ORF13-07 - A Novel Humanized CYP21A2 -I173N Mouse Model for Congenital Adrenal Hyperplasia (CAH) Mimics Phenotypic Features of Human CAH Patients

21-hydroxylase deficiency (21OHD) is the most common form of congenital adrenal hyperplasia (CAH), and is caused by mutations in the CYP21A2 gene. 21OHD causes a wide array of clinical symptoms that result from gluco- and mineralocorticoid deficiency and adrenal androgen excess. Despite advances in hormone replacement therapy and neonatal screening improving survival rates, long-term hormone treatment continues to remain difficult with risks to growth, metabolism, fertility, and cardiovascular health. New therapies aim to mimic physiological cortisol rhythms and to reduce androgen while minimizing complications arising from standard glucocorticoid therapy. However, current efforts to evaluate new treatment strategies are hindered by a lack of a suitable animal model for testing. Here we present a viable and humanized mouse model in which the murine gene Cyp21a1 is replaced by its human orthologue CYP21A2, carrying the common and clinically relevant human point mutation I173N causing classic or simple virilizing CAH.
Homozygous I173N embryos required dexamethasone treatment during pregnancy of heterozygous females and until weaning of pups which are viable without treatment afterwards. Twenty weeks old homozygous mice show marked adrenocortical hyperplasia with a thickened zona fasciculata, enhanced expression of the CYP21A2 gene while lacking Cyp21a1 expression, and an increase of StaR, Cyp11a1 and Cyp11b2 gene expression. LC-MS/MS measurements of plasma steroids at 20 weeks show decreased corticosterone and 11-deoxycorticosterone levels (p ≤ 0.0001) in both sexes in the presence of highly significantly elevated progesterone and ACTH levels (p ≤ 0.0001) in comparison to the wildtype mice. We also observed increased aldosterone levels in female mutants while the mean arterial blood pressure was reduced in mutant mice (p ≤ 0.001). While mutant male mice are fertile with normal appearing testes, females are infertile and remain in the diestrus phase. Ovarian sections from mutant females show lack of corpora lutea and impaired follicular development compatible with ovulatory failure. Bulk RNA sequencing of I173N adrenals revealed an upregulation of steroidogenic and ACTH response pathways and downregulation of certain circadian and metabolic pathways indicating possible adrenal overactivation and disrupted rhythmicity. Overall, CYP21A2-I173N mutant mice exhibit a stronger phenotype than the CYP21A2-R484Q mice published recently, and show various features of sexual dimorphism in adrenal and gonadal function in CAH.
In conclusion, this new mouse strain exhibits features which align well with human CAH providing a valuable in vivo model to study CAH and test novel therapeutic approaches. Thus, this model will facilitate the transition from basic research into clinical application.