Evaluation of Amino Acid Polymorphisms within the PEDF Protein in the Normal Population and in Patients with AMD

Abstract

Abstract: : Purpose: Pigment epithelium-derived factor (PEDF) is a neurotrophic factor with proposed antiangiogenic properties. Intravitreal levels of PEDF are lower in patients with AMD and Diabetic Retinopathy than in patients without proliferative disease. The purpose of this study is to determine the frequency of DNA polymorphic variations within the PEDF gene, that result in amino acid changes within the PEDF protein, between patients with and without AMD. Methods: SNP and EST database searches were performed to determine the number of nucleotide polymorphisms and cDNA clones with variant PEDF sequences. Primers were designed within intronic sequences to amplify exons containing nucleotide polymorphisms with resulting amino acid changes. PCR products from 183 AMD patients and 116 age-matched controls were either sequenced or characterized by RFLP analysis. Predictions of phosphorylation sites were ascertained using the PhosphoBase database. Evolutionary conservation of amino acid sequence was determined via alignment of the various putative human PEDF proteins with other nonhuman mammalian PEDF sequences. Results: Three SNPs within exons 3, 4 and 5 can result in amino acid changes, Met-72-Thr, Pro-132-Arg and Thr-196-Ile, respectively. Pro-132 and Thr-196 are conserved in bovine and murine PEDF proteins, whereas Glu-72 and Ala-72 are present in bovine and murine, respectively. A putative phosphorylation site at Ser-93 is lost in 2 PEDF isoforms. At amino acid position 72, of 183 individuals with AMD, 25 are homozygous for Thr (T/T-72), 64 are homozygous for Met (M/M-72) and 94 are heterozygous (M/T-72), whereas of the 116 controls 13 are T/T-72, 52 are M/M-72 and 51 are M/T-72. Of 115 patients diagnosed with exudative AMD, 16 are T/T-72, 36 are M/M-72 and 63 are M/T-72 and for 58 individuals diagnosed with dry AMD, 6 are T/T-72, 24 are M/M-72 and 28 are M/T-72. Chi-squared analysis implies that there is not a single genotype associated with AMD, nor is there a genotype preference between exudative AMD and dry AMD at amino acid position-72. Conclusions: The amino acid changes at all three positions are non-conservative, which may indicate a modulation of the function of the protein. Although at position 72, there is no statistically significant difference in the proportions of genotype between AMD patients and controls according to the Chi-square test of homogeneity (p-value=.233) nor is there statistically significant association between genotype and disease type according to the Chi-square test of independence (p-value=.402).

Keywords: age-related macular degeneration • candidate gene analysis • genetics