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1.
Infect Genet Evol ; 84: 104498, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32771700

RESUMO

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.


Assuntos
Infecções por Coronavirus/genética , Resistência à Doença/genética , Predisposição Genética para Doença , Interações Hospedeiro-Patógeno/genética , Metagenômica , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Alelos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Furina/genética , Furina/imunologia , Frequência do Gene , Variação Genética , Genoma Humano , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Pandemias , Peptidil Dipeptidase A/imunologia , Plasminogênio/genética , Plasminogênio/imunologia , Pneumonia Viral/imunologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Tripsina/genética , Tripsina/imunologia
2.
J Pharmacol Sci ; 144(2): 89-93, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763057

RESUMO

l-3,4-dihydroxyphenylalanine (l-DOPA) is a candidate neurotransmitter. l-DOPA is released by nicotine through nicotinic receptors. Recently, G-protein coupled receptor GPR143, was identified as a receptor for l-DOPA. In this study, genetic association studies between GPR143 genetic polymorphisms and smoking behaviors revealed that the single-nucleotide polymorphism rs6640499, in the GPR143 gene, was associated with traits of smoking behaviors in Japanese individuals. In Gpr143 gene-deficient mice, nicotine-induced hypolocomotion and rewarding effect were attenuated compared to those in wild-type mice. Our findings suggest the involvement of GPR143 in the smoking behaviors.


Assuntos
Proteínas do Olho/genética , Deleção de Genes , Estudos de Associação Genética , Glicoproteínas de Membrana/genética , Nicotina/efeitos adversos , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas-G/genética , Receptores de Neurotransmissores/genética , Reforço Psicológico , Transtornos Relacionados ao Uso de Substâncias/genética , Animais , Grupo com Ancestrais do Continente Asiático , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença
3.
PLoS One ; 15(7): e0234792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614850

RESUMO

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues.


Assuntos
Proteínas Angiogênicas/biossíntese , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Substituição de Aminoácidos , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Encéfalo/citologia , Proteínas de Transporte/análise , Linhagem da Célula , Epitopos/imunologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Desenvolvimento Muscular , Proteína MyoD/análise , Especificidade de Órgãos , Conformação Proteica , Domínios Proteicos , Coelhos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/imunologia , Sequências Repetitivas de Aminoácidos , Pele/citologia , Especificidade da Espécie , Tatuagem , Adulto Jovem
4.
Dev Genes Evol ; 230(4): 305-314, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32671457

RESUMO

Crinoids are considered as the most basal extant echinoderms. They retain aboral nervous system with a nerve center, which has been degraded in the eleutherozoan echinoderms. To investigate the evolution of patterning of the nervous systems in crinoids, we examined temporal and spatial expression patterns of three neural patterning-related homeobox genes, six3, pax6, and otx, throughout the development of a feather star Anneissia japonica. These genes were involved in the patterning of endomesodermal tissues instead of the ectodermal neural tissues in the early planktonic stages. In the stages after larval attachment, the expression of these genes was mainly observed in the podia and the oral nervous systems instead of the aboral nerve center. Our results indicate the involvement of these three genes in the formation of oral nervous system in the common ancestor of the echinoderms and suggest that the aboral nerve center is not evolutionally related to the brain of other bilaterians.


Assuntos
Equinodermos/crescimento & desenvolvimento , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX6/metabolismo , Animais , Padronização Corporal/genética , Equinodermos/genética , Equinodermos/metabolismo , Evolução Molecular , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Larva/genética , Larva/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Neurônios , Fatores de Transcrição Otx/genética , Fator de Transcrição PAX6/genética
5.
Am J Hum Genet ; 107(2): 278-292, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32707085

RESUMO

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains. A third dominant Best disease iPSC-RPE model did not respond to gene augmentation, but showed normalization of BEST1 channel activity following CRISPR-Cas9 editing of the mutant allele. We then subjected all three dominant Best disease iPSC-RPE models to gene editing, which produced premature stop codons specifically within the mutant BEST1 alleles. Single-cell profiling demonstrated no adverse perturbation of retinal pigment epithelium (RPE) transcriptional programs in any model, although off-target analysis detected a silent genomic alteration in one model. These results suggest that gene augmentation is a viable first-line approach for some individuals with dominant Best disease and that non-responders are candidates for alternate approaches such as gene editing. However, testing gene editing strategies for on-target efficiency and off-target events using personalized iPSC-RPE model systems is warranted. In summary, personalized iPSC-RPE models can be used to select among a growing list of gene therapy options to maximize safety and efficacy while minimizing time and cost. Similar scenarios likely exist for other genotypically diverse channelopathies, expanding the therapeutic landscape for affected individuals.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/genética , Mutação/genética , Alelos , Bestrofinas/genética , Cálcio/metabolismo , Linhagem Celular , Canalopatias/genética , Proteínas do Olho/genética , Edição de Genes/métodos , Terapia Genética/métodos , Genótipo , Células HEK293 , Humanos , Epitélio Pigmentado da Retina/fisiologia
6.
Gene ; 754: 144890, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32534057

RESUMO

PURPOSE: Stargardt disease (STGD) is the most frequent cause of hereditary macular dystrophy in childhood. Variants in the ABCA4, ELOVL4, PROM1, BEST1, and PRPH2 genes have been detected in patients with autosomal recessive or dominant STGD. This study was aimed at identifying the novel disease-associated variants in Chinese patients with STGD. METHODS: Ten Chinese families and two sporadic cases with STGD (n = 32) were enrolled in the study. All subjects underwent genetic analysis with next-generation sequencing (NGS), which was based on a specially customized capture panel targeting exons and untranslated regions (UTRs) of 792 genes related to common hereditary ophthalmopathy. Variants were analyzed to assess possible pathogenicity. RESULTS: Fourteen disease-associated variants of ABCA4 were detected in 9 Chinese families with autosomal recessive STGD, including 11 pathogenic variants and 3 likely pathogenic variants. Variant c.4253 + 4C > T in ABCA4 was identified as one de novo variant. Of the 14 distinct variants in ABCA4, 7 novel variants were found. In addition, one known variant of PROM1, c.1117C > T (p.Arg373Cys), was detected in one family and one sporadic case with autosomal dominant STGD, respectively. One novel missense variant of ELOVL4, c.59A > G (p.Asn20Ser), was found in one sporadic case with autosomal dominant STGD. The potential deleterious effects of these novel variants were confirmed through intensive analysis. CONCLUSION: By panel-based NGS, 8 novel disease-associated variants are identified in two genes responsible for STGD, including ABCA4 and ELOVL4. Our results further extend the mutation spectrum of these two genes in Chinese patients with STGD. One ABCA4 c.4253 + 4C > T variant is identified as a de novo splicing variant.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Marcadores Genéticos , Variação Genética , Mutação , Doença de Stargardt/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Criança , Proteínas do Olho/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Linhagem , Adulto Jovem
7.
PLoS One ; 15(6): e0232111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579557

RESUMO

Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) ß2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFß2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFß2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM.


Assuntos
Dexametasona/farmacologia , Limbo da Córnea/metabolismo , Malha Trabecular/efeitos dos fármacos , Fator de Crescimento Transformador beta2/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma de Ângulo Aberto/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/efeitos dos fármacos , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Regulação para Cima/efeitos dos fármacos
8.
Proc Natl Acad Sci U S A ; 117(26): 15293-15304, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541062

RESUMO

Organisms possess photoperiodic timing mechanisms to detect variations in day length and temperature as the seasons progress. The nature of the molecular mechanisms interpreting and signaling these environmental changes to elicit downstream neuroendocrine and physiological responses are just starting to emerge. Here, we demonstrate that, in Drosophila melanogaster, EYES ABSENT (EYA) acts as a seasonal sensor by interpreting photoperiodic and temperature changes to trigger appropriate physiological responses. We observed that tissue-specific genetic manipulation of eya expression is sufficient to disrupt the ability of flies to sense seasonal cues, thereby altering the extent of female reproductive dormancy. Specifically, we observed that EYA proteins, which peak at night in short photoperiod and accumulate at higher levels in the cold, promote reproductive dormancy in female D. melanogaster Furthermore, we provide evidence indicating that the role of EYA in photoperiodism and temperature sensing is aided by the stabilizing action of the light-sensitive circadian clock protein TIMELESS (TIM). We postulate that increased stability and level of TIM at night under short photoperiod together with the production of cold-induced and light-insensitive TIM isoforms facilitate EYA accumulation in winter conditions. This is supported by our observations that tim null mutants exhibit reduced incidence of reproductive dormancy in simulated winter conditions, while flies overexpressing tim show an increased incidence of reproductive dormancy even in long photoperiod.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas do Olho/metabolismo , Fotoperíodo , Estações do Ano , Temperatura , Animais , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica/fisiologia , Reprodução
9.
Medicine (Baltimore) ; 99(25): e20793, 2020 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-32569224

RESUMO

OBJECTIVE: To study the relationship between single nucleotide polymorphism (SNP) of the 3 primer untranslated region (UTR) variants of the cell fate determination factor Dachshund 1(DACH1) gene and the susceptibility of patients with endometrial cancer (EC). METHODS: Genomic DNA was extracted from the peripheral venous blood of 235 EC patients and 235 healthy controls, and the DACH1 gene rs9285274, rs9529895, rs17088351, and rs59352399 loci were analyzed by Sanger sequencing. Patients progression-free survival (PFS) was recorded after 3 years follow-up from October 2016 to October 2019. RESULTS: Carriers of the C allele of the DACH1 gene rs9529895 locus had a significantly lower risk for EC than T allele carriers (odds ratio = 0.56, 95%confidence interval: 0.38-0.84, P < .01). The correlation between DACH1 gene rs9529895 locus SNP and the risk for EC was affected by age, body mass index, smoking, drinking, and diabetes. Age, and rs9285274, rs9529895, and rs59352399 locus SNP were the best models for predicting the risk for EC. The accuracy rate was 57.02%, and the Cross-validation Consistency was 10/10 (x = 4.33, P = .04). The DACH1 gene rs9529895 locus C allele (TC+CC) carriers had significantly higher PFS than the TT genotype carriers (P = .04). The DACH1 gene was expressed in decreased amounts in the cancer tissues of EC patients, and the DACH1 mRNA expression level in the CC genotype, TC genotype, and TT genotype of rs9529895 locus was also decreased (P = .02). CONCLUSION: DACH1 gene rs9529895 locus SNP is significantly related to the risk for EC and PFS of EC patients. The possible mechanism behind this relationship is that the DACH1 gene rs9529895 locus SNP affects DACH1 expression level.


Assuntos
Neoplasias do Endométrio/genética , Proteínas do Olho/genética , MicroRNAs/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Western Blotting , Estudos de Casos e Controles , Proteínas do Olho/metabolismo , Feminino , Predisposição Genética para Doença/genética , Humanos , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo
10.
Nature ; 582(7811): 240-245, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499647

RESUMO

Meta-analyses of genome-wide association studies (GWAS) have identified more than 240 loci that are associated with type 2 diabetes (T2D)1,2; however, most of these loci have been identified in analyses of individuals with European ancestry. Here, to examine T2D risk in East Asian individuals, we carried out a meta-analysis of GWAS data from 77,418 individuals with T2D and 356,122 healthy control individuals. In the main analysis, we identified 301 distinct association signals at 183 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 61 loci that are newly implicated in predisposition to T2D. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. Previously undescribed associations include signals in or near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect the differentiation of muscle and adipose cells3. At another locus, expression quantitative trait loci at two overlapping T2D signals affect two genes-NKX6-3 and ANK1-in different tissues4-6. Association studies in diverse populations identify additional loci and elucidate disease-associated genes, biology, and pathways.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Aldeído-Desidrogenase Mitocondrial/genética , Alelos , Anquirinas/genética , Índice de Massa Corporal , Estudos de Casos e Controles , Europa (Continente)/etnologia , Proteínas do Olho/genética , Extremo Oriente/etnologia , Feminino , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Fatores de Transcrição/genética , Transcrição Genética
11.
Ren Fail ; 42(1): 463-473, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32408786

RESUMO

Objective: This report was designed to assess the functional role of miR-218/dachshund family transcription factor 1 (DACH1) in diabetic kidney disease (DKD) and investigate its possible molecular mechanism.Materials and Methods: From the GEO database, we downloaded different datasets for analyzing the expression of miR-218 and DACH1 in DKD. TargetScan was adopted to predict the binding sites between miR-218 and DACH1, which was further verified by dual-luciferase reporter assays. The renal proximal tubule cells (HK-2) treated with high glucose (HG) were used as an in vitro model. QRT-PCR and western blot were used to determine the expression of DACH1 and other relative factors. Cell counting kit-8 and flow cytometer were applied to detect cell viability and apoptosis. The levels of inflammatory cytokines were determined by an ELISA assay.Results: A prominent raise of miR-218 was observed in DKD through bioinformatics analysis, which was further confirmed in the HG-induced model. DACH1 is a target of miR-218. miR-218 reduced cell viability and induced apoptosis by negatively regulating DACH1. Moreover, upregulating miR-218 in HG models increased the concentrations of pro-inflammatory cytokines TNF-α and IL-1ß, reduced the level of anti-inflammatory cytokine IL-10, and promoted the epithelial-mesenchymal transition (EMT) process, which is possibly achieved by targeting DACH1. While downregulating miR-218 showed the opposite results.Conclusion: These data demonstrated that, under an in vitro HG environment, miR-218 suppressed the HK-2 cells proliferation, promoted apoptosis, caused an inflammatory response, and facilitated the EMT process largely by targeting DACH1, providing an insight into the therapeutic intervention of DKD.


Assuntos
Apoptose/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Proteínas do Olho/metabolismo , Glucose/farmacologia , MicroRNAs/genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Olho/genética , Humanos , Inflamação , Rim , Fatores de Transcrição/genética
13.
Invest Ophthalmol Vis Sci ; 61(5): 30, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32428231

RESUMO

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.


Assuntos
Dependovirus/genética , Proteínas do Olho/genética , Terapia Genética , Amaurose Congênita de Leber/terapia , Proteínas Associadas aos Microtúbulos/genética , Retina/fisiopatologia , Adulto , Animais , Criança , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Terapia Genética/métodos , Vetores Genéticos , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Fenótipo , Pupila/fisiologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Testes de Campo Visual , Campos Visuais/fisiologia , Adulto Jovem
14.
Invest Ophthalmol Vis Sci ; 61(4): 42, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32343782

RESUMO

Purpose: To study cone structure and function in patients with retinitis pigmentosa (RP) owing to mutations in rhodopsin (RHO), expressed in rod outer segments, and mutations in the RP-GTPase regulator (RPGR) gene, expressed in the connecting cilium of rods and cones. Methods: Four eyes of 4 patients with RHO mutations, 5 eyes of 5 patients with RPGR mutations, and 4 eyes of 4 normal subjects were studied. Cone structure was studied with confocal and split-detector adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral-domain optical coherence tomography. Retinal function was measured using a 543-nm AOSLO-mediated adaptive optics microperimetry (AOMP) stimulus. The ratio of sensitivity to cone density was compared between groups using the Wilcoxon rank-sum test. Results: AOMP sensitivity/cone density in patients with RPGR mutations was significantly lower than normal (P < 0.001) and lower than patients with RHO mutations (P < 0.015), whereas patients with RHO mutations were similar to normal (P > 0.9). Conclusions: Retinal sensitivity/cone density was lower in patients with RPGR mutations than normal and lower than patients with RHO mutations, perhaps because cones express RPGR and degenerate primarily, whereas cones in eyes with RHO mutations die secondary to rod degeneration. High-resolution microperimetry can reveal differences in cone degeneration in patients with different forms of RP.


Assuntos
Proteínas do Olho/genética , Células Fotorreceptoras Retinianas Cones/patologia , Retinite Pigmentosa/diagnóstico por imagem , Retinite Pigmentosa/genética , Rodopsina/genética , Tomografia de Coerência Óptica/métodos , Adulto , Fatores Etários , Estudos de Casos e Controles , Eletrorretinografia/métodos , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Mutação/genética , Oftalmoscopia/métodos , Medição de Risco , Polarimetria de Varredura a Laser/métodos , Fatores Sexuais , Estatísticas não Paramétricas , Adulto Jovem
15.
Proc Natl Acad Sci U S A ; 117(18): 9922-9931, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32312818

RESUMO

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare -/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.


Assuntos
Cílios/genética , Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Segmento Externo da Célula Bastonete/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Animais , Cílios/patologia , Distrofias de Cones e Bastonetes/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , RNA Interferente Pequeno/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Segmento Externo da Célula Bastonete/patologia
16.
Orv Hetil ; 161(18): 738-746, 2020 05 01.
Artigo em Húngaro | MEDLINE | ID: mdl-32338487

RESUMO

Introduction and aim: We were looking for altered gene expression on peripheral blood cells significant to type 2 diabetes causing the world epidemic. Method: Muscle biopsy samples of healthy volunteers with (n = 6) or without (n = 6) first degree type 2 diabetic relatives were analyzed by mRNS microarray. After confirmation of microarrays results by quantitative real-time PCR, the expression of eight differently expressed genes were further investigated on peripheral blood cells of 58 healthy volunteers without diabetic relatives and 58 healthy ones with first-degree type 2 diabetic relatives. Results: The expressions of SERPINF1 gene were significantly lover in blood cells both from females (relative quantification: FC - female: = 0.69, p<6*10-3) and males (FC - male: = 0.65, p<2*10-3) with diabetic relatives. This change may not be the consequence of worsening metabolic state as it was identical in cells of type 2 diabetic patients and in healthy volunteers with diabetic relatives. We suggest that the altered SERPINF1 gene expression in peripheral mononuclear blood cells could be a genetic definiteness. Conclusion: With the help of SERPINF1 gene expression in white blood cells and lipid and biochemical blood parameters we suggest a mathematical formula for the augury of type 2 diabetes that should be checked on a larger population, but we hope it could be used as a diabetic marker. The expression of LAMP2 gene did not differ between the two healthy groups, but it showed a maternal parent of origin effect. In the case of maternal inheritance, we found higher LAMP2 expression suggesting that gene from the mother has a determining effect. Orv Hetil. 2020; 161(18): 738-746.


Assuntos
Células Sanguíneas , Diabetes Mellitus Tipo 2/genética , Expressão Gênica , Biomarcadores , Proteínas do Olho/genética , Feminino , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Masculino , Fatores de Crescimento Neural/genética , Serpinas/genética
17.
J Neurosci ; 40(20): 4059-4072, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32265257

RESUMO

Photoreceptor neurons are surrounded by an extracellular matrix, called the interphotoreceptor matrix (IPM). Activities crucial to vision occur within the IPM, including trafficking of nutrients and metabolites, retinal attachment, and interactions needed for normal outer segment phagocytosis. The IPM includes the following two unique proteoglycans: IPM proteoglycan 1 (IMPG1) and IMPG2. Patients with mutations in IMPG1/IMPG2 develop visual deficits with subretinal material accumulation, highlighting the critical role of the IPM in vision. To determine the role of these proteoglycans in retinal physiology and the pathologic mechanisms that lead to vision loss, we generated mouse models lacking IMPG1/IMPG2. In normal retina, IMPG1 and IMPG2 occupy distinct IPM compartments, represent the main source of chondroitin sulfate and are fundamental for the constitution of the cone-specific glycocalyx stained by the PNA (peanut agglutinin) lectin marker. No evident morphologic or functional deficits were found in mice lacking IMPG1. In the absence of IMPG2, IMPG1 abnormally accumulated at the subretinal space need, likely leading to the formation of subretinal lesions and reduced visual function. Interestingly, mice lacking both IMPG1 and IMPG2, regardless of sex, showed normal retinal structure and function, demonstrating that the aberrant IMPG1 distribution is the main cause of the visual alterations observed in the absence of IMPG2. In conclusion, our results show the dependence of secreted proteoglycans such as IMPG1 on the extracellular environment to properly integrate into the matrix, demonstrate the role of IMPG2 in shaping the IPM, and shed light on the potential mechanisms leading to the development of subretinal lesions and vision loss.SIGNIFICANCE STATEMENT The photoreceptors are specialized neurons that drive phototransduction in the mammalian retina. These cells are organized and surrounded by an extracellular matrix, the interphotoreceptor matrix (IPM). Mutations in IPM proteoglycans are associated with blindness in humans. Our studies show that two specific proteoglycans of the IPM, IPM proteoglycan 1 (IMPG1) and IMPG2, form a dynamic structure with distinct localization and dependency. When IMPG2 is absent, IMPG1 cannot integrate into the IPM, leading to abnormal proteoglycan accumulation and visual deficits. This work adds a new layer of understanding to IPM physiology and describes the pathologic events following deficits in proteoglycans, providing novel possibilities for visual restoration in patients with IMPG-related pathologies.


Assuntos
Células Fotorreceptoras de Vertebrados/fisiologia , Proteoglicanas/genética , Visão Ocular/fisiologia , Animais , Sulfatos de Condroitina/metabolismo , Eletrorretinografia , Proteínas da Matriz Extracelular/genética , Espaço Extracelular , Proteínas do Olho/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Mutação , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Transtornos da Visão/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-32244061

RESUMO

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-ß and the thromboxane A2 receptor-ß (TPß receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPß receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCß, WDR36 augmented TPß receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPß receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPß towards PLC and SphK1, most likely by scavenging Gαq and PLCß. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.


Assuntos
Proteínas do Olho/metabolismo , Fosfolipase C beta/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Tromboxanos/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Proteínas do Olho/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Mioblastos/metabolismo
19.
Invest Ophthalmol Vis Sci ; 61(3): 10, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32176261

RESUMO

Purpose: To investigate the clinical and genetic characteristics of occult macular dystrophy (OMD) based on a Chinese patient cohort. Methods: Fifteen Chinese OMD patients from nine unrelated families underwent genetic testing, and all of them harbored a pathogenic RP1L1 variant. Comprehensive ophthalmic examinations were performed in nine probands, including spectral-domain optical coherence tomography (SD-OCT), near-infrared reflectance (NIR), fundus autofluorescence (AF), and multifocal electroretinography. Results: The RP1L1 variants p.R45W and p.S1199C were identified in 13 patients and two patients, respectively, and one was a de novo mutation. Among the nine probands, the median ages at onset and examination were 25.0 years (range, 6-51 years) and 27.0 years (range, 14-55 years), respectively. The median decimal visual acuity was 0.20 (range, 0.04-0.5). Foveal photoreceptor thickness and visual acuity showed a significant correlation (r = 0.591; P = 0.01). All eyes presented with an absent interdigitation zone and blurred ellipsoid zone of photoreceptors when examined by SD-OCT. In addition, central round lesions with low NIR reflectance were observed in 66.7% (12/18) of eyes by NIR reflectance imaging, corresponding to the regions with abnormal photoreceptor microstructures observed by SD-OCT. Of the 18 eyes, only four eyes showed ring-like faint hyperfluorescence around the macula by AF. Conclusions: To the best of our knowledge, this is the largest study in a cohort of Chinese OMD patients with RP1L1 mutations. Our findings revealed that the two recurrent RP1L1 variants are related to OMD in the Chinese population. Furthermore, multimodal imaging combined with genetic testing is valuable for diagnosing and monitoring OMD progression.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteínas do Olho/genética , Degeneração Macular/genética , Mutação , Adolescente , Adulto , Idade de Início , Criança , Estudos de Coortes , Eletrorretinografia , Feminino , Angiofluoresceinografia , Predisposição Genética para Doença , Humanos , Degeneração Macular/diagnóstico por imagem , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Imagem Multimodal/métodos , Linhagem , Tomografia de Coerência Óptica , Acuidade Visual/genética , Adulto Jovem
20.
Invest Ophthalmol Vis Sci ; 61(3): 11, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32176262

RESUMO

Purpose: To define remodeling of photoreceptor synaptic terminals and second-order retinal neurons in canine X-linked progressive retinal atrophy 1 caused by a five-nucleotide deletion in the RPGR exon ORF15. Methods: Retinas of normal and mutant dogs were used for gene expression, Western blot, and immunohistochemistry. Cell-specific markers were used to examine disease-dependent retinal remodeling. Results: In mutant retinas, a number of rod axon terminals retract into the outer nuclear layer. This neuritic atrophy preceded significant loss of rods and was evident early in disease. Rod bipolar and horizontal cell processes were found to extend into the outer nuclear layer, where they seemed to form contacts with the spherules of rod photoreceptors. No ectopic rewiring was observed. Because cytoskeletal reorganization was previously shown to underlie photoreceptor axon retraction, we examined normal and mutant retinas for expression of axon guidance receptors ROBO1 and ROBO2, which are known to regulate actin cytoskeleton dynamics. We found that the overall expression of both ROBO1 and ROBO2 is retained at the same level in premature and fully developed normal retinas. However, analysis of predisease and early disease retinas identified markedly decreased levels of ROBO1 in rod spherules compared with controls. In contrast, no differences in ROBO1 signals were noted in cone pedicles in normal and mutant retinas, where ROBO1 levels remained similarly low. Conclusions: Depletion of ROBO1 in rod synaptic terminals correlates with the remodeling of axonal and dendritic processes in the outer retina of dogs with X-linked progressive retinal atrophy 1 and may play a role in the retraction of rod axons.


Assuntos
Doenças do Cão/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptores Imunológicos/metabolismo , Degeneração Retiniana/metabolismo , Animais , Orientação de Axônios/fisiologia , Axônios/patologia , Doenças do Cão/genética , Doenças do Cão/patologia , Cães , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Doenças Genéticas Ligadas ao Cromossomo X/veterinária , Microscopia Confocal , Proteínas do Tecido Nervoso/deficiência , Plasticidade Neuronal/fisiologia , Terminações Pré-Sinápticas/patologia , Receptores Imunológicos/deficiência , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/veterinária , Neurônios Retinianos/metabolismo , Neurônios Retinianos/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia
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