Abstract 233: Molecular and Electrophysiological Analyses of Gene Variants in Bilateral Adrenal Hyperaldosteronism

Abstract

Primary aldosteronism (PA) is the most common cause of secondary hypertension with a high prevalence amongst patients with resistant hypertension. Recently somatic mutations have been defined in aldosterone-producing adenomas (APAs)-associated PA. Herein, we analyzed a single hereditary case and in 30 sporadic cases of bilateral adrenal hyperaldoteronism (BHA) using whole exome sequencing and generated a candidate gene list using an allelic frequency ≤1%. Unique variants were identified in the gene ATP2B4 [coding for a Ca-ATPase Pump, PMCA4], in the affected individuals in the familial case and in two sporadic BHA samples. These variants were chosen for in vitro analyses due the potential role on PMCA4 in regulating intracellular calcium and due to the causal role of mutations in the family member ATP2B3 in APA-related PA. The ATP2B4 variants were functionally analyzed testing the hypothesis that it causes an inability to pump out intracellular Ca 2+ , causing elevated intracellular Ca 2+ , net cell depolarization and increased aldosterone synthase ( CYP11B2 ) expression. Results from in vitro molecular analyses: 1) Real time quantitative PCR (qPCR) confirmed ATP2B4 mRNA in the various tissue including the human adult adrenal gland, APAs, and the human adrenocortical cell line HAC15. 2) Immunohistochemistry indicated PMCA4 expression throughout the adrenal cortex. 3) Knock down of ATP2B4 in HAC15 exhibited reduced Angiotensin II-stimulation in one of four shRNA clones. 4) Stable HAC15 cell lines for doxycycline (dox) - inducible wildtype and variant forms of ATP2B4 , were generated. Q-PCR and immunostaining confirmed dox-induced upregulation of ATP2B4 mRNA and protein. However, upregulation of the PMCA4 variants did not have a differential effect on basal or agonist-stimulated CYP11B2 expression. Whole cell recordings in HAC15 and HEK cells indicated robust PMCA4 conductance in native cells, but reduced conductance in the WT and variant PMCA4. In conclusion, while this study did not define a pathogenic role for ATP2B4 variants in BHA, we describe an approach of WES analysis for familial and sporadic BHA and outline a template for the thorough in vitro characterization of gene variants in the pathogenesis of BHA.