Abstract
Primary Aldosteronism (PA) is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60–80% of aldosterone-producing adenomas (APA). We undertook WES on 39 consecutive APAs in search of further variants. 1 APA revealed a somatic mutation (Val380Asp) within the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). An adjacent mutation (Gly379Asp) was discovered on WES from a PA patient in Munich.
Both short and long isoforms (442 & 453 residues) of wild-type (WT) and both mutant CADM1 genes were cloned into lentivirus vectors and each transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion and other parameters. Previous studies in pancreatic islet cells suggested a role of CADM1 in regulating gap junction (GJ) communication. To assess this we microinjected single WT or mutant H295R cells with the GJ permeable dye calceinAM and counted the dye-positive cells after 1 hour. The effect of inhibiting or silencing GJs in H295R cells using peptide gap27 or a Dharmacon smartpool was assessed. H295R cells were also co-transfected with WT or mutant CADM1 and the GJ protein CX43, tagged with the mApple fluorophore. These were mixed with cells transfected with CX43-Venus, allowing confocal visualisation of GJ formation. Protein modelling was undertaken to determine whether Asp in the intramembranous domain changes angulation of CADM1.
All mutant isoforms had consistently different effects, shown as a range compared to WT. Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (p<0.01) and 10-20-fold increase in CYP11B2 expression (p<0.001) compared to WT. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. CX43 inhibition increased aldosterone secretion 2-fold (p<0.01), and CYP11B2 expression 3 to 8-fold (<0.001). Knock-down of GJ proteins increased aldosterone secretion 1.5-fold (p<0.01) and CYP11B2 expression 1.7-fold (p<0.001).
Protein modelling showed mutations to increase the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2nm to 24.7 and 27.9nm. Mixing of Venus and mApple-tagged CX43 transfected cells showed fewer intact GJ channels in cells co-transfected with mutant compared to WT CADM1 [mutant 42/291 (14.4%) VS WT 68/212 (32.1%) p<0.001].
The CADM1 mutations shows the importance of membrane proteins in aldosterone regulation to extend beyond ion channels and transporters. A key role may be to bring opposing CX43 hemichannels close enough to form GJ channels, permitting the oscillating Ca2+ currents which regulate aldosterone in intact adrenal slices.
OR34-02 Somatic Transmembrane Domain Mutations of a Cell Adhesion Molecule, CADM1, Cause Primary Aldosteronism by Preventing Gap Junction Communication Between Adrenocortical Cells
