Polycystic ovary syndrome (PCOS) is characterized by excessive theca cell androgen secretion, dependent upon LH, which acts through the intermediacy of adenosine 3', 5'-cyclic monophosphate (cAMP). cAMP signaling pathways are controlled through regulation of its synthesis by adenylyl cyclases, and degradation by phosphodiesterases (PDEs). PDE 8A is a high-affinity cAMP-specific PDE, which is highly expressed in steroidogenic cells. We evaluated the human PDE8A gene as a PCOS candidate gene based on the hypothesis that reduced PDE8A activity or expression would contribute to excessive ovarian androgen production. 5 PCOS and 5 normal theca cell preparations from different subjects were used to amplify the PDE8A coding sequence. 10 PCOS and 10 normal women genomic DNA were used to amplify promoter sequences. Here we report the discovery of a rare variant 596A>G (R136Q) and a novel SNP 1391A>G (N401S) in the PDE8A coding sequence causing non-synonymous amino acid substitutions, and a new -576bp A>G SNP in the promoter region. We tested the variants for functional significance and association with PCOS. We assayed PDE8A activity in theca cells with the two coding sequence variants and compared activity from normal theca cell preparatons without variant. We found that the PDE8A activity was 5.4-fold and 2-fold reduced in the 136Q and 401S variant-containing cells compared to the normal theca cells without this variant. Although PDE8A kinetics were consistent with reduced activity (lower Vmax) in theca cell lysates, study of the expressed variants did not confirm reduced activity in cell-free assays. We also studied protein localization using green fluorescent protein -tagged proteins and found that the tagged proteins were localized to the cell periphery at the plasma membrane. There appeared to be no differences among the coding sequence variants in localization. The PDE8A promoter SNP and a previously described promoter -1235 A>G SNP (rs12900078) in linkage disequilibrium with the new SNP did not affect promoter activity in vitro promoter assays. SNPs were genotyped in 455 families with PCOS. Association and linkage between SNPs and PCOS was tested with the transmission disequilibrium test (TDT). The more common coding sequence SNP (N401S), and the promoter SNPs were not associated with PCOS in our TDT-based analysis. We conclude that although polymorphism exists in the PDE8A promoter and coding sequence, the sequence variants are not a major contributor to the PCOS phenotype. However, our study documented that PDE8A is expressed in human theca cells and that it is localized at a strategic position in the cell, the plasma membrane, which positions PDE8A to control cAMP levels generated in response to tropic stimulation of cell surface receptors coupled to adenylate cyclase. This research was support by National Institutes of Health grant 2U54 HD034449.(poster)