Novel gain-of-function CLCN2 germline mutation associated with nonclassical lateralizing primary aldosteronism

Primary aldosteronism (PA) is commonly caused by somatic and germline mutations in various genes. Patients with unilateral PA may display classical or nonclassical histopathologic features in adrenal tumors. Somatic and germline mutations are linked to classical unilateral and familial PA, respectively. In this study, we aim to search for novel gene mutations causing nonclassical lateralizing PA. Surgically resected adrenal tumor tissues from patients enrolled in a PA cohort were subjected to histopathological evaluation. Genetic analyses were performed for genomic DNA isolated from peripheral blood leukocytes and CYP11B2 immunohistochemistry-guided macrodissected adrenal tissues to identify novel variants. A PA patient with a 1.8 cm mass in the right adrenal gland underwent laparoscopic right adrenalectomy and achieved complete biochemical success at 12 months, but partial clinical improvement. Post-adrenalectomy tissues displayed the nonclassical histopathologic feature with multiple aldosterone-producing micronodules (mAPMs), but no solitary aldosterone-producing adenoma. Genetic analyses identified a heterozygous CLCN2 germline variant, c.1087C>T (p.R363C), that was likely pathogenic based on bioinformatic evaluations. In vitro biochemical, electrophysiological, and hormone assays revealed that the human ClC-2 R363C Cl- channel displayed significant gain-of-function phenotypes, including enhanced protein stability and cell surface expression, higher channel open probability at physiological membrane potentials, and increased aldosterone production in human adrenocortical cells. The dominant gain-of-function effect of ClC-2 R363C was also observed when we co-expressed the variant with its wild-type counterpart. Altogether, we report the first association of gain-of-function CLCN2 germline mutation with nonclassical mAPMs, providing new insights into the pathogenic mechanisms of PA.