RESUMO
PURPOSE: To identify the genes expressed in normal human trabecular meshwork tissue, a tissue critical to the pathogenesis of glaucoma. METHODS: Total RNA was extracted from human trabecular meshwork (HTM) harvested from 3 different donors. Extracted RNA was used to synthesize individual SAGE (serial analysis of gene expression) libraries using the I-SAGE Long kit from Invitrogen. Libraries were analyzed using SAGE 2000 software to extract the 17 base pair sequence tags. The extracted sequence tags were mapped to the genome using SAGE Genie map. RESULTS: A total of 298,834 SAGE tags were identified from all HTM libraries (96,842, 88,126, and 113,866 tags, respectively). Collectively, there were 107,325 unique tags. There were 10,329 unique tags with a minimum of 2 counts from a single library. These tags were mapped to known unique Unigene clusters. Approximately 29% of the tags (orphan tags) did not map to a known Unigene cluster. Thirteen percent of the tags mapped to at least 2 Unigene clusters. Sequence tags from many glaucoma-related genes, including myocilin, optineurin, and WD repeat domain 36, were identified. CONCLUSIONS: This is the first time SAGE analysis has been used to characterize the gene expression profile in normal HTM. SAGE analysis provides an unbiased sampling of gene expression of the target tissue. These data will provide new and valuable information to improve understanding of the biology of human aqueous outflow.
Assuntos
Mapeamento Cromossômico/métodos , Etiquetas de Sequências Expressas/química , Expressão Gênica , Malha Trabecular/metabolismo , Adulto , Idoso , Proteínas de Ciclo Celular , Análise por Conglomerados , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Genoma , Glaucoma/genética , Glaucoma/metabolismo , Glaucoma/patologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Masculino , Proteínas de Membrana Transportadoras , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Malha Trabecular/citologia , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismoRESUMO
PURPOSE: To investigate whether recently described polymorphisms in the optic atrophy 1 gene (OPA1) are associated with primary open-angle glaucoma (POAG) with elevated intraocular pressure in the Caucasian, African-American, and Ghanaian (West African) populations. METHODS: POAG was defined as the presence of glaucomatous optic nerve damage, associated visual field loss, and elevated intraocular pressure (>21 mm of mercury in both eyes). We used TaqMan allelic discrimination assays to genotype two single nucleotide polymorphisms (SNPs, rs10451941 and rs166850) in OPA1 in the Caucasian (279 cases, 227 controls), African American (193 cases, 97 controls), and Ghanaian (170 cases, 138 controls) populations. Allele, genotype, and haplotype frequencies were compared between the cases and controls from each population. RESULTS: There was no significant difference in OPA1 allele or genotype frequencies between POAG patients and controls at the rs10451941 and rs166850 SNPs in any population (p>0.05). Haplotype analysis also failed to demonstrate a significant association with POAG. The age-of-onset distribution in the Caucasian POAG patients was independent from genotypes at rs10451941. CONCLUSIONS: There was no association between two previously implicated OPA1 polymorphisms and a POAG phenotype that includes elevated intraocular pressure. This represents the first association analysis of OPA1 in high tension glaucoma in the African American and Ghanaian populations and is the largest study to date on the investigation of the potential association between OPA1 and POAG with elevated intraocular pressure. OPA1 association with POAG may be limited to patients with normal tension glaucoma in these populations.