Human aromatase: Gene resequencing and functional genomics

Aromatase [cytochrome P450 19 (CYP19)] is a critical enzyme for estrogen biosynthesis, and aromatase inhibitors are of increasing importance in the treatment of breast cancer. We set out to identify and characterize genetic polymorphisms in the aromatase gene, CYP19, as a step toward pharmacogenomic studies of aromatase inhibitors. Specifically, we "resequenced" all coding exons, all upstream untranslated exons plus their presumed core promoter regions, all exonintron splice junctions, and a portion of the 3′-untranslated region of CYP19 using 240 DNA samples from four ethnic groups. Eighty-eight polymorphisms were identified, resulting in 44 haplotypes. Functional genomic studies were done with the four nonsynonymous coding single nucleotide polymorphisms (cSNP) that we observed, two of which were novel. Those cSNPs altered the following amino acids: Trp³⁹Arg, Thr ²⁰¹Met, Arg²⁶⁴Cys, and Met³⁶⁴Thr. The Cys ²⁶⁴, Thr³⁶⁴, and double variant Arg³⁹Cys ²⁶⁴ allozymes showed significant decreases in levels of activity and immunoreactive protein when compared with the wild-type (WT) enzyme after transient expression in COS-1 cells. A slight decrease in protein level was also observed for the Arg³⁹ allozyme, whereas Met²⁰¹ displayed no significant changes in either activity or protein level when compared with the WT enzyme. There was also a 4-fold increase in apparent K_m value for Thr³⁶⁴ with androstenedione as substrate. Of the recombinant allozymes, only the double mutant (Arg³⁹CyS²⁶⁴) displayed a significant change from the WT enzyme in inhibitor constant for the aromatase inhibitors exemestane and letrozole. These observations indicate that genetic variation in CYP19 might contribute to variation in the pathophysiology of estrogen-dependent disease.