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Santiago Delgado-Tirado, Salvador Pastor, Irene Rodriguez-Hernandez, Jimena Rojas, Lucia Gonzalez-Buendia, Rogelio Gonzalez-Sarmiento, Jose-Carlos Pastor; Functionality Characterization of rs2229094 (T>C) polymorphism and LTα Expression in Human Retinas. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6402.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the expression of LTα in healthy human retinas, the localization in human samples with previous retinal detachment (RD) and investigate the functionality of the LTα rs2229094 (T>C) polymorphism, previously described (Rojas et al Ophthalmology. 2010) and associated to PVR development in a two phases study.
Phase I: Total RNA from 3 healthy human retinas and 2 peripheral blood samples were extracted and subjected to reverse transcription polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTα cDNA. In addition, 3 human eyes with RD and one healthy control eye were subjected to immunohistochemistry (IHC) with specific antibodies against LTα. Phase II: Functionality of the T and C alleles was assessed by using pCEFL-Flag expression vector (co-expressing the Flag epitope and either the full-length Arg13-LTα or Cys13-LTα human cDNA) and transient transfection assays in the COS-1 kidney fibroblast cell line. In addition, expression analysis by RT-PCR, western-blotting and subcellular localization of both T and C alleles, by immunofluorescence assays were performed.
Phase I: RT-PCR analysis revealed signals of mRNA LTα below the significance levels in human healthy retinas. The analysis of peripheral blood showed different LTα transcripts with premature stop codons and full length LTα transcript. Sequential IHC staining revealed no differences between human healthy and RD retinas. Phase II: No differences in mRNA and protein expression levels and in the subcellular localization between both alleles were found. Both alleles were located in the cytoplasm.
Although results suggest lack of functionality, and it appears to be no differences regarding ICH analysis, this polymorphism could remain as a valid biomarker to identify patients at high risk to develop PVR after RD surgery, due to the strong association between PVR and LTα rs2229094 polymorphism previously identified by our group.
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