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Funcionality characterization of RS2229094 (T>C) polymorphism and LT[Alpha] expression in human retinas
Author(s) -
DELGADO TIRADO S,
PASTOR IDOATE S,
RODRIGUEZ HERNANDEZ I,
ROJAS J,
GONZALEZ BUENDIA L,
GONZALEZ SARMIENTO R,
PASTOR JC
Publication year - 2014
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2014.t066.x
Subject(s) - western blot , microbiology and biotechnology , biology , subcellular localization , immunohistochemistry , allele , messenger rna , reverse transcription polymerase chain reaction , immunofluorescence , antibody , gene , immunology , genetics
Purpose To determine the expression and localization of lymphotoxin alpha (LTα) in human retinas, and investigate the functionality of the LTα rs2229094 (T>C) polymorphism, previously associated to proliferative vitreoretinopathy (PVR) development. Methods Phase I: RNA from 3 healthy human retinas and 2 peripheral blood samples was subjected to reverse transcription polymerase chain reaction (RT‐PCR) analysis. In addition, 3 human eyes with chronic retinal detachment (RD) and one healthy control were subjected to immunohistochemistry (IHC) with specific antibody against LTα. Phase II: Functionality of T and C alleles was assessed by using pCEFL‐Flag expression vector, and transient transfection assays. Besides, expression analysis by RT‐PCR, Western‐blot and subcellular localization of both alleles, by immunofluorescence (IF) assay were performed. Results Phase I: Signals of mRNA LTα below significance levels were detected in healthy human retinas. Peripheral blood analysis showed different LTα transcripts with premature stop codons. IHC staining revealed no differences between human healthy and RD retinas. Phase II: No differences in mRNA and protein expression levels and subcellular localization between both alleles were found. Both alelles showed cytoplasmic LTα localization. Conclusion Although results suggest lack of functionality, and it appears to be no differences regarding LTα expression and localization, this polymorphism could remain as a valid biomarker to identify patients at high‐risk for developing PVR after RD, due to the strong association between PVR and LTα rs2229094 polymorphism previously identified by our group.