Molecular scanning of the beta-3-adrenergic receptor gene in Pima Indians and Caucasians

Background. The beta-3-adrenergic receptor (β3AR) stimulates lipolysis and thermogenesis in adipocytes. The Trp64Arg β3AR variant is associated in some, but not all, studies with an earlier onset of Type 2 diabetes mellitus and features of the insulin resistance syndrome. Functional studies as to the role of the Trp64Arg variant have been inconclusive. Earlier studies screened the β3AR gene in only ten obese, diabetic Pima Indians. Potentially another yet to be identified polymorphism in the β3AR gene in linkage disequilibrium with the Trp64Arg polymorphism could explain the findings in the association and functional studies. Methods. We scanned the β3AR gene in 20 diabetic Pima subjects and 20 Caucasian subjects using single stranded conformational polymorphism (SSCP) analysis. Variants were sequenced using dideoxy sequence analysis and further characterized using allele specific oligonucleotide hybridization (ASO) and RNA template specific-polymerase chain reaction (RS-PCR) assays. Results. We found a guanine to thymidine substitution in the first intron, 14 bases from the splice donor site in both groups. In virtually all subjects, only two haplotypes were detected, Trp64/g1856 and Arg64/t1856, indicating that the g1856t polymorphism is in linkage disequilibrium with the Trp64Arg polymorphism. The g1856t substitution introduces a new consensus splice donor site which, if used, would encode a truncated protein. RNA levels of the two β3AR alleles were approximately equal in omental adipose tissue of heterozygotes. No aberrantly spliced β3AR mRNA was detected, indicating that the new consensus splice donor site is not used in vivo. Conclusion. The g1856t polymorphism is in linkage disequilibrium with the Trp64Arg variant, but does not appear to have a functional role.