ESPE Abstracts (2016) 86 RFC1.5

aUniversity of Birmingham, Birmingham, West Midlands, UK; bStellenbosch University, Stellenbosch, South Africa; cUniversity of Sheffield, Sheffield, South Yorkshire, UK

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is caused by mutations in the CYP21A2 gene. Steroid 21-hydroxylase deficiency results in impaired synthesis of mineralcorticoids and glucocorticoids (GC), plus androgen excess. Hormonal imbalances in 21OHD are postulated to result in systemic transcriptomic and metabolomic alterations. Such perturbations are likely to be underlying co-morbidities, which are increasingly observed in individuals with CAH. Due to the lack of suitable animal models, Cyp21a2 ko mice are not viable, systemic consequences of 21OHD are poorly understood. Therefore, we generated several zebrafish cyp21a2 null-alleles by genome editing using a TALEN (transcription activator-like effector nuclease) approach. These lines serve as an in vivo model to study whole-organism consequences due to 21OHD. Zebrafish Cyp21a2 is highly conserved and converts 17-hydroxyprogesterone into 11-deoxycolesterol in vitro. Furthermore, we found that cyp21a2 expression is largely restricted to the interrenal gland (adrenal counterpart), the brain and ovary. Two different null-allele strains were used to characterise the cyp21a2 mutant phenotype. Homozygous cyp21a2 mutants display a phenotype with altered visual background adaption (similar to hyperpigmentation) that has been described for zebrafish with defective GC synthesis or action. Deficient cortisol synthesis in cyp21a2 mutants was confirmed by steroid measurements using ultra-performance convergence chromatography tandem mass spectrometry. The expression of the GC regulated target genes fkbp5 and pck1 is reduced in mutants, validating systemic cortisol deficiency. The consequential up-regulation of the hypothalamus-pituitary interrenal axis in cyp21a2 mutants was shown by increased pomca expression. Whole mount in situ hybridisation demonstrated interrenal hyperplasia in cyp21a2 mutants. In conclusion, we have shown that cyp21a2 mutant zebrafish represent a valid model to study the consequences of 21OHD. The whole-organism analysis of this model will provide urgently warranted insights into dysregulated pathways important for the pathophysiology of 21-hydroxylase deficiency and associated co-morbidities in patients with CAH.

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