Functional characterization of rs2229094 (T>C) polymorphism in the tumor necrosis factor locus and lymphotoxin alpha expression in human retina: the Retina 4 project
Authors Pastor-Idoate S, Rodríguez-Hernández I, Rojas J, Gonzalez-Buendia L, Delgado-Tirado S, Lopez JC, González-Sarmiento R, Pastor JC
Received 20 February 2017
Accepted for publication 19 April 2017
Published 22 May 2017 Volume 2017:11 Pages 973—981
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Amy Norman
Peer reviewer comments 2
Editor who approved publication: Dr Scott Fraser
Salvador Pastor-Idoate,1,2 Irene Rodríguez-Hernández,2,3 Jimena Rojas,1 Lucia Gonzalez-Buendia,1 Santiago Delgado-Tirado,1,4 Jose Carlos López,1 Rogelio González-Sarmiento,2,3 Jose C Pastor1,4
1IOBA Eye Institute, University of Valladolid, Valladolid, 2Molecular Medicine Unit, Department of Medicine, 3Molecular and Cellular Cancer Biology Institute, High Council of Scientific Research, Biomedical Research Institute of Salamanca, University of Salamanca, Salamanca, 4Department of Ophthalmology, Hospital Clínico Universitario, Valladolid, Spain
Purpose: The objective of this study is to determine the expression and localization of lymphotoxin alpha (LTA) in human retinas and the functionality of one of its polymorphisms rs2229094 (C13R) (T>C), previously associated with proliferative vitreoretinopathy (PVR) development.
Materials and methods: Total RNA from three healthy human retinas were extracted and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis, using flanking primers of LTA cDNA. In addition, three human eyes with retinal detachment (RD) and three healthy control eyes were subjected to immunohistochemistry (IHC) with a specific antibody against LTA. The functionality of T and C alleles was assessed by using pCEFL-Flag expression vector and transient transfection assays in COS-1 cell line. In addition, expression analysis by RT-PCR, Western blot and subcellular localization of both alleles and by immunofluorescence assay was performed.
Results: RT-PCR analysis revealed no significant levels of messenger RNA (mRNA) LTA in healthy human retinas. Sequential IHC staining showed differences between healthy human and RD retinas. No differences in mRNA and protein expression levels and in subcellular localization between both alleles were found. Both alleles were located in the cytoplasm of COS-1 cells.
Conclusion: Although results suggest lack of functionality, the differences found in IHC study and its strong association with PVR and its relationship with tumor necrosis factor locus, warrant further studies and could justify the use of this polymorphism as a valid biomarker to identify high-risk patients to develop PVR after RD.
Keywords: proliferative vitreoretinopathy, lymphotoxin alpha, tumor necrosis factor alpha, inflammation, cytokines, polymorphism
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