a correlation was not seen, suggesting a different mechanism of symptom development. Those F. oxysporum isolates that carried the Secreted In Xylem (SIX) genes, CRX genes and C5 gene were significantly more aggressive on both seedlings and mature bulbs. However, the SIX‐negative F. oxysporum isolates also significantly reduced the emergence of onion seedlings, and thus a division into pathogenic and nonpathogenic F. oxysporum isolates was not obvious. On mature bulbs, those SIX‐negative isolates that carried the gene CRX2 were significantly more virulent than those without CRX2. The F. proliferatum isolates were all pathogenic to onion and carried one SIX2 gene homologue, SIX2‐1. The majority of F. proliferatum isolates also carried another homologue, SIX2‐2. Several isolates negative for SIX2‐2 had another potential virulence gene, CRX2, suggesting that these two genes could encode overlapping functions. The SIX2‐2 sequence was identical in all those isolates harbouring it, while SIX2‐1 showed variation that agreed with the phylogeny based on the translation elongation factor 1‐α sequence. This suggests that the two SIX2 genes of F. proliferatum might have been acquired separately.

Association analyses of rare variants identify two genes associated with refractive error.

PurposeGenetic variants identified through population-based genome-wide studies are generally of high frequency, exerting their action in the central part of the refractive error spectrum. However, the power to identify associations with variants of lower minor allele frequency is greatly reduced, requiring considerable sample sizes. Here we aim to assess the impact of rare variants on genetic variation of refractive errors in a very large general population cohort.MethodsGenetic association analyses of non-cyclopaedic autorefraction calculated as mean spherical equivalent (SPHE) used whole-exome sequence genotypic information from 50,893 unrelated participants in the UK Biobank of European ancestry. Gene-based analyses tested for association with SPHE using an optimised SNP-set kernel association test (SKAT-O) restricted to rare variants (minor allele frequency < 1%) within protein-coding regions of the genome. All models were adjusted for age, sex and common lead variants within the same locus reported by previous genome-wide association studies. Potentially causal markers driving association at significant loci were elucidated using sensitivity analyses by sequentially dropping the most associated variants from gene-based analyses.ResultsWe found strong statistical evidence for association of SPHE with the SIX6 (p-value = 2.15 x 10−10, or Bonferroni-Corrected p = 4.41x10-06) and the CRX gene (p-value = 6.65 x 10−08, or Bonferroni-Corrected p = 0.001). The SIX6 gene codes for a transcription factor believed to be critical to the eye, retina and optic disc development and morphology, while CRX regulates photoreceptor specification and expression of over 700 genes in the retina. These novel associations suggest an important role of genes involved in eye morphogenesis in refractive error.ConclusionThe results of our study support previous research highlighting the importance of rare variants to the genetic risk of refractive error. We explain some of the origins of the genetic signals seen in GWAS but also report for the first time a completely novel association with the CRX gene.

Late-Onset Autosomal Dominant Macular Degeneration Caused by Deletion of the CRX Gene

To characterize the phenotype observed in a case series with macular disease and determine the cause. Multicenter case series. Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

Change of transcription level of photoreceptor-specific CRX gene in the peripheral blood of the participants of an arctic world oceanic international flight

To reveal the dynamics of CRX expression level in humans after a prolonged temporary exposure to polar day conditions. The study included 6 pilots (males from 39 to 69 y.o.) who participated in the Arctic World Oceanic International Flight Sever Vash (West to East, from 62°N 74°E to 72°N 114°E). Samples of peripheral blood for RNA isolation were collected at the start and at the end of the flight. The level of mRNA in the samples was evaluated based on the data of quantitative real-time PCR of the CRX gene, as well as the b2M and TBP housekeeping genes (reference). Expression of the CRX gene in the studied group (p<0.01) was revealed; the total average level of mRNA was about 3 times higher prior to, and approximately 7 times higher after normalization to the b2M gene. Five pilots had increased expression of the CRX gene within the range of -1.53 to -3.07 (Z-score of <0 before the flight and >0 after the flight). In one pilot, the level of CRX expression was higher at the start, but it reduced by the end of the circumnavigation flight. The results confirm the hypothesis that the CRX gene is expressed after a prolonged temporary exposure to polar day conditions. It was also revealed that during rapid adaptation, equal changes in the illumination of retinal photoreceptors lead to different individual dynamics of CRX expression.

The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia

Abstract. Herlina L, Istiaji B. 2020. The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia. Biodiversitas 21: 4677-4685. One of the most important diseases and become challenge in breeding resistant variety of shallots in Indonesia is Fusarium disease, caused by Fusarium oxysporum f.sp. cepae (FOC). To discriminate the F. oxysporum into forma speciales is uneasy, often managed through laborious and time-intensive disease assays., therefore molecular approaches become the most relevant choice. FOCs from Indonesia were examined by molecular criteria based on putative and effector genes-based markers, i.e.: SIX-genes, C5-gene, and CRX1-2-genes. The alignment analysis discovered some regions which sequences highly contained conserved bases, while amplification result bands vary in size, between 400 - 500 bp. The combined of 7 SIX-genes primers and 3 effector primers (C5 and CRX1-2) used in the clustering analysis of 15 FOC isolates in this study showed that they succeeded in separating 15 FOC isolates into 4 groups through NTSys. Clustering analysis showed that those markers succeeded in grouping 15 FOC isolates into different clades (by coefficient of similarity: 0.69). Phylogenetic analysis based on CRX genes sequence as putative effector genes confirmed that CRX1 and CRX2 genes were able to classify the FOC into their forma speciales. Those effectors genes are potential to serve as marker-templates to facilitate identification of FOC which infested shallots in Indonesia.

Impacts of ciliary neurotrophic factor on the retinal transcriptome in a mouse model of photoreceptor degeneration

Ciliary neurotrophic factor (CNTF) has been tested in clinical trials for human retinal degeneration due to its potent neuroprotective effects in various animal models. To decipher CNTF-triggered molecular events in the degenerating retina, we performed high-throughput RNA sequencing analyses using the Rds/Prph2 (P216L) transgenic mouse as a preclinical model for retinitis pigmentosa. In the absence of CNTF treatment, transcriptome alterations were detected at the onset of rod degeneration compared with wild type mice, including reduction of key photoreceptor transcription factors Crx, Nrl, and rod phototransduction genes. Short-term CNTF treatments caused further declines of photoreceptor transcription factors accompanied by marked decreases of both rod- and cone-specific gene expression. In addition, CNTF triggered acute elevation of transcripts in the innate immune system and growth factor signaling. These immune responses were sustained after long-term CNTF exposures that also affected neuronal transmission and metabolism. Comparisons of transcriptomes also uncovered common pathways shared with other retinal degeneration models. Cross referencing bulk RNA-seq with single-cell RNA-seq data revealed the CNTF responsive cell types, including Müller glia, rod and cone photoreceptors, and bipolar cells. Together, these results demonstrate the influence of exogenous CNTF on the retinal transcriptome landscape and illuminate likely CNTF impacts in degenerating human retinas.

Association of CRX genotypes and retinal phenotypes confounded by variable expressivity and electronegative electroretinogram.

A framework for understanding the phenotypic features of CRX retinopathy was established. To perform a phenotype-genotype correlation analysis in two groups of patients with heterozygous mutations in distinct locations of the CRX gene, encoding the cone-rod homeobox. Multicentre retrospective study. Twenty-one Japanese patients from 14 families with a heterozygous CRX mutation. Retrospective data analysis. Clinical records on CRX mutation, symptoms, best-corrected visual acuity (BCVA), visual field, fundus photography, fundus auto-fluorescence, optical coherence tomography and electroretinograms (ERGs). Six different CRX heterozygous mutations were identified in the subjects. Twelve patients from 9 families shared the p.R41W mutation and 1 patient had the p.R43C mutation, both of which affect the homeobox domain of CRX. These patients often displayed adult-onset retinal dystrophy with macular degeneration. In contrast, five patients with downstream mutations (p.S204fs, p.S213fs, p.G243X and p.L299F) displayed retinal degeneration or macular degeneration with bone-spicule pigmentation. Three asymptomatic carriers with different mutations (p.R41W, p.S213fs and p.G243X) were present in both groups. Nearly all patients and carriers had an electronegative ERG in response to a bright flash under dark adaptation. There was no cross-sectional association between patients' age and BCVA, despite progressive decline in BCVA. Heterozygous mutations within or downstream of the homeobox domain in CRX relate to the difference associated retinal phenotypes, which was confounded by variable expressivity and electronegative ERGs. CRX mutations should be considered in patients with an electronegative ERG with minimal or no macular changes.

Generation of a human iPSC line, INMi003-A, with a missense mutation in CRX associated with autosomal dominant cone-rod dystrophy

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a 53 year-old patient with autosomal dominant cone-rod dystrophy (CRD) caused by a missense mutation, c.121C > T, in the CRX gene. Patient fibroblasts were reprogrammed using the non-integrative Sendai virus reprogramming system and the human OSKM transcription factor cocktail. The generated iPSCs contained the congenital mutation in exon 3 of CRX and were pluripotent and genetically stable. This iPSC line will be an important tool for retinal differentiation studies to better understand the CRD phenotype caused by the mutant p.Arg41Trp CRX protein.

Generation of a human iPSC line, INMi004-A, with a point mutation in CRX associated with autosomal dominant Leber congenital amaurosis

The human induced pluripotent stem cell (iPSC) line, INMi004-A, was generated using dermal fibroblasts from a 6 year-old patient with autosomal dominant Leber Congenital Amaurosis (LCA) caused by the point mutation c.695delC (p.Pro232Argfs*139) in the CRX gene. We used non-integrative Sendai virus vectors containing the human OSKM transcription factor cocktail to reprogram patient fibroblasts. The generated iPSC line contained the congenital deletion c.695delC in exon 4 of CRX, had a normal karyotype, and was capable of differentiation into all three germ layers. This cell line represents an important tool to study the pathophysiology of CRX-associated LCA.

SUN-050 The Evolutionarily Conserved Function of COUP-TF Genes in the Differentiation of Photoreceptor Cells in the Retina

Central nervous system including brain and retina is composed of appropriate number and subtypes of neurons derived from neural progenitor cells. However, our understanding of the proliferation and differentiation of neural progenitor cells is limited. COUP-TFI and -TFII genes, encoding two nuclear receptors, are associated with neurodevelopmental disease and heart defects respectively. Seven-up, the homologous gene of COUP-TF gene in Drosophila, is expressed and required in photoreceptor cell precursors. We observed that along dorso-ventral axis, COUP-TFI and -TFII genes are differentially expressed in mouse retinal stem cells. Minor defects in the eye are generated in either COUP-TFI or -TFII single gene knockout mouse with RXCre. Interestingly, loss of both genes in the retinal stem cells leads to failed differentiation of photoreceptors and thinner retina. The reduced bipolar cells, amacrine cells, horizontal cells, and ganglionic cells are also displayed in the double mutant retina. In addition, the development of Muller cells is repressed accompanying with fibrosis. Mechanistic studies reveal that COUP-TF genes may regulate the differentiation of photoreceptors through Crx and Nrl genes, and modulate the proliferation of retinal stem cells through P27 and P57 genes. Thus, our study shed new light on the understanding of evolutionarily conserved function of COUP-TF genes in the differentiation of photoreceptor cells.