Analysis of Polymorphisms in the Cart Gene (Cocaine and Amphetamine Receptor Transcript) as Risk Factors for Neovascular Age–Related Macular Degeneration

Abstract

Abstract: : Purpose: To examine the candidate gene CART, chosen for its expression pattern and its association with obesity, as a possible risk factor for neovascular age–related macular degeneration (AMD). Methods: We ascertained 66 extremely discordant sibpairs in which one member had the neovascular form of AMD (mean age = 72.59, SD = 7.66) and another member had normal maculae and was past the age of diagnosis for the affected sibling (mean age = 77.01, SD = 7.83). If more than one unaffected sibling were available, we selected the oldest unaffected sibling for this study. Disease status was confirmed by fundus photography in all subjects, except in 4 cases where a home visit was conducted. Leukocyte DNA samples were purified, and five fragments of the CART gene were amplified by PCR (2 regions of the promoter, and all 3 exons). Genotypes were determined through agarose gel electrophoresis of DNA fragments resulting from restriction enzyme digestion and/or through direct sequencing. All statistical analyses were performed with McNemar’s test. Results: Restriction digestion of two known polymorphisms in the promoter region: G>C at –929, which destroys a StyI site; and T>C at –390, which destroys an HphI site (Yamada et al. Int Journal Obesity, 26:132, 2002) showed no statistically significant association with neovascular AMD (–929: n=12 informative pairs, p=0.39; 390: n=8, p=0.72). The two non–synonymous polymorphisms in exons 1 and 3 reported in the NCBI SNP database were also analyzed using restriction digest assays: C82A creates a StyI site in exon 1, and C337A creates an NlaIII site in exon 3. There was no statistically significant association for C82A: n=8, p=0.29; no informative pairs were found in our cohort with the C337A change. CART exon 2 was sequenced in the forward direction only to search for two variants evaluated by others as obesity risk factors (del Giudice et al. Diabetes, 51:2157, 2001; Challis et al. ibid, 49:872, 2000). Neither change was found in our sibpairs, however a previously unreported change (A212C) was found. This variant is non–synonymous (Gln78Pro), but was only found in 3 informative pairs. Conclusions:Our preliminary analysis revealed no statistically significant association between any of the CART polymorphisms studied and neovascular AMD.

Keywords: age-related macular degeneration • genetics • neovascularization