Abstract
Abstract: :
Purpose: Studies of candidate gene association and linkage disequilibrium suggest that age–related macular degeneration (AMD) has a genetic component. The associations of single nucleotide polymorphisms (SNPs) in oxidative stress, apolipoprotein E, and angiotensin converting enzyme genes with AMD have previously been reported. Recently, the role of the immune system in the pathology of AMD has been brought into question. CX3CR1 is the receptor of fractalkine (CX3CL1), a CX3C chemokine. There exist two non–synonymous SNPs in the CX3CR1 open reading frame, V249I and T280M. The allele frequency of T280M ranges 10.3–20.2% in North American Caucasians. SNPs, notably the T280M SNP, in CX3CR1, have been correlated with other aged–related diseases such as atherosclerosis. This study investigates these SNPs and the expression of CX3CR1 in eyes with a histopathological diagnosis of AMD. Methods: Archived, paraffin–embedded, ocular slides from 40 patients with pathological diagnosis of AMD were collected. Non–retinal, peripheral retinal and macular retinal cells on the hematoxylin–eosin stained slides were carefully microdissected manually under a light microscope or using the PixCell IIe laser capture microscope. The non–retinal cells were used for DNA extraction and SNP analysis. SNP assay was performed through PCR amplification followed by restriction fragment length polymorphism analysis. The retinal cells of selected cases were used for RNA extraction and RT–PCR. Immunohistochemistry was also performed on the unstained slides of the selected cases with rabbit anti–human CX3CR1 polyclonal antibody following the avidin–biotin–complex technique. Results: DNA was successfully extracted from the microdissected ocular cells in 32 of the 40 AMD cases. Fourteen of the 32 AMD cases (43.8%) were found to be carriers of the T280M polymorphism in genotype, with an allele frequency of 29.7%. The transcripts of CX3CR1 were much lower in the macular lesion as compared with the normal peripheral retina in the AMD eyes. The CX3CR1 protein was also less expressed in the degenerative photoreceptors and RPE cells. Conclusions: Polymorphism in the T280M allele could result in a decreased number of CX3CR1 receptor binding sites for fractalkine as well as reduce fractalkine–binding affinity on peripheral blood monocytes. Our data suggest that insufficient interaction between CX3CR1 and fractalkine, as well as lower expression of CX3CR1 in the macular lesion, may potentially promote the development of AMD.
Keywords: age–related macular degeneration • cytokines/chemokines • genetics