OR05-07 Somatic Mutations in MCOLN3 Drives Dysregulated Aldosterone Production in Aldosterone-Producing Adenomas

Abstract Disclosure: D. van Rooyen: None. S. Bandulik: None. G. Coon: None. M. Laukemper: None. C. Kumar-Sinha: None. A. Udager: None. A.M. Lerario: None. C. Lee: None. H. Wachtel: None. D. Cohen: None. J.M. Luther: None. T.J. Giordano: None. F. Butz: None. B. Popp: part-time employed by Labor Berlin - Charité Vivantes GmbH. A. Turcu: None. R. Warth: None. W.E. Rainey: None. J. Rege: None. Primary aldosteronism (PA), the most prevalent cause of endocrine hypertension, is characterized by renin-independent aldosterone excess originating from lesions in one or both adrenal glands. Aldosterone-driver somatic mutations in genes encoding cell surface ion pumps/transporters/channels have been found in approximately 95% of adrenal aldosterone-producing adenomas (APAs). These mutations facilitate intracellular calcium (Ca2+) influx, which eventually leads to increased aldosterone synthase (CYP11B2) expression and elevated aldosterone production. Using next-generation sequencing, we identified three novel variants in MCOLN3 in APAs from four male patients with PA (p.Y391D, n=2; p.F415I, n=1; p.N411_V412delinsI, n=1). MCOLN3 encodes TRPML3 —a highly conserved inwardly-rectifying Ca2+-permeable cation channel. The identified variants are located in close proximity to the highly conserved ion transport pore and selectivity filter of TRPML3 and likely cause abnormal ion transport. Notably, our single-cell RNAseq and immunohistochemical assessments of normal adrenals showed MCOLN3 expression localized to the zona glomerulosa of the adrenal cortex, but not confined to CYP11B2-expressing cells. All APAs demonstrated moderate to substantial immunoreactivity for MCOLN3 and CYP11B2. The functional effects of mutated MCOLN3 were investigated using HAC15 adrenocortical cells overexpressing the mutant MCOLN3 variants (HAC15-MCOLN3Y391D and HAC15-MCOLN3N411_V412delinsI). Transcript and steroid data were assessed after 48h and compared to cells expressing wild-type (WT) MCOLN3 (HAC15-MCOLN3WT). A robust elevation in NR4A2 (NURR1, an upstream regulator of CYP11B2 transcription) and CYP11B2 transcription was observed in HAC15-MCOLN3Y391D (18- and 5-fold; P<0.0001) and HAC15-MCOLN3N411_V412delinsI (11- and 4-fold; P<0.000) vs. HAC15-MCOLN3WT. Aldosterone production was increased in both MCOLN3 mutant-expressing cells (2-fold vs. WT; P<0.0001). In addition, we observed increased Ca2+ influx in both mutants vs. WT. Electrophysiological studies of the mutants and WT demonstrated that the mutation-induced pathologic Ca2+ influx occurs via two mechanisms (direct and indirect). First, the mutations cause a gain-of-function phenotype leading to a constitutively active MCOLN3 Ca2+-permeable channel. Second, the mutations cause abnormal sodium influx, which leads to depolarization of the membrane potential, prompting the opening of voltage-gated Ca2+ channels and an increase in cytosolic Ca2+ activity. Both the direct and indirect mechanisms causing Ca2+ influx trigger an increase in CYP11B2 expression and dysregulated aldosterone production. This is the first report of disease-causing MCOLN3 mutations in humans, and collectively, these data implicate mutated MCOLN3 as a driver of aldosterone excess in PA. Presentation: Saturday, July 12, 2025