Effects of Novel Polymorphisms in the RAGE Gene on Transcriptional Regulation and Their Association With Diabetic Retinopathy
- Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds General Infirmary, Leeds, U.K.
Interactions between advanced glycation end products (AGEs) and the receptor for AGE (RAGE) are implicated in the vascular complications in diabetes. We have identified eight novel polymorphisms, of which the −1420 (GGT)n, −1393 G/T, −1390 G/T, and −1202 G/A were in the overlapping PBX2 3′ untranslated region (UTR), and the −429 T/C (66.5% TT, 33.5% TC/CC), −407 to –345 deletion (99% I, 1% I/D, 0% D), −374 T/A (66.4% TT, 33.6% TA/AA), and +20 T/A were in the RAGE promoter. To evaluate the effects on transcriptional activity, we measured chloramphenicol acetyl transferase (CAT) reporter gene expression, driven by variants of the –738 to +49 RAGE gene fragment containing the four polymorphisms identified close to the transcriptional start site. The –429 C, −374 A, and 63-bp deletion alleles resulted in a mean increase of CAT expression of twofold (P < 0.0001), threefold (P < 0.001), and fourfold (P < 0.05), respectively, with the –374 T and A alleles yielding highly differential binding of nuclear protein extract from both monocyte- and hepatocyte-derived cell lines. The prevalence of the functional polymorphisms were investigated in subjects with type 2 diabetes (106 with and 109 without retinopathy), with the –429 C allele showing an increase in the retinopathy group (P < 0.05). These data suggest that the polymorphisms involved in differences in RAGE gene regulation may influence the pathogenesis of diabetic vascular complications.
Address correspondence and reprint requests to Dr Barry I. Hudson, Academic Unit of Molecular Vascular Medicine, Research School of Medicine, G Floor, Martin Wing, Leeds General Infirmary, Leeds, LS1 3EX, UK. E-mail:.
Received for publication 30 May 2000 and accepted in revised form 2 March 2001.
AGE, advanced glycation end products; CAT, chloramphenicol acetyl transferase; DHPLC, denaturing high-performance liquid chromatography; EMSA, electrophoretic mobility shift assays; PCR, polymerase chain reaction; RAGE, receptor for AGE; RFLP, restriction fragment–length polymorphism; SSCP, single-strand conformation polymorphism; TEAA, tri-ethyl ammonium acetate; UTR, untranslated region.