Endocrine Abstracts

Introduction: FGD is a rare autosomal recessive disorder associated with isolated glucocorticoid deficiency. Loss-of-function mutations in MC2R (ACTH receptor) and MC2R accessory protein (MRAP) cause FGD. Missense MC2R variants represent the commonest type of MC2R variant in our registry (78.5%) whereas missense MRAP variants represent the least common type of MRAP variant (14.3%).

Methods: Three variants, MC2R p.L225R(c.674T>G), MRAP p.L53P(c.158T>C) and MRAP p.E28K(c.82G>A) were studied. DNA variant constructs were created using site directed mutagenesis and confirmed by sequencing. HEK293T cells were transiently transfected with the constructs and cAMP bioluminescence assay performed to determine ACTH response. GraphPad Prism v.10.4.1 was used to generate dose response curves and two-way ANOVA analysis.

Results: All variants demonstrated significantly reduced or absent activity compared to WT MC2R and MRAP. When exposed to ACTH concentrations (1x10^-7 and 1x10^-8 M), compared to WT, MRAP variants showed a much decreased cAMP response (p.L53P mounting a higher response than p.E28K) whereas the MC2R variant showed no response. When exposed to ACTH concentrations (<1x10^-9 M), there was minimal or no response from all variants.

Discussion: For MC2R p.L225R, residue 225 is located on the 6^th transmembrane domain of MC2R, important for ACTH binding affinity and signalling, explaining why in the respective patient, symptom onset was in the neonatal period. For MRAP variants, residue 28 is located in the N terminus and residue 53 in the transmembrane domain. The N terminus is critical for the trafficking of MRAP/MC2R to the cell surface and interaction with MC2R, which could explain why there is a lower response to ACTH of MRAP p.E28K compared to the MRAP p.L53P variant.

Conclusion: We characterised one MC2R and two MRAP missense variants causing FGD. Such in-vitro analysis can improve understanding of genotype-phenotype correlations in FGD.