Elke E. W. Verploegen1,2, Hedi L. Claahsen-van der Grinten1, Antonius E. van Herwaarden2, Paul N. Span3, Fred C.G.J. Sweep2, Margo Dona4,5
(1) Department of Pediatrics, Division of Pediatric Endocrinology, Amalia Children’s Hospital, Radboud University Medical Center, Nijmegen, Netherlands.
(2) Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, Netherlands.
(3) Department of Radiation Oncology, Radboud University Medical Center, Nijmegen, Netherlands.
(4) Department of Animal sciences & health, Institute of Biology, Leiden, Netherlands.
(5) Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands
Background
Congenital adrenal hyperplasia (CAH) is a group of rare autosomal recessive disorders, most commonly caused by 21-hydroxylase (CYP21A2) deficiency. This enzymatic defect results in impaired cortisol synthesis and the accumulation of precursors steroids and adrenal androgens. Lifelong glucocorticoid therapy is required to suppress ACTH and androgen excess, but both under- and overtreatment cause significant long-term complications, and treatment responses vary widely between individuals. To better understand CAH pathophysiology and variability in glucocorticoid responses, reliable experimental model systems are needed. Zebrafish may serve as a promising model for studying CAH pathophysiology due to their conserved endocrine pathways, rapid development, and suitability for genetic manipulation.
Methods
We generated a cyp21a2-deficient zebrafish model using CRISPR/Cas9. Genotypes of homozygous, heterozygous, and wild-type zebrafish were confirmed by polymerase chain reaction (PCR) followed by Sanger sequencing. The steroid profile of the cyp21a2-deficient zebrafish larvae was determined using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS).
Results
We successfully generated founder fish with a frameshift mutation in exon 6 of cyp21a2 (c.del810-820). Phenotypic characterization of cyp21a2-deficient zebrafish larvae demonstrated cortisol deficiency, elevated androgen levels, and reduced fertility, reflecting the hallmark features of CAH.
Conclusion
This in vivo model enables the study of CAH pathophysiology and factors contributing to variability in glucocorticoid response.