RESUMEN
Pigmentary glaucoma has recently been associated with missense mutations in PMEL that are dominantly inherited and enriched in the protein's fascinating repeat domain. PMEL pathobiology is intriguing because PMEL forms functional amyloid in healthy eyes, and this PMEL amyloid acts to scaffold melanin deposition. This is an informative contradistinction to prominent neurodegenerative diseases where amyloid formation is neurotoxic and mutations cause a toxic gain of function called "amyloidosis". Preclinical animal models have failed to model this PMEL "dysamyloidosis" pathomechanism and instead cause recessively inherited ocular pigment defects via PMEL loss of function; they have not addressed the consequences of disrupting PMEL's repetitive region. Here, we use CRISPR to engineer a small in-frame mutation in the zebrafish homolog of PMEL that is predicted to subtly disrupt the protein's repetitive region. Homozygous mutant larvae displayed pigmentation phenotypes and altered eye morphogenesis similar to presumptive null larvae. Heterozygous mutants had disrupted eye morphogenesis and disrupted pigment deposition in their retinal melanosomes. The deficits in the pigment deposition of these young adult fish were not accompanied by any detectable glaucomatous changes in intraocular pressure or retinal morphology. Overall, the data provide important in vivo validation that subtle PMEL mutations can cause a dominantly inherited pigment pathology that aligns with the inheritance of pigmentary glaucoma patient pedigrees. These in vivo observations help to resolve controversy regarding the necessity of PMEL's repeat domain in pigmentation. The data foster an ongoing interest in an antithetical dysamyloidosis mechanism that, akin to the amyloidosis of devastating dementias, manifests as a slow progressive neurodegenerative disease.
Asunto(s)
Glaucoma de Ángulo Abierto , Enfermedades Neurodegenerativas , Animales , Humanos , Adulto Joven , Amiloide/metabolismo , Ojo/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Antígeno gp100 del Melanoma/genética , Melanosomas/genética , Melanosomas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Pez CebraRESUMEN
Pigmentary glaucoma (PG) is a common glaucoma subtype that results from release of pigment from the iris, called pigment dispersion syndrome (PDS), and its deposition throughout the anterior chamber of the eye. Although PG has a substantial heritable component, no causative genes have yet been identified. We used whole exome sequencing of two independent pedigrees to identify two premelanosome protein (PMEL) variants associated with heritable PDS/PG. PMEL encodes a key component of the melanosome, the organelle essential for melanin synthesis, storage and transport. Targeted screening of PMEL in three independent cohorts (n = 394) identified seven additional PDS/PG-associated non-synonymous variants. Five of the nine variants exhibited defective processing of the PMEL protein. In addition, analysis of PDS/PG-associated PMEL variants expressed in HeLa cells revealed structural changes to pseudomelanosomes indicating altered amyloid fibril formation in five of the nine variants. Introduction of 11-base pair deletions to the homologous pmela in zebrafish by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method caused profound pigmentation defects and enlarged anterior segments in the eye, further supporting PMEL's role in ocular pigmentation and function. Taken together, these data support a model in which missense PMEL variants represent dominant negative mutations that impair the ability of PMEL to form functional amyloid fibrils. While PMEL mutations have previously been shown to cause pigmentation and ocular defects in animals, this research is the first report of mutations in PMEL causing human disease.
Asunto(s)
Glaucoma de Ángulo Abierto/genética , Antígeno gp100 del Melanoma/genética , Antígeno gp100 del Melanoma/fisiología , Adulto , Amiloide/metabolismo , Animales , Femenino , Células HeLa , Humanos , Iris/metabolismo , Masculino , Melanosomas/genética , Persona de Mediana Edad , Mutación Missense/genética , Linaje , Pigmentación/genética , Secuenciación del Exoma/métodos , Adulto Joven , Pez CebraRESUMEN
Molecular mechanisms governing the development of the human cochlea remain largely unknown. Through genome sequencing, we identified a homozygous FOXF2 variant c.325A>T (p.I109F) in a child with profound sensorineural hearing loss (SNHL) associated with incomplete partition type I anomaly of the cochlea. This variant is not found in public databases or in over 1000 ethnicity-matched control individuals. I109 is a highly conserved residue in the forkhead box (Fox) domain of FOXF2, a member of the Fox protein family of transcription factors that regulate the expression of genes involved in embryogenic development as well as adult life. Our in vitro studies show that the half-life of mutant FOXF2 is reduced compared to that of wild type. Foxf2 is expressed in the cochlea of developing and adult mice. The mouse knockout of Foxf2 shows shortened and malformed cochleae, in addition to altered shape of hair cells with innervation and planar cell polarity defects. Expressions of Eya1 and Pax3, genes essential for cochlear development, are reduced in the cochleae of Foxf2 knockout mice. We conclude that FOXF2 plays a major role in cochlear development and its dysfunction leads to SNHL and developmental anomalies of the cochlea in humans and mice.
Asunto(s)
Cóclea/embriología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/fisiología , Adulto , Animales , Niño , Cóclea/metabolismo , Cóclea/fisiología , Desarrollo Embrionario , Femenino , Células Ciliadas Auditivas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Organogénesis , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/fisiología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Transducción de Señal/genética , Secuenciación Completa del GenomaRESUMEN
The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.
Asunto(s)
Melanocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutación , Enfermedades Neurodegenerativas/patología , Antígeno gp100 del Melanoma/metabolismo , Secuencia de Aminoácidos , Proteínas Amiloidogénicas/metabolismo , Animales , Biología Computacional/métodos , Humanos , Glicoproteínas de Membrana/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Filogenia , Pigmentación , Dominios Proteicos , Homología de Secuencia , Antígeno gp100 del Melanoma/genéticaRESUMEN
In this special issue of the American Journal of Medical Genetics, Part C, we explore the ever-expanding field of Ophthalmic Genetics. The eye is unique among organs for its accessibility to physical examination, permitting exploration of every tissue by slit lamp microscopy, ophthalmoscopy, and imaging including color and autofluorescent photography, ultrasound, optical coherence tomography (OCT), electrophysiology, and adaptive optics confocal and scanning laser ophthalmoscopy. This accessibility permits a variety of surgical and nonsurgical treatments, including the first FDA-approved gene therapy, voretigene neparvovec-rzyl for RPE65-associated Leber Congenital Amaurosis. In this issue, we sought to provide a survey highlighting how heritable ophthalmic disorders are recognizable and accessible to clinical geneticists as well as ophthalmologists.
Asunto(s)
Oftalmopatías/terapia , Terapia Genética , Oftalmología/tendencias , Tomografía de Coherencia Óptica/métodos , Oftalmopatías/diagnóstico por imagen , Oftalmopatías/genética , Oftalmopatías/patología , HumanosRESUMEN
Current genetic screening methods for inherited eye diseases are concentrated on the coding exons of known disease genes (gene panels, clinical exome). These tests have a variable and often limited diagnostic rate depending on the clinical presentation, size of the gene panel and our understanding of the inheritance of the disorder (with examples described in this issue). There are numerous possible explanations for the missing heritability of these cases including undetected variants within the relevant gene (intronic, up/down-stream and structural variants), variants harbored in genes outside the targeted panel, intergenic variants, variants undetectable by the applied technology, complex/non-Mendelian inheritance, and nongenetic phenocopies. In this article we further explore and review methods to investigate these sources of missing heritability.
Asunto(s)
Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/genética , Genoma Humano/genética , Genómica/métodos , Enfermedades Hereditarias del Ojo/epidemiología , Enfermedades Hereditarias del Ojo/terapia , Humanos , Oftalmología/tendenciasRESUMEN
Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of this structural variant. Deletion in zebrafish reduced pitx2 expression during development and resulted in shallow anterior chambers. We screened additional patients for copy number variation of the putative regulatory elements and found an overlapping deletion in a second family and in a potentially-ancestrally-related index patient with ASD and glaucoma. These data suggest that mutations affecting conserved non-coding elements of PITX2 may constitute an important class of mutations in patients with ASD for whom the molecular cause of their disease have not yet been identified. Improved functional annotation of the human genome and transition to sequencing of patient genomes instead of exomes will be required before the magnitude of this class of mutations is fully understood.
Asunto(s)
Anomalías del Ojo/genética , Glaucoma/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Segmento Anterior del Ojo/metabolismo , Secuencia Conservada , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Anomalías del Ojo/metabolismo , Eliminación de Gen , Glaucoma/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Intrones , Músculos , Mutación , Linaje , Eliminación de Secuencia , Factores de Transcripción/metabolismo , Pez Cebra/genética , Proteína del Homeodomínio PITX2RESUMEN
Aniridia and Axenfeld-Rieger Syndrome are related, human ocular disorders that are typically inherited in an autosomal dominant manner. Both result from incorrect development of the eye and have, as their most serious consequences, elevated risk to develop the blinding condition glaucoma. This review will focus on describing the clinical presentations of Aniridia and Axenfeld-Rieger Syndrome as well as the molecular genetics and current and emerging therapies used to treat patients.
Asunto(s)
Anomalías Múltiples , Aniridia/genética , Segmento Anterior del Ojo/anomalías , Anomalías del Ojo/genética , Enfermedades Hereditarias del Ojo/genética , Proteínas de Homeodominio/genética , Aniridia/diagnóstico , Aniridia/metabolismo , Segmento Anterior del Ojo/metabolismo , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/metabolismo , Genotipo , Proteínas de Homeodominio/metabolismo , Humanos , MutaciónRESUMEN
Mutations in the forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger syndrome (ARS). Here, we investigated the effect of four ARS missense variants on FOXC1 structure and function, and examined the predictive value of four in silico programs for all 31 FOXC1 missense variants identified to date. Molecular modeling of the FOXC1 forkhead domain predicts that c.402G> A (p.C135Y) alters FOXC1's structure. In contrast, c.378A> G (p.H128R) and c.481A> G (p.M161V) are not predicted to change FOXC1's structure. Functional analysis indicates that p.H128R reduced DNA binding, transactivation, nuclear localization, and has a longer protein half-life than normal. p.C135Y significantly disrupts FOXC1's DNA binding, transactivation, and nuclear localization. p.M161V reduces transactivation capacity without affecting other FOXC1 functions. C.1103C> A (p.T368N) is indistinguishable from wild-type FOXC1 in all tests, consistent with being a rare benign variant. Comparison of these four variants, plus 18 previously characterized FOXC1 missense variants, with predictions from four commonly used in silico bioinformatics programs indicated that sorting intolerant from tolerant (SIFT), polymorphism phenotyping (PolyPhen-2), and MutPred can sensitively identify as pathogenic only FOXC1 mutations with significant functional defects. This information was used to predict, as disease-causing, nine additional FOXC1 missense variations. Importantly, our results indicate SIFT, PolyPhen-2, and MutPred can reliably be used to predict missense variant pathogenicity for forkhead transcription factors.
Asunto(s)
Segmento Anterior del Ojo/anomalías , Biología Computacional , Anomalías del Ojo/genética , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Modelos Moleculares , Mutación , Alelos , Secuencia de Aminoácidos , Biología Computacional/métodos , Anomalías del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Genotipo , Células HeLa , Humanos , Mutación Missense , Conformación Proteica , Programas Informáticos , Relación Estructura-Actividad , Transactivadores/metabolismoRESUMEN
The forkhead box (Fox) superfamily of transcription factors has essential roles in organogenesis and tissue differentiation. Foxa1 and Foxa2 are expressed during prostate budding and ductal morphogenesis, whereas Foxa1 expression is retained in adult prostate epithelium. Previous characterization of prostatic tissue rescued from embryonic Foxa1 knockout mice revealed Foxa1 to be essential for ductal morphogenesis and epithelial maturation. However, it is unknown whether Foxa1 is required to maintain the differentiated status in adult prostate epithelium. Here, we employed the PBCre4 transgenic system and determined the impact of prostate-specific Foxa1 deletion in adult murine epithelium. PBCre4/Foxa1(loxp/loxp) mouse prostates showed progressive florid hyperplasia with extensive cribriform patterning, with the anterior prostate being most affected. Immunohistochemistry studies show mosaic Foxa1 KO consistent with PBCre4 activity, with Foxa1 KO epithelial cells specifically exhibiting altered cell morphology, increased proliferation, and elevated expression of basal cell markers. Castration studies showed that, while PBCre4/Foxa1(loxp/loxp) prostates did not exhibit altered sensitivity in response to hormone ablation compared with control prostates, the number of Foxa1-positive cells in mosaic Foxa1 KO prostates was significantly reduced compared with Foxa1-negative cells following castration. Unexpectedly, gene expression profile analyses revealed that Foxa1 deletion caused abnormal expression of seminal vesicle-associated genes in KO prostates. In summary, these results indicate Foxa1 expression is required for the maintenance of prostatic cellular differentiation.
Asunto(s)
Diferenciación Celular/genética , Epitelio/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Hiperplasia Prostática/genética , Animales , Epitelio/patología , Factor Nuclear 3-alfa del Hepatocito/deficiencia , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Inmunohistoquímica , Integrasas/genética , Integrasas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Seminales/metabolismo , Transcriptoma/genéticaRESUMEN
Anterior segment dysgenesis refers to a spectrum of disorders affecting structures in the anterior segment of the eye including the iris, cornea and trabecular meshwork. Approximately 50% of patients with anterior segment dysgenesis develop glaucoma. Traditional genetic methods using linkage analysis and family-based studies have identified numerous disease-causing genes such as PAX6, FOXC1 and PITX2. Despite these advances, phenotypic and genotypic heterogeneity pose continuing challenges to understand the mechanisms underlying the complexity of anterior segment dysgenesis disorders. Genomic methods, such as genome-wide association studies, are potentially an effective tool to understand anterior segment dysgenesis and the individual susceptibility to the development of glaucoma. In this review, we provide the rationale, as well as the challenges, to utilizing genomic methods to examine anterior segment dysgenesis disorders.
Asunto(s)
Segmento Anterior del Ojo/anomalías , Anomalías del Ojo/genética , Genómica , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , FenotipoRESUMEN
The PITX2 'homeobox' and FOXC1 and FOXC2 'forkhead box' transcription factors are critical for eye development and cause human ocular diseases when mutated. We have identified biochemical and genetic links between these transcription factors and a transcriptional regulator protein PRKC apoptosis Wilms' tumor 1 regulator (PAWR) that we propose to functionally connect all these proteins in a common pathway critically involved in eye development. We discovered all binary physical interactions between FOXC1, PITX2, FOXC2 and PAWR. Importantly, PAWR modulates the abilities of PITX2, FOXC1 and FOXC2 to activate their genetic targets. Together with either FOXC1 or FOXC2, PAWR increases PITX2 activity. PAWR reduces PITX2 activity in the absence of FOXC1 or FOXC2. At the same time, PAWR also exerts different regulatory effects on different FOXC target sites. Furthermore, morpholino knockdown of pitx2, foxc1 and pawr in zebrafish indicate that PAWR, FOXC1 and PITX2 genetically interact, and are in the same developmental pathway. These data for the first time tie PITX2, FOXC1, FOXC2 and PAWR into a common regulatory pathway. We have therefore identified a functional link between three transcription factors, modulated by PAWR, which we propose underlies the similar ocular phenotypes and glaucoma pathology caused by mutations of these genes.
Asunto(s)
Oftalmopatías/genética , Ojo/embriología , Ojo/crecimiento & desarrollo , Redes Reguladoras de Genes , Factores de Transcripción/genética , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Larva/crecimiento & desarrollo , Larva/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Pez Cebra/genética , Proteína del Homeodomínio PITX2RESUMEN
Ocular mal-development results in heterogeneous and frequently visually disabling phenotypes that include coloboma and microphthalmia. Due to the contribution of bone morphogenetic proteins to such processes, the function of the paralogue Growth Differentiation Factor 3 was investigated. Multiple mis-sense variants were identified in patients with ocular and/or skeletal (Klippel-Feil) anomalies including one individual with heterozygous alterations in GDF3 and GDF6. These variants were characterized, individually and in combination, through integrated biochemical and zebrafish model organism analyses, demonstrating appreciable effects with western blot analyses, luciferase based reporter assays and antisense morpholino inhibition. Notably, inhibition of the zebrafish co-orthologue of GDF3 accurately recapitulates patient phenotypes. By demonstrating the pleiotropic effects of GDF3 mutation, these results extend the contribution of perturbed BMP signaling to human disease and potentially implicate multi-allelic inheritance of BMP variants in developmental disorders.
Asunto(s)
Anomalías del Ojo/genética , Factor 3 de Diferenciación de Crecimiento/genética , Músculo Esquelético/anomalías , Mutación , Secuencia de Aminoácidos , Animales , Línea Celular , Anomalías del Ojo/metabolismo , Femenino , Factor 3 de Diferenciación de Crecimiento/química , Factor 3 de Diferenciación de Crecimiento/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Linaje , Alineación de Secuencia , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
PURPOSE: Mutations in the homeobox transcription factor paired-like homeodomain transcription factor 2 (PITX2) cause Axenfeld-Reiger syndrome (ARS), which is associated with anterior segment dysgenesis (ASD) and glaucoma. To understand ARS pathogenesis, it is essential to know the normal functions of PITX2 and the proteins with which PITX2 interacts in the eye. Therefore, we used a unique cDNA library that we created from human trabecular meshwork (TM) primary cells to discover PITX2-interacting proteins (PIPs). METHODS: A human TM cDNA library was created from primary cells in the ProQuest Two-Hybrid prey vector: pEXP-AD502. Human PITX2A and PITX2C isoforms were used independently as "bait" to identify novel PIPs. A total of 1.25×106 clones were screened by yeast two-hybrid (Y2H) analyses. PIPs obtained from each Y2H experiment were confirmed by yeast retransformation and mammalian co-immunoprecipitation assays. RESULTS: EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) was identified by both PITX2A and PITX2C isoforms as a novel PIP from Y2H analyses. EFEMP2 is 443 amino acids long with six epidermal growth factor (EGF)-like modules and one fibulin-like module. The PITX2-interaction domain in EFEMP2 lies between the second EGF-like module and the COOH-terminal fibulin-like module. Co-immunoprecipitation assays in COS-7 cells confirmed the interaction between PITX2 and EFEMP2. CONCLUSIONS: We discovered EFEMP2 as a novel PITX2-interacting protein. Further, our cDNA library made from human TM primary cells is a unique and effective resource to identify novel interacting proteins for glaucoma and ASD candidates. This resource could be used both for discovery and validation of interactomes identified from in silico analysis.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Proteínas de Homeodominio/metabolismo , Malla Trabecular/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Proteínas de la Matriz Extracelular/genética , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Biblioteca de Genes , Vectores Genéticos , Glaucoma/diagnóstico , Glaucoma/genética , Proteínas de Homeodominio/genética , Humanos , Inmunoprecipitación , Cultivo Primario de Células , Unión Proteica , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Malla Trabecular/citología , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Proteína del Homeodomínio PITX2RESUMEN
PURPOSE: Nuclear receptor 2E1 (NR2E1) is a transcription factor with many roles during eye development and thus may be responsible for the occurrence of certain congenital eye disorders in humans. To test this hypothesis, we screened NR2E1 for candidate mutations in patients with aniridia and other congenital ocular malformations (anterior segment dysgenesis, congenital optic nerve malformation, and microphthalmia). METHODS: The NR2E1 coding region, 5' and 3' untranslated regions (UTRs), exon flanking regions including consensus splice sites, and six evolutionarily conserved non-coding candidate regulatory regions were analyzed by sequencing 58 probands with aniridia of whom 42 were negative for PAX6 mutations. Nineteen probands with anterior segment dysgenesis, one proband with optic nerve malformation, and two probands with microphthalmia were also sequenced. The control population comprised 376 healthy individuals. All sequences were analyzed against the GenBank sequence AL078596.8 for NR2E1. In addition, the coding region and flanking intronic sequences of FOXE3, FOXC1, PITX2, CYP1B1, PAX6, and B3GALTL were sequenced in one patient and his relatives. RESULTS: Sequencing analysis showed 17 NR2E1 variants including two novel rare non-coding variants (g.-1507G>A, g.14258C>T), and one novel rare coding variant (p.Arg274Gly). The latter was present in a male diagnosed with Peters' anomaly who subsequently was found to have a known causative mutation for Peters' plus syndrome in B3GALTL (c.660+1G>A). In addition, the NR2E1 novel rare variant Arg274Gly was present in the unaffected mother of the patient but absent in 746 control chromosomes. CONCLUSIONS: We eliminated a major role for NR2E1 regulatory and coding mutations in aniridia and found a novel rare coding variant in NR2E1. In addition, we found no coding region variation in the control population for NR2E1, which further supports its previously reported high level of conservation and low genetic diversity. Future NR2E1 studies in ocular disease groups such as those involving retinal and optic nerve abnormalities should be undertaken to determine whether NR2E1 plays a role in these conditions.
Asunto(s)
Aniridia/genética , Proteínas del Ojo/genética , Polimorfismo de Nucleótido Simple , Receptores Citoplasmáticos y Nucleares/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Análisis Mutacional de ADN , Anomalías del Ojo/genética , Femenino , Pruebas Genéticas , Humanos , Masculino , Microftalmía/genética , Mutación , Sistemas de Lectura Abierta , Nervio Óptico/anomalías , Receptores Nucleares HuérfanosRESUMEN
Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. POAG is associated with a characteristic progression of changes to ocular morphology and degeneration at the optic nerve head with the loss of visual fields. Physical mapping efforts identified genomic loci in which to search for causative POAG gene mutations. WDR36, at locus GLC1G, was initially identified as a gene with a low frequency of non-synonymous sequence variations that were exclusive to adult-onset POAG patients. It has since been shown that rare WDR36 sequence variants are also present in the normal population at similarly low frequencies. The lack of a consistent genotype:phenotype correlation prompted us to investigate the functional consequences of WDR36 sequence variations. WDR36 is involved in rRNA processing, a critical step in ribosome biogenesis, and is very similar to yeast Utp21p which is a member of the small subunit (SSU) processome complex responsible for maturation of 18S rRNA. We, therefore, developed a yeast model system to test the functional and phenotypic consequences of POAG-associated sequence variants introduced into UTP21. Alone, the POAG variants did not produce any significant defects in cell viability or rRNA processing. However, when combined with disruption of STI1 (which synthetically interacts with UTP21), 5 of the 11 tested variants had increased or decreased cell viability which corresponded to reduced or elevated levels of pre-rRNA, respectively. These results demonstrate that, in the correct genetic background, WDR36 sequence variants can lead to an altered cellular phenotype, supporting the theory that WDR36 participates in polygenic forms of glaucoma.
Asunto(s)
Proteínas del Ojo/metabolismo , Glaucoma de Ángulo Abierto/genética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Ojo/metabolismo , Ojo/patología , Proteínas del Ojo/química , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estructura Secundaria de Proteína , Ratas , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
PURPOSE: To investigate the role of multigenic variation in primary open-angle glaucoma (POAG) involving the rRNA processing gene WD repeat domain 36 (WDR36). METHODS: We examined the heat shock protein 70/90 (HSP70/90)-organizing co-chaperone stress-induced-phosphoprotein 1 (STI1) as a potential co-modifying gene in glaucoma patients found to harbor WDR36 amino acid variation. The STI1 gene was sequenced and its POAG-associated amino acid variant K434R, as well as the single nucleotide polymorphism (SNP) P173T, were tested for functional defects in a yeast model system previously used to characterize WDR36 variants (using the homologous yeast gene U3 protein 21 [UTP21]). RESULTS: A POAG patient heterozygous for the WDR36 variant L25P was discovered to also carry the STI1 variant K434R in a heterozygous state. Variant K434R, located at an evolutionarily-conserved site, was not found in a pool of clinically-examined individuals lacking WDR36 variation which included 55 normal controls and 20 patients with normal tension glaucoma (NTG). STI1 (K434R) and the homologous yeast variant K470R were able to rescue yeast growth-inhibition by the HSP90-inhibitor radicicol. Double mutant haploid strains expressing human STI1 (K434R) and recombinant yeast UTP21 variants did not have significantly different levels of 18S rRNA from the corresponding hSTI1 (WT) strains. However, specific double mutant K434R strains exhibited significantly slower culture growth at 37 °C. Double mutant P173T strains also displayed altered growth rates at 37 °C. CONCLUSIONS: STI1 variation does not play a significant direct role in the genetics of POAG. However, as previously found for the STI1 null allele, non-synonymous variants of human STI1 confer growth dysregulation in the context of specific yeast UTP21 mutations and heat stress. Based on the genetic association of two co-heterozygous STI1 and WDR36 variants in a POAG patient and the functional analyses performed in a model system for basic eukaryotic cellular processes, these experiments point to a conserved molecular pathway involving STI1 and WDR36.
Asunto(s)
Proteínas del Ojo/genética , Glaucoma de Ángulo Abierto/genética , Proteínas de Choque Térmico/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adulto , Secuencia de Aminoácidos , Estudios de Casos y Controles , Proliferación Celular , Exones , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Glaucoma de Ángulo Abierto/fisiopatología , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Heterocigoto , Humanos , Macrólidos/farmacología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , Inhibidores de Proteínas Quinasas/farmacología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
PURPOSE: Forkhead box Q1 (FOXQ1) has been shown to contribute to the development and progression of cancers, including ovarian and breast cancer (BC). However, research exploring FOXQ1 expression, copy number variation (CNV), and prognostic value across different BC subtypes is limited. Our purpose was to evaluate FOXQ1 mRNA expression, CNV, and prognostic value across BC subtypes. MATERIALS AND METHODS: We determined FOXQ1 expression and CNV in BC patient tumors using RT-qPCR and qPCR, respectively. We also analyzed FOXQ1 expression and CNV in BC cell lines in the CCLE database using K-means clustering. The prognostic value of FOXQ1 expression in the TCGA-BRCA database was assessed using univariate and multivariate Cox's regression analysis as well as using the online tools OncoLnc, GEPIA, and UALCAN. RESULTS: Our analyses reveal that FOXQ1 mRNA is differentially expressed between different subtypes of BC and is significantly decreased in luminal BC and HER2 patients when compared to normal breast tissue samples. Furthermore, analysis of BC cell lines showed that FOXQ1 mRNA expression was independent of CNV. Moreover, patients with low FOXQ1 mRNA expression had significantly poorer overall survival compared to those with high FOXQ1 mRNA expression. Finally, low FOXQ1 expression had a critical impact on the prognostic values of BC patients and was an independent predictor of overall survival when it was adjusted for BC subtypes and to two other FOX genes, FOXF2 and FOXM1. CONCLUSION: Our study reveals for the first time that FOXQ1 is differentially expressed across BC subtypes and that low expression of FOXQ1 is indicative of poor prognosis in patients with BC.
RESUMEN
Mutations in the homeobox transcription factor PITX2 result in Axenfeld-Rieger syndrome (ARS), which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from PITX2 mutations, it is essential to know the normal functions of PITX2 and its interaction with the network of proteins in the eye. Yeast two-hybrid screening was performed using a cDNA library from a human trabecular meshwork primary cell line to detect novel PITX2-interacting proteins and study their role in ARS pathogenesis. After screening of approximately 1 x 10(6) clones, one putative interacting protein was identified named PRKC apoptosis WT1 regulator (PAWR). This interaction was further confirmed by retransformation assay in yeast cells as well as co-immunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a proapoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Our analysis indicates that the homeodomain and the adjacent inhibitory domain in PITX2 interact with the C-terminal leucine zipper domain of PAWR. Endogenous PAWR and PITX2 were found to be located in the nucleus of ocular cells and to co-localize in the mesenchyme of the iridocorneal angle of the developing mouse eye, consistent with a role in the development of the anterior segment of the eye. PAWR was also found to inhibit PITX2 transcriptional activity in ocular cells. These data suggest PAWR is a novel PITX2-interacting protein that regulates PITX2 activity in ocular cells. This information sheds new light in understanding ARS and associated glaucoma pathogenesis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas del Ojo/metabolismo , Ojo , Proteínas de Homeodominio/metabolismo , Malla Trabecular , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular , Ojo/anatomía & histología , Ojo/metabolismo , Proteínas del Ojo/genética , Femenino , Proteínas de Homeodominio/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Malla Trabecular/citología , Malla Trabecular/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Proteína del Homeodomínio PITX2RESUMEN
Mutations in the human FOXC1 transcription factor gene underlie Axenfeld-Rieger (AR) syndrome, a disorder characterized by anterior segment malformations in the eye and glaucoma. Through the use of an inducible FOXC1 protein, along with an intermediate protein synthesis blocker, we have determined direct targets of FOXC1 transcriptional regulation. FOXC1 regulates the expression of FOXO1A and binds to a conserved element in the FOXO1A promoter in vivo. The zebrafish foxO1a orthologs exhibit a robust expression pattern in the periocular mesenchyme. Furthermore, FOXO1A expression is reduced in cultured human trabecular meshwork (TM) cells and in the zebrafish developing eye when FOXC1 expression is knocked down by siRNAs and morpholino antisense oliognucleotides, respectively. We also demonstrate that reduced FOXC1 expression increases cell death in cultured TM cells in response to oxidative stress, and increases cell death in the developing zebrafish eye. These studies have uncovered a novel role for FOXC1 as an essential mediator of cellular homeostasis in the eye and indicate that a decreased resistance to oxidative stress may underlie AR-glaucoma pathogenesis. Given that FOXO1A influences cellular homeostasis when positively or negatively regulated; the dysregulation of FOXO1A activities in the eye through FOXC1 loss of function mutations and FOXC1 gene duplications provides an explanation into how seemingly similar human disorders can arise from both increases and decreases in FOXC1 gene dose.