Wnt/β-catenin signaling is essential for adrenocortical development. Zinc and ring finger 3 (ZNRF3), an E3 ubiquitin ligase that attenuates Wnt/β-catenin signaling, is negatively regulated by R-spondin via an extracellular domain that is partially encoded by exon 2 of ZNRF3. We recently identified ZNRF3 exon 2 deletions in three individuals with congenital adrenal hypoplasia. ZNRF3 exon 2 deletion impairs R-spondin binding, thereby attenuating β-catenin expression, eventually developing congenital adrenal hypoplasia. To elucidate the influence of ZNRF3/Znrf3 exon 2 deletion on adrenocortical development, we generated homozygous Znrf3 exon 2 deletion (Znrf3 ^Δ2/Δ2) mice. The adrenal glands of Znrf3 ^Δ2/Δ2 mice did not show gross morphological changes at birth but became enlarged with age. Moderate hyperplasia of the zona fasciculata (ZF), dispersed medulla arrangement, and a radially spreading zone comprised of cells with large nuclei between the ZF and medulla were observed at 6 weeks of age. Immunohistochemistry revealed low levels of 20α-hydroxysteroid dehydrogenase, a marker of the adrenal X-zone, in Znrf3 ^Δ2/Δ2 mice. Plasma ACTH and serum corticosterone levels in Znrf3 ^Δ2/Δ2 mice did not differ significantly from those in wild-type mice. Transcriptome analyses of the adrenal glands revealed substantial downregulation of X-zone markers but no significant changes in the expression of genes involved in the Wnt/β-catenin signaling pathway. These results show that a species-specific difference in the effects of ZNRF3/Znrf3 exon 2 deletions in humans and mice; Znrf3 ^Δ2/Δ2 mice do not develop congenital adrenal hypoplasia but instead exhibit moderate ZF hyperplasia, dispersed medulla arrangement, and X-zone dysplasia.

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for two weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient over-expression of mouse Star. We found that human STAR can also restore defective steroidogenesis of Star-edited Y1 cells, and were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia, and can be used to evaluate the functionality of human STAR variants.