RESUMEN
The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields.
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Pruebas Genéticas , Enfermedades de la Retina , Secuenciación Completa del Genoma , Humanos , Enfermedades de la Retina/genética , Enfermedades de la Retina/diagnóstico , Pruebas Genéticas/métodos , Secuenciación Completa del Genoma/métodos , Masculino , Femenino , Suiza , Estudios de Cohortes , Adulto , Variaciones en el Número de Copia de ADN , Secuenciación del Exoma/métodos , Biología Computacional/métodos , Persona de Mediana Edad , Niño , Adolescente , LinajeRESUMEN
Congenital cataract (CC), the most prevalent cause of childhood blindness and amblyopia, necessitates prompt and precise genetic diagnosis. The objective of this study is to identify the underlying genetic cause in a Swiss patient with isolated CC. Whole exome sequencing (WES) and copy number variation (CNV) analysis were conducted for variant identification in a patient born with a total binocular CC without a family history of CC. Sanger Sequencing was used to confirm the variant and segregation analysis was used to screen the non-affected parents. The first de novo missense mutation at c.391T>C was identified in exon 3 of CRYGC on chromosome 2 causing the substitution of a highly conserved Tryptophan to an Arginine located at p.Trp131Arg. Previous studies exhibit significant changes in the tertiary structure of the crystallin family in the following variant locus, making CRYGC prone to aggregation aggravated by photodamage resulting in cataract. The variant can be classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria (PP3 + PM1 + PM2 + PS2; scoring 10 points). The identification of this novel variant expands the existing knowledge on the range of variants found in the CRYGC gene and contributes to a better comprehension of cataract heterogeneity.
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Catarata , gamma-Cristalinas , Humanos , Triptófano/genética , gamma-Cristalinas/química , Variaciones en el Número de Copia de ADN , Linaje , Mutación , Catarata/genética , Catarata/congénito , Mutación MissenseRESUMEN
Optic nerve hypoplasia (ONH) is a congenital optic nerve abnormality caused by underdevelopment of retinal ganglion cells (RGCs). Despite being a rare disease, ONH is the most common optic disk anomaly in ophthalmological practice. So far, mutations in several genes have been identified as causative; however, many cases of ONH remain without a molecular explanation. The early transcription factor atonal basic-helix-loop-helix (bHLH) transcription factor 7 (ATOH7) is expressed in retinal progenitor cells and has a crucial role in RGC development. Previous studies have identified several mutations in the ATOH7 locus in cases of eye developmental diseases such as non-syndromic congenital retinal non-attachment and persistent hyperplasia of the primary vitreous. Here we present two siblings with a phenotype predominated by bilateral ONH, with additional features of foveal hypoplasia and distinct vascular abnormalities, where whole-exome sequencing identified two compound heterozygous missense mutations affecting a conserved amino acid residue within the bHLH domain of ATOH7 (NM_145178.3:c.175G>A; p.(Ala59Thr) and c.176C>T; p.(Ala59Val)). ATOH7 expression constructs with patient single nucleotide variants were cloned for functional characterization. Protein analyses revealed decreased protein amounts and significantly enhanced degradation in the presence of E47, a putative bHLH dimerization partner. Protein interaction assays revealed decreased heterodimerization and DNA-binding of ATOH7 variants, resulting in total loss of transcriptional activation of luciferase reporter gene expression. These findings strongly support pathogenicity of the two ATOH7 mutations, one of which is novel. Additionally, this report highlights the possible impact of altered ATOH7 dimerization on protein stability and function.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Enfermedades del Nervio Óptico/congénito , Hipoplasia del Nervio Óptico/metabolismo , Hipoplasia del Nervio Óptico/patología , Adolescente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Niño , Femenino , Pruebas Genéticas , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Mutación Missense/genética , Enfermedades del Nervio Óptico/genética , Enfermedades del Nervio Óptico/metabolismo , Enfermedades del Nervio Óptico/patología , Hipoplasia del Nervio Óptico/genética , Linaje , Células Ganglionares de la Retina/metabolismoRESUMEN
Basic helix-loop-helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas del Tejido Nervioso , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , ADN , Células HEK293 , Humanos , Proteínas Mutantes , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.
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Transportadoras de Casetes de Unión a ATP/genética , Bestrofinas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Periferinas/genética , Enfermedades de la Retina/genética , Análisis de Secuencia de ADN , Adolescente , Adulto , Anciano , Niño , Preescolar , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Variaciones en el Número de Copia de ADN , Proteínas del Ojo/genética , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proteínas de la Membrana/genética , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Mutación , Proteínas del Tejido Nervioso/genética , Reacción en Cadena de la Polimerasa , Enfermedades de la Retina/congénito , Enfermedades de la Retina/diagnóstico , Adulto JovenRESUMEN
Mutations in Norrin, the ligand of a receptor complex consisting of FZD4, LRP5 and TSPAN12, cause severe developmental blood vessel defects in the retina and progressive loss of the vascular system in the inner ear, which lead to congenital blindness and progressive hearing loss, respectively. We now examined molecular pathways involved in developmental retinal angiogenesis in a mouse model for Norrie disease. Comparison of morphometric parameters of the superficial retinal vascular plexus (SRVP), including the number of filopodia, vascular density and number of branch points together with inhibition of Notch signaling by using DAPT, suggest no direct link between Norrin and Notch signaling during formation of the SRVP. We noticed extensive vessel crossing within the SRVP, which might be a loss of Wnt- and MAP kinase-characteristic feature. In addition, endomucin was identified as a marker for central filopodia, which were aligned in a thorn-like fashion at P9 in Norrin knockout (Ndp(y/-)) mice. We also observed elevated mural cell coverage in the SRVP of Ndp(y/-) mice and explain it by an altered expression of PDGFß and its receptor (PDGFRß). In vivo cell proliferation assays revealed a reduced proliferation rate of isolectin B4-positive cells in the SRVP from Ndp(y/-) mice at postnatal day 6 and a decreased mitogenic activity of mutant compared with the wild-type Norrin. Our results suggest that the delayed outgrowth of the SRVP and decreased angiogenic sprouting in Ndp(y/-) mice are direct effects of the reduced proliferation of endothelial cells from the SRVP.
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Proliferación Celular , Proteínas del Ojo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vasos Retinianos/citología , Vasos Retinianos/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Dipéptidos/farmacología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Proteínas del Ojo/genética , Células HEK293 , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Seudópodos/efectos de los fármacos , Receptor Notch1/metabolismo , Retina/metabolismo , Retina/patología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacosRESUMEN
The proneural transcription factor atonal basic helix-loop-helix transcription factor 7 (ATOH7) is expressed in early progenitors in the developing neuroretina. In vertebrates, this is crucial for the development of retinal ganglion cells (RGCs), as mutant animals show an almost complete absence of RGCs, underdeveloped optic nerves, and aberrations in retinal vessel development. Human mutations are rare and result in autosomal recessive optic nerve hypoplasia (ONH) or severe vascular changes, diagnosed as autosomal recessive persistent hyperplasia of the primary vitreous (PHPVAR). To better understand the role of ATOH7 in neuroretinal development, we created ATOH7 knockout and eGFP-expressing ATOH7 reporter human induced pluripotent stem cells (hiPSCs), which were differentiated into early-stage retinal organoids. Target loci regulated by ATOH7 were identified by Cleavage Under Targets and Release Using Nuclease with sequencing (CUT&RUN-seq) and differential expression by RNA sequencing (RNA-seq) of wildtype and mutant organoid-derived reporter cells. Additionally, single-cell RNA sequencing (scRNA-seq) was performed on whole organoids to identify cell type-specific genes. Mutant organoids displayed substantial deficiency in axon sprouting, reduction in RGCs, and an increase in other cell types. We identified 469 differentially expressed target genes, with an overrepresentation of genes belonging to axon development/guidance and Notch signaling. Taken together, we consolidate the function of human ATOH7 in guiding progenitor competence by inducing RGC-specific genes while inhibiting other cell fates. Furthermore, we highlight candidate genes responsible for ATOH7-associated optic nerve and retinovascular anomalies, which sheds light to potential future therapy targets for related disorders.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Madre Pluripotentes Inducidas , Retina , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Retina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Diferenciación Celular/genética , Transducción de Señal , Células Ganglionares de la Retina/metabolismo , Organoides/metabolismo , Regulación del Desarrollo de la Expresión GénicaRESUMEN
X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is one of the most severe forms of RP due to its early onset and intractable progression. Most cases have been associated with genetic variants within the purine-rich exon ORF15 region of this gene. RPGR retinal gene therapy is currently being investigated in several clinical trials. Therefore, it is crucial to report and functionally characterize (all novel) potentially pathogenic DNA sequence variants. Whole-exome sequencing (WES) was performed for the index patient. The splicing effects of a non-canonical splice variant were tested on cDNA from whole blood and a minigene assay. WES revealed a rare, non-canonical splice site variant predicted to disrupt the wildtype splice acceptor and create a novel acceptor site 8 nucleotides upstream of RPGR exon 12. Reverse-transcription PCR analyses confirmed the disruption of the correct splicing pattern, leading to the insertion of eight additional nucleotides in the variant transcript. Transcript analyses with minigene assays and cDNA from peripheral blood are useful tools for the characterization of splicing defects due to variants in the RPGR and may increase the diagnostic yield in RP. The functional analysis of non-canonical splice variants is required to classify those variants as pathogenic according to the ACMG's criteria.
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Proteínas del Ojo , Retinitis Pigmentosa , Humanos , Proteínas del Ojo/genética , ADN Complementario , Mutación , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/diagnóstico , RetinaRESUMEN
Coloboma and microphthalmia (C/M) are related congenital eye malformations, which can cause significant visual impairment. Molecular diagnosis is challenging as the genes associated to date with C/M account for only a small percentage of cases. Overall, the genetic cause remains unknown in up to 80% of patients. High throughput DNA sequencing technologies, including whole-exome sequencing (WES), are therefore a useful and efficient tool for genetic screening and identification of new mutations and novel genes in C/M. In this study, we analyzed the DNA of 19 patients with C/M from 15 unrelated families using singleton WES and data analysis for 307 genes of interest. We identified seven novel and one recurrent potentially disease-causing variants in CRIM1, CHD7, FAT1, PTCH1, PUF60, BRPF1, and TGFB2 in 47% of our families, three of which occurred de novo. The detection rate in patients with ocular and extraocular manifestations (67%) was higher than in patients with an isolated ocular phenotype (46%). Our study highlights the significant genetic heterogeneity in C/M cohorts and emphasizes the diagnostic power of WES for the screening of patients and families with C/M.
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Coloboma/genética , Secuenciación del Exoma , Microftalmía/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Coloboma/diagnóstico , Variaciones en el Número de Copia de ADN , Femenino , Heterogeneidad Genética , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Masculino , Tamizaje Masivo/métodos , Microftalmía/diagnóstico , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
IMPORTANCE: Identification of geographic population-based differences in genotype and phenotype heterogeneity are important for targeted and patient-specific diagnosis and treatment, counseling, and screening strategies. OBJECTIVE: To report disease-causing variants and their detailed phenotype in patients with bilateral congenital cataract from a single center in Switzerland and thereby draw a genetic map and perform a genotype-phenotype comparison of this cohort. DESIGN, SETTING, AND PARTICIPANTS: This clinical and molecular-genetic cohort study took place through the collaboration of the Department of Ophthalmology at the University Hospital Zurich and the Institute of Medical Molecular Genetics, University of Zurich, Schlieren, Switzerland. Thirty-seven patients from 25 families with different types of bilateral congenital cataract were included. All participating family members received a comprehensive eye examination. Whole exome sequencing was performed in the index patients, followed by a filtering process to detect possible disease-associated variants in genes previously described in association with congenital cataract. Probable disease-causing variants were confirmed by Sanger sequencing in available family members. All data were collected from January 2018 to June 2020, and the molecular-genetic analyses were performed from January 2019 to July 2020. MAIN OUTCOMES AND MEASURES: Identification of the underlying genetic causes of bilateral congenital cataract, including novel disease-causing variants and phenotype correlation. RESULTS: Among the 37 patients (18 [49%] male and 19 [51%] female; mean [SD] age, 17.3 [15.9] years) from 25 families, pathogenic variants were detected in 20 families (80% detection rate), which included 13 novel variants in the following genes: BCOR, COL4A1, CRYBA2, CRYBB2, CRYGC, CRYGS, GJA3, MAF, NHS, and WFS1. Putative disease-causing variants were identified in 14 of 20 families (70%) as isolated cases and in 6 of 20 families (30%) with syndromic cases. A recessive variant in the CRYBB2 gene in a consanguineous family with 2 affected siblings showing a nuclear and sutural cataract was reported in contrast to previously published reports. In addition, the effect on splicing in a minigene assay of a novel splice site variant in the NHS gene (c.[719-2A>G]) supported the pathogenicity of this variant. CONCLUSIONS AND RELEVANCE: This study emphasizes the importance of genetic testing of congenital cataracts. Known dominant genes need to be considered for recessive inheritance patterns. Syndromic types of cataract may be underdiagnosed in patients with mild systemic features.
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Catarata , Catarata/congénito , Estudios de Cohortes , Femenino , Pruebas Genéticas , Humanos , Masculino , Linaje , Suiza/epidemiologíaRESUMEN
PURPOSE: To (i) describe a series of patients with isolated or syndromic nanophthalmos with the underlying genetic causes, including novel pathogenic variants and their functional characterization and (ii) to study the association of retinal dystrophy in patients with MFRP variants, based on a detailed literature review of genotype-phenotype correlations. METHODS: Patients with nanophthalmos and available family members received a comprehensive ophthalmological examination. Genetic analysis was based on whole-exome sequencing and variant calling in core genes including MFRP, BEST1, TMEM98, PRSS56, CRB1, GJA1, C1QTNF5, MYRF and FAM111A. A minigene assay was performed for functional characterization of a splice site variant. RESULTS: Seven patients, aged between three and 65 years, from five unrelated families were included. Novel pathogenic variants in MFRP (c.497C>T, c.899-3C>A, c.1180G>A), and PRSS56 (c.1202C>A), and a recurrent de novo variant in FAM111A (c.1706G>A) in a patient with Kenny-Caffey syndrome type 2, were identified. In addition, we report co-inheritance of MFRP-related nanophthalmos and ADAR-related Aicardi-Goutières syndrome. CONCLUSION: Nanophthalmos is a genetically heterogeneous condition, and the severity of ocular manifestations appears not to correlate with variants in a specific gene. However, retinal dystrophy is only observed in patients harbouring pathogenic MFRP variants. Furthermore, heterozygous carriers of MFRP and PRSS56 should be screened for the presence of high hyperopia. Identifying nanophthalmos as an isolated condition or as part of a syndrome has implications for counselling and can accelerate the interdisciplinary care of patients.
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ADN/genética , Proteínas de la Membrana/genética , Microftalmía/genética , Mutación , Adolescente , Adulto , Anciano , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Pruebas Genéticas , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Microftalmía/metabolismo , Persona de Mediana Edad , Linaje , Fenotipo , Adulto JovenRESUMEN
Purpose: The aim of this study was to investigate the molecular basis of childhood glaucoma in Switzerland to recommend future targeted genetic analysis in the Swiss population. Methods: Whole-exome sequencing and copy number variation (CNV) analysis was performed in a Swiss cohort of 18 patients from 14 unrelated families. Identified variants were validated by Sanger sequencing and multiplex ligation-dependent probe amplification. Breakpoints of structural variants were determined by a microarray. A minigene assay was conducted for functional analysis of a splice site variant. Results: A diagnosis of primary congenital glaucoma was made in 14 patients, of which six (43%) harbored pathogenic variants in CYP1B1, one (7%) a frameshift variant in FOXC1, and seven (50%) remained without a genetic diagnosis. Three patients were diagnosed with glaucoma associated with nonacquired ocular anomalies, of which two patients with mild ocular features of Axenfeld-Rieger syndrome harbored a FOXC1 duplication plus an additional FOXC1 missense variant, and one patient with a Barkan membrane remained without genetic diagnosis. A diagnosis of juvenile open-angle glaucoma was made in one patient, and genetic analysis revealed a FOXC1 duplication. Conclusions: Sequencing of CYP1B1 and FOXC1, as well as analysis of CNVs in FOXC1, should be performed before extended gene panel sequencing. Translational Relevance: The identification of the molecular cause of childhood glaucoma is a prerequisite for genetic counseling and personalized care for patients and families.
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Exoma , Glaucoma , Citocromo P-450 CYP1B1/genética , Variaciones en el Número de Copia de ADN/genética , Factores de Transcripción Forkhead/genética , Glaucoma/genética , Humanos , Suiza , Secuenciación del ExomaRESUMEN
Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/ß-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR.
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Barrera Hematorretinal/metabolismo , Células Endoteliales/metabolismo , Vitreorretinopatías Exudativas Familiares/genética , Neovascularización Fisiológica/genética , Proteínas Serina-Treonina Quinasas/genética , Vasos Retinianos/crecimiento & desarrollo , Animales , Células Endoteliales/citología , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Proteínas de Microfilamentos/genética , Fenotipo , Vía de Señalización Wnt/genéticaRESUMEN
X-linked Norrie disease, familial exudative vitreoretinopathy (FEVR), Coat's disease and retinopathy of prematurity are severe human eye diseases and can all be caused by mutations in the Norrie disease pseudoglioma gene. They all show vascular defects and characteristic features of retinal hypoxia. Only Norrie disease displays additional neurological symptoms, which are sensorineural hearing loss and mental retardation. In the present study, we analysed transcript levels of the ligand Norrin (Ndph) and its two receptors Frizzled-4 (Fzd4) and LDL-related protein receptor 5 (Lrp5) in six different brain regions (cerebellum, cortex, hippocampus, olfactory bulb, pituitary and brain stem) of 6- to 8-month-old wild-type and Ndph knockout mice by quantitative real-time PCR. No effect of the Ndph knockout allele on Fzd4 or Lrp5 receptor expression was found. Furthermore, no alterations of the transcript levels of three hypoxia-regulated angiogenic factors (Vegfa, Itgrb3 and Tie1) were observed in the absence of Norrin. Interestingly, we identified significant differences in Ndph, Fzd4 and Lrp5 transcript levels in brain regions of wild-type mice and observed highest expression of Norrin and frizzled-4 in cerebellum. Transcript analyses were correlated with morphological data obtained from cerebellum and immunohistochemical studies of blood vessels in different brain regions. Vessel density was reduced in the cerebellum of Ndph knockout mice but the number of Purkinje and granular cells was not altered. This provides the first description of a brain phenotype in Ndph knockout mice, which will help to elucidate the role of Norrin in the brain.
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Cerebelo/anomalías , Cerebelo/irrigación sanguínea , Arterias Cerebrales/anomalías , Proteínas del Ojo/genética , Receptores Frizzled/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Moduladores de la Angiogénesis , Proteínas Angiogénicas/genética , Proteínas Angiogénicas/metabolismo , Animales , Cerebelo/metabolismo , Arterias Cerebrales/metabolismo , Femenino , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/genética , Células de Purkinje/citología , Células de Purkinje/metabolismoRESUMEN
PURPOSE: The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. METHODS: In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. RESULTS: This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3'-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. CONCLUSIONS: RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.
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Enfermedades Genéticas Ligadas al Cromosoma X/diagnóstico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación/genética , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/genética , Exones/genética , Proteínas del Ojo/genética , Familia , Femenino , Proteínas de Unión al GTP , Genes Dominantes , Heterocigoto , Humanos , Patrón de Herencia/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Proteínas de la Membrana/genética , Linaje , Polimorfismo Genético , Eliminación de SecuenciaRESUMEN
Mutations in the GRM6 gene, which encodes the metabotropic glutamate receptor 6 (mGluR6), lead to autosomal recessive congenital stationary night blindness (CSNB), which is characterized by loss of night vision due to a defect in signal transmission from photoreceptor to the adjacent ON-bipolar cells in the retina. So far, the sequence variations that have been described in six different families include nonsense, frameshift, and missense mutations. Here we investigated the impact of missense mutations in the ligand-binding domain, a conserved cysteine-rich domain, and the intracellular domain on the localization of the protein. We visualized and discriminated between surface and intracellular protein. Here we demonstrate that the wild-type (wt) protein localizes to the cell surface, and to endoplasmic reticulum (ER) and Golgi compartments. This also holds true for a mGluR6 variant containing a polymorphic, nondisease-associated amino acid exchange in the ligand-binding domain. In contrast, all disease-associated missense mutations lead to retention of the protein in the ER, while dimerization seems not to be affected. This is the first report that shows that CSNB-associated mutations in three different domains of mGluR6 abolish proper protein trafficking. We propose that the ligand-binding and the poorly characterized cysteine-rich domains, in addition to the intracellular domains, have a pivotal role in correct trafficking of metabotropic glutamate receptors to the cell surface.
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Cisteína/metabolismo , Mutación Missense/genética , Ceguera Nocturna/genética , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Biología Computacional , Dimerización , Humanos , Ligandos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
PURPOSE: In a spontaneous mutant substrain of C57BL/10 mice, severely affected retinal ribbon-type synapses have been described. The retinopathy was accompanied by a substantial loss in the activities of the second-order neurons. Rod photoreceptor responses were maintained with reduced amplitude, whereas cone activities were absent. This study was conducted to identify the genetic defect underlying this hitherto unknown autosomal recessive cone-rod dysfunction. METHODS: Genome-wide linkage analysis and screening of positional candidate genes were used to identify the causative mutation. Tissue-specific transcriptional activity of the defective gene was determined by Northern blot analysis and RT-PCR approaches. The number of cone photoreceptors was estimated by immunohistochemistry. RESULTS: The mutation was localized to a 275-kb region of chromosome 6. Within this candidate interval, a homozygous frameshift mutation (c.2367insC) was identified in the Cacna2d4 gene of affected animals. This gene codes for an L-type calcium channel auxiliary subunit of the alpha2delta type. The mutation introduces a premature stop codon that truncates one third of the predicted Cacna2d4 protein. A severe reduction in Cacna2d4 transcript levels observed in mutant retinas probably results in the lack of Cacna2d4 protein. The mutation leads to significant loss of rods, whereas the number of cone cells remains unaffected until 6 weeks of age. CONCLUSIONS: The Cacna2d4 mutation underlies a novel channelopathy leading to cone-rod dysfunction in the visual system of mice and provides a new candidate gene for human retinal disorders including night blindness, retinitis pigmentosa, and cone-rod dystrophies.
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Canales de Calcio Tipo L/genética , Mutación del Sistema de Lectura , Retina/fisiopatología , Degeneración Retiniana/genética , Degeneración Retiniana/fisiopatología , Sinapsis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Análisis Mutacional de ADN , Electrorretinografía , Ligamiento Genético , Genotipo , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transmisión SinápticaRESUMEN
PURPOSE: To characterize developmental defects and the time course of Norrie disease in retinal and hyaloid vasculature during retinal development and to identify underlying molecular angiogenic pathways that may be affected in Norrie disease, exudative vitreoretinopathy, retinopathy of prematurity, and Coats' disease. METHODS: Norrie disease pseudoglioma homologue (Ndph)-knockout mice were studied during retinal development at early postnatal (p) stages (p5, p10, p15, and p21). Histologic techniques, quantitative RT-PCR, ELISA, and Western blot analyses provided molecular data, and scanning laser ophthalmoscopy (SLO) angiography and electroretinography (ERG) were used to obtain in vivo data. RESULTS: The data showed that regression of the hyaloid vasculature of Ndph-knockout mice occurred but was drastically delayed. The development of the superficial retinal vasculature was strongly delayed, whereas the deep retinal vasculature did not form because of the blockage of vessel outgrowth into the deep retinal layers. Subsequently, microaneurysm-like lesions formed. Several angiogenic factors were differentially transcribed during retinal development. Increased levels of hypoxia inducible factor-1alpha (HIF1alpha) and VEGFA, as well as a characteristic ERG pattern, confirmed hypoxic conditions in the inner retina of the Ndph-knockout mouse. CONCLUSIONS: These data provide evidence for a crucial role of Norrin in hyaloid vessel regression and in sprouting angiogenesis during retinal vascular development, especially in the development of the deep retinal capillary networks. They also suggest an early and a late phase of Norrie disease and may provide an explanation for similar phenotypic features of allelic retinal diseases in mice and patients as secondary consequences of pathologic hypoxia.
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Proteínas del Ojo/fisiología , Proteínas del Tejido Nervioso/fisiología , Arteria Oftálmica/fisiopatología , Neovascularización Retiniana/metabolismo , Vasos Retinianos/crecimiento & desarrollo , Retinopatía de la Prematuridad/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Cuerpo Vítreo/irrigación sanguínea , Animales , Western Blotting , Proteínas de Unión al ADN/metabolismo , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Angiografía con Fluoresceína , Expresión Génica/fisiología , Humanos , Hipoxia/metabolismo , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Recién Nacido , Ratones , Ratones Noqueados , Proteínas Nucleares/metabolismo , Oftalmoscopía , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Retinopatía de la Prematuridad/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Vitreorretinopatía Proliferativa/patologíaRESUMEN
PURPOSE: To describe the clinical features and genetic analysis of eight X-linked congenital stationary night blindness (XLCSNB) Dutch patients. METHODS: Electroretinogram (ERG) measurements were assessed in Dutch patients. Molecular genetic testing by denaturing high performance liquid chromatography (DHPLC), single stranded conformation polymorphism (SSCP) analysis, and direct sequencing of the CACNA1F and NYX genes were performed in the patients possessing a negative Schubert Bornschein ERG. RESULTS: Molecular genetic testing of CACNA1F and NYX revealed three novel and two known CACNA1F sequence variants as well as two novel sequence alterations in the NYX gene. While one of the CACNA1F sequence variants (5756G>A, R1919H) has been previously described as a common polymorphism in Japanese families, we did not found this transition in 100 European control alleles. CONCLUSIONS: In a pool of eight diagnosed XLCSNB patients, five showed a sequence variation in the CACNA1F and two in the NYX gene. In only one of the eight patients no sequence alteration could be detected. This might be explained by a mutation in other, as yet unidentified coding or regulatory sequences of NYX or CACNA1F or additional genes.
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Canales de Calcio Tipo L/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Mutación , Ceguera Nocturna/congénito , Proteoglicanos/genética , Adulto , Anciano , Niño , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , Electrooculografía , Electrorretinografía , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Humanos , Países Bajos , Ceguera Nocturna/fisiopatología , Estimulación Luminosa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
PURPOSE: Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exudative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot-containing extracellular signaling molecule of unknown function. In the current study, gene expression was determined in a mouse model of ND, to unravel disease-associated mechanisms at the molecular level. METHODS: Gene transcription in the eyes of 2-year-old Ndp knockout mice was compared with that in the eyes of age-matched wild-type control animals, by means of cDNA subtraction and microarrays. Clones (n = 3072) from the cDNA subtraction libraries were spotted onto glass slides and hybridized with fluorescently labeled RNA-derived targets. More than 230 differentially expressed clones were sequenced, and their expression patterns were verified by virtual Northern blot analysis. RESULTS: Numerous gene transcripts that are absent or downregulated in the eye of Ndp knockout mice are photoreceptor cell specific. In younger Ndp knockout mice (up to 1 year old), however, all these transcripts were found to be expressed at normal levels. CONCLUSIONS: The identification of numerous photoreceptor cell-specific transcripts with a reduced expression in 2-year-old, but not in young, Ndp knockout mice indicates that normal gene expression in these light-sensitive cells of mutant mice is established and maintained over a long period and that rods and cones are affected relatively late in the mouse model of ND. Obviously, the absence of the Ndp gene product is not compatible with long-term survival of photoreceptor cells in the mouse.