Functional analysis of the susceptibility haplotype in the Receptor for Advanced Glycation End-products gene and its role in the hyperglycemia-driven pathology



Year of publication 2008
Type Conference abstract
MU Faculty or unit

Faculty of Medicine

Description Background and Aims: Receptor for Advanced Glycation End-products (RAGE) belongs to the class of membrane receptors recognizing AGEs produced in accelerated rate during hyperglycemia and contributing to the development of diabetic complications. Our previous studies have shown that common haplotype in the RAGE gene (denoted RAGE2) conferred susceptibility to the development and accelerated onset of diabetic nephropathy (frequency 21.7% in nephropathy vs. 12.8% in control diabetic, P=0.015, OR=1.65 [95% CI 1.08-2.5]). The aim of the study was to elucidate the functional role of the RAGE2 susceptibility haplotype in the modulation of RAGE gene transcriptional activity. Materials and Methods: A 788bp PCR fragment of the RAGE promoter region (-738 to +49 relative to the transcription start containing the -429T/C and -374T/A polymorphic variants) was amplified using genomic DNA of homozygous subjects to create all possible haplotypes present in population (i.e. T/T, T/A, C/T) and cloned into reporter vector pGL3-Basic. Additionally, a 303bp sequence of the RAGE intron 8 was amplified from homozygotes for the 2184A/G and cloned into the enhancer site of the same vector in the combination with the promoter variants. Constructs were propagated in the competent E. coli and subsequently used for transfection of BAEC cell line. After incubation for 48 hrs at 37C at 5% CO2 at varying concentration of D-glucose (5 and 30mmol/L) luciferase level was determined. Results: Luciferase activity (expressed in RLU) significantly differed between the three promotor haplotypes (p<0.05, ANOVA) with -429C/-374T RAGE2 haplotype giving the highest activity in both normo- and hyperglycemic conditions. Intron variant did not significantly influenced observed transcriptional activity of promoter variants in this assay system (p>0.05, ANOVA). Conclusions: Using reporter gene assays we were able to elucidate functional impact of the RAGE promoter variants constituting previously identified risk haplotype for diabetic nephropathy. Speculated additional effect of the intron 8 variant as an enhancer factor was not proved. Functional effect of the RAGE variants further supports its significance in pathogenesis of diabetic complications.
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