ESPE Abstracts

Background: The steroidogenic enzyme aromatase is encoded by the CYP19A1 gene. Aromatase activity is required for estrogen biosynthesis from androgen precursors in the ovary and several extragonadal tissues. The role of aromatase and thus estrogens for human biology is best illustrated by disease states, both deficiency and excess which might be caused by genetic disorders.

Aim: A novel deletion-insertion mutation spanning from intron 10 to the 3′ UTR of the CYP19A1 gene was found in a 46,XX girl presenting with ambiguous genitalia at birth. The mother virilized during pregnancy and parents were first cousins. We investigated this novel mutation genetically and performed functional and structural studies to characterize the role of the C-terminus of the aromatase protein.

Methods and results: Direct sequencing of the CYP19A1 gene revealed a deletion of 2081 nt starting in intron 10 to the 3′ UTR corresponding to c.1263+354_*922del. Minigene experiments confirmed that this deletion prevented splicing leading to a shorter protein of 426 aa, namely p.P423_H503delinsRALP. Aromatase activity of WT and mutant CYP19A1 was assessed in transiently transfected HEK293 cells using radiolabeled androstenedione as substrate and the tritiated water release assay to measure conversion to estrone. Compared to WT, the mutant aromatase enzyme showed complete loss of activity. Structure analysis suggested that the C-terminal membrane anchor and heme binding cysteine residue were deleted in the mutated protein. The mutated protein was predicted not to bind heme and would therefore have no enzymatic activity.

Conclusion: The c.1263+354_*922del CYP19A1 mutation codes for a C-terminally truncated aromatase protein which causes a severe phenotype of aromatase deficiency in humans. In line with the phenotype, this aromatase mutation has no activity in vitro indicating that the C-terminus of the aromatase protein is crucial for its activity.