Endocrine Abstracts

Introduction: Germline inactivating ARMC5 (Armadillo repeat containing 5) mutations are responsible for Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH). ARMC5 presents the characteristics of a tumor suppressor gene. Mutations are observed in more than 85% of patients with a clear familial presentation and 20 to 25% of apparently sporadic cases. Genetic alterations are spread all over ARMC5 coding sequence and more than two-third of them are missense variants. In this case the pathogenicity prediction is mostly based on bioinformatics in silico analysis. We have previously developed an apoptosis assay using immunochemistry caspase staining on cells lines overexpressing transiently ARMC5 wild type (wt) or its missense variants to demonstrate the deleterious effects of newly identified missense variants. However this approach is time consuming and therefore difficult to transfer for routine use in the oncogenetic laboratory of our hospital. We have previously observed that the stability of the ARMC5 mutant is altered.

Aim: To develop a simple functional test to confirm bioinformatic predictions of pathogenicity of newly identified ARMC5 variants.

Method: HeLa cells are transiently transfected with vectors expressing Flag-tagged ARMC5 wt or ARMC5 missenses. Cell lysates are prepared after 14 and 24 hours of transfection and proteins are resolved by SDS-PAGE. Flag-ARMC5 protein levels are analyzed by Western blot experiment using an antibody against the Flag tag (Sigma^®).

Results: Similar protein levels were observed for ARMC5 wt and all its variants tested at 14 hours of overexpression, demonstrating similar expression of all the vectors. By contrast, at 24h of overexpression we observed a marked protein level decrease for ARMC5 wt and for two benign polymorphisms (p.F14Y and p.507L) compared to all other mutants tested (p.R898W, p.L548F, C657R, I664S p,.F700del, p.Y736P, p.L754P, p.L778P, p.C139R, p. R315W, p.L331P, p.R362L, p.R454W).

Conclusion and perspectives: Using a simple ARMC5 protein expression assay, we managed to distinguish pathogenic missense mutants from benign polymorphisms. ARMC5 non-pathogenic polymorphisms have the same protein expression profile than ARMC5 wt. This study shows the potency of this simple assay to confirm in routine practice the pathogenicity of new ARMC5 variants identified in PBMAH patients. The mechanisms of this observation – stability or apoptosis- is an interesting research question that might help to progress in the understanding of PBMAH pathophysiology.