Single-exon deletions of ZNRF3 exon 2 cause congenital adrenal hypoplasia

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/{beta}-catenin signaling. R-spondin 1 (RSPO1) enhances Wnt/{beta}-catenin signaling via binding and removal of ZNRF3 from the cell surface. To explore a novel genetic form of PAI, we analyzed nine childhood-onset patients with PAI of biochemically and genetically unknown etiology using array comparative genomic hybridization. We identified various-sized single-exon deletions encompassing of ZNRF3 exon 2 in three patients, who showed neonatal-onset adrenal hypoplasia with glucocorticoid and mineralocorticoid deficiencies. RT-PCR analysis showed that the three distinct single exon deletions were commonly transcribed into a 126-nucleotide deleted mRNA and translated into 42-amino acid deleted protein ({Delta}Ex2-ZNRF3). Three-dimensional structure modeling predicted that interaction between ZNRF3 and RSPO1 was disturbed in {Delta}Ex2-ZNRF3, suggesting loss of RSPO1-dependent activation of Wnt/{beta}-catenin signaling. Cell-based functional assays with the TCF-LEF reporter showed that RSPO1-dependent activation of Wnt/{beta}-catenin signaling was attenuated in cells expressing {Delta}Ex2-ZNRF3 as compared with those expressing wild-type ZNRF3. In summary, we provided genetic evidence linking deletions encompassing ZNRF3 exon 2 and congenital adrenal hypoplasia, which might be related to constitutive inactivation of Wnt/{beta}-catenin signaling by {Delta}Ex2-ZNRF3.