Identification of a novel 12-nucleotide insertion polymorphism in the promoter region of ADRA2B: full linkage with the 9-nucleotide deletion in the coding region and influence on transcriptional activity.

The alpha(2B)-adrenoceptor (alpha(2B)-AR) mediates vasoconstriction and a common polymorphism (+901 Ins/Del), located in the coding region of the human alpha(2B)-AR gene (ADRA2B), has been demonstrated to affect receptor function in vitro. In this study, we have identified a novel polymorphism corresponding to the insertion of 12-nucleotides (GGGACGGCCCTG) at position -4825 relative to the start codon (-4825 del/ins) in the far upstream region of the ADRA2B promoter. The genotyping of 71 unrelated Finnish individuals showed that the -4825 ins polymorphism is common and in complete linkage with the Del polymorphism at position +901 and a G/C substitution at position -98. Transfection of various cell lines with luciferase constructs containing a 5.5 kb fragment of the ADRA2B promoter region indicated that the 12-nucleotide insertion at -4825 resulted in a large reduction of transcriptional activity. Electrophoretic mobility shift assays with oligonucleotide probes corresponding to the two ADRA2B alleles demonstrated that the region where the -4825 del/ins variation occurs binds nuclear proteins and that the 12-nucleotide insertion affects the pattern of bound transcription factors. Altogether, the present findings show that the previously identified +901 Del polymorphism is linked with a variation in the ADRA2B promoter that affects transcriptional activity in vitro. The molecular mechanisms underlying this effect are still unclear but a possible impact of the -4825 ins polymorphism on alpha(2B)-AR expression would merit to be examined in vivo as a diminution of promoter activity may limit the functional consequences of the +901 Del polymorphism.