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John A. Earle, Usha Chakravarthy, Graeme R. Clark, Giuliana Silvestri, Gareth J. McKay; Assessment Of Polymorphic Variation In Toll-like Receptor 3 (TLR3) And Potential Implication For Age-related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2011;52(14):5226.
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Variation within the Toll-like receptor 3 (TLR3) gene has previously been reported to be associated with AMD (Yang et al, 2008). We have examined polymorphic variation across this region to evaluate potential anomalies in gene expression profiles.
Retinal DNA and RNA was extracted from 13 donor eyes (Bristol eye bank) using Trizol. Isolated genomic DNA was PCR amplified using in house primers and nucleotide sequenced. DNA sequence data was analyzed using DNAStar to enable contig assembly of sense and antisense sequences in overlapping fragments against a Genbank reference sequence (NG_007278). Assembled contigs were examined for polymorphic variation and those detected annotated with information amalgamating gene expression data and mammalian conservation derived from the UCSC Genome Browser.
Thirty-seven single nucleotide polymorphisms (SNPs) were identified in the 13 donor eyes. Of these, 35 were variants that had been previously reported in the dbSNP repository (NCBI) while 2 were novel. SNP annotation indicated that 32 were intronic, 2 were coding (1 synonymous and 1 non-synonymous) and 2 were located in the 5’ or 3’ untranslated regions. In addition, 1 SNP was located upstream of exon 1 and has the potential to influence gene transcription. The synonomous SNP was highly conserved among different species and the non-synonomous SNP less so (9 species identified through UCSC). Intronic SNPs showed less interspecific conservation (<30%).
Thirty-seven SNPs have been identified across the TLR3 gene, some of which may play an important role in the functionality of this gene. This compares well to the sequenced genomic data available through UCSC for subjects of European descent (5 genomes) within which 38 SNPs are identifiable for this region. Studies of gene expression to characterise functionality are ongoing in order to examine possible contribution to the pathogenesis of AMD.
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