The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues

Summary Objective The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. Design To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (–Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT‐PCR) and by splicing assays performed in Y1 cells transfected with a P450aro –Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the –Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. Patients An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8·4 years, spontaneous breast development and a 194·6 pmol/l serum oestradiol level was observed. Results The –Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, –Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the –Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. Conclusions Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full‐length protein might explain the phenotype of partial aromatase deficiency.