Endocrine Abstracts

Melanocortin-2-receptor (MC2R) accessory protein (MRAP) is a small single transmembrane protein, highly expressed in adrenal glands and essential for MC2R expression and function. ACTH binds to the MRAP-MC2R complex and activates the adenylyl cyclase cascade, leading to cAMP production, phosphorylation of cAMP-dependent PKA, activation of the steroidogenesis pathway and production of adrenal glucocorticoids (GC). Loss-of-function mutations in MRAP give rise to isolated GC deficiency aka FGD2. FGD2 presents early in childhood with symptoms and signs of hypocortisolaemia and excessive ACTH: hyperpigmentation, failure-to-thrive, hypoglycaemic attacks or coma and death. Mutations of MRAP account for 20% of FGD. In 2005, Metherell et al reported patients carried mutations either in exon 3 (c.3G>A, c.106+1G>A, c.106+1G>T, c.106+1G>C, c.106+1delG, c.106+3insT) or exon 4 V44X (c.128delG) of MRAP, leading to non-existent or non-functional protein. Since then, more than ten other MRAP mutations have been reported. However, like many conditions variants of uncertain significance (VUS) remains the bottleneck to diagnosis and reporting back to patients. To be able to study FGD2 and test the functionality of novel variants, we used CRISPR-Cas9 to remove MRAP expression at the genomic level in adrenocortical carcinoma cell-line NCI-H295R. Targeted cells were single cell sorted and single clones of MRAP-KO H295R cells were assessed. Genomic DNA of the KO clones were purified and the locus of MRAP were amplified by PCR. KO of MRAP at the genomic level was confirmed by Sanger sequencing of three successfully created MRAP-KO clones. MRAP-KO clones showed decreased cAMP level when stimulated by ACTH by a pGlo assay (CLARIOstar®), which confirmed the lack of MRAP. The established MRAP-KO H295R cells will be a new tool for us to test novel variants and seek drugs and therapeutics to treat FGD2.