ESPE Abstracts (2018) 89 FC7.4

Altered Substrate Specificities and Metabolite Production by Aromatase (CYP19A1) Due to the R192H Mutation

Sameer S Udhanea,b, Bernhard Dickc & Amit V Pandeya,d

aUniversity Children’s Hospital Bern, Bern, Switzerland; bMedical College of Wisconsin Cancer Center, Milwaukee, Wisconsin, USA; cDepartment of Nephrology and Hypertension, University of Bern, Bern, Switzerland; dDepartment of Biomedical Research, University of Bern, Bern, Switzerland

Background: Aromatase (CYP19A1) a member of cytochrome P450 protein family is a major steroid metabolizing enzyme which converts androgens to estrogens. Mutations in aromatase can lead to autosomal recessive aromatase deficiency. An R192H mutation in CYP19A1 described earlier caused severe phenotype of aromatase deficiency with regressive virilization of the 46,XX new-born, but without signs of androgen excess during pregnancy. Computational studies suggested that R192H disrupts substrate access channel in the CYP19A1 that may affect binding of the substrates and exit of catalytic products. In current study we explored the specificity of different substrates towards aromatase to find out if the R192H mutation in aromatase affects any particular substrate and metabolite more than others.

Methods: WT and R192H variants of CYP19A1 were cloned into a pcDNA3 vector for expression in COS1 cells. Three different conditions were used for cell experiments. In first case endogenous steroids produced natively by cells were used as substrates. In second case androstenedione was added to cells and in third case, testosterone (T) was added to transformed cells for use as a substrate. Steroids were measured by GC-MS analysis. Computational calculations of substrate binding were done using X-ray crystal structure of human placental aromatase available from the protein databank.

Results: We observed a range of differences between WT and R192H mutation of aromatase towards metabolism of different substrates. Without any external substrate, the level of testosterone was not affected. However, when androstenedione was added externally, an 18 fold increase in T and 7.4 fold increase in DHT was observed, while 17-β Estradiol levels were reduced to 32% of WT. With the R192H mutation the 11β-hydroxy-Androsterone was increased 2 fold, Etiocholanolone was increased 4 fold and androsterone increased by 2 fold. When T was added externally for use as substrate, the observed T levels after incubation were 16 fold higher for the R192H mutation compared to WT while DHT was elevated by 7 fold. In addition, when using T as substrate, for the R192H mutation Etiocholanolone levels increased 7 fold but Estriol dropped to 54% of the WT and 17-β Estradiol was 31% of WT.

Conclusion: Selective effects on specific substrates has been demonstrated by the R192H mutation in human aromatase. This provides an important mechanism of disease causing effect in human aromatase where metabolism of different substrates of aromatase could be affected to varying degrees.

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