Single-Exon Deletions of ZNRF3 Exon 2 Cause Congenital Adrenal Hypoplasia

• Published in: The journal of clinical endocrinology and metabolism 2024-02, Vol.109 (3), p.641-648
• Main Authors: Amano, Naoko, Narumi, Satoshi, Aizu, Katsuya, Miyazawa, Mari, Okamura, Kohji, Ohashi, Hirofumi, Katsumata, Noriyuki, Ishii, Tomohiro, Hasegawa, Tomonobu
• Format: Article
• Language: English
• ISSN: 0021-972X; 1945-7197
• DOI: 10.1210/clinem/dgad627

Abstract Context Primary adrenal insufficiency (PAI) is a life-threatening condition characterized by the inability of the adrenal cortex to produce sufficient steroid hormones. E3 ubiquitin protein ligase zinc and ring finger 3 (ZNRF3) is a negative regulator of Wnt/β-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/β-catenin signaling via binding and removal of ZNRF3 from the cell surface. Objective This work aimed to explore a novel genetic form of PAI. Methods We analyzed 9 patients with childhood-onset PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. To examine the functionality of the identified single-exon deletions of ZNRF3 exon 2, we performed three-dimensional (3D) structure modeling and in vitro functional studies. Results We identified various-sized single-exon deletions encompassing ZNRF3 exon 2 in 3 patients who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the 3 distinct single-exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein (ΔEx2-ZNRF3). Based on 3D structure modeling, we predicted that interaction between ZNRF3 and RSPO1 would be disturbed in ΔEx2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/β-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/β-catenin signaling was attenuated in cells expressing ΔEx2-ZNRF3 as compared with those expressing wild-type ZNRF3. Conclusion We provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/β-catenin signaling by ΔEx2-ZNRF3.