Somatic mutations in SLC30A1 lead to aldosterone-producing adenomas  and primary aldosteronism
Primary aldosteronism (PA), the most common form of endocrine hypertension, is characterized by inappropriately elevated aldosterone production via renin-independent mechanisms. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. Herein, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected two closely-stationed somatic variants in SLC30A1 in five APAs (p.L51_A57del, n=3; p.L49_L55del, n=2) that were devoid of any of the known aldosterone-driver mutations. SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). PA cases with SLC30A1 mutations showed male dominance and demonstrated increased serum aldosterone concentration compared with age-matched male controls. We tested functional effects of the variant SLC30A1L51_A57del in a doxycycline-inducible system using the human adrenocortical HAC15 cell line. Functional in vitro studies following doxycycline treatment indicated increased adrenal cell aldosterone production, CYP11B2 mRNA expression, CYP11B2 promoter activity, depolarization of the resting membrane potential and increased cytosolic Ca2+ levels in the SLC30A1L51_A57del cells. Collectively, these data support a pathological role for mutant SLC30A1 on the development of PA. Overall design: The human adrenocortical carcinoma cell line HAC15 containing a CYP11B2 promoter-driven secreted Gaussia luciferase reporter (HAC15-B2Luc)17,18, was used as parental cells. The HAC15-B2Luc cells were grown in DMEM-F12 containing 10% cosmic calf serum (CCS), 1% insulin-transferrin-selenium, and antibiotics (penicillin, streptomycin and gentamicin) and maintained in a 37°C humidified atmosphere (5% CO2), as previously described18. These cells were transduced8,18,19 with the SLC30A1WT and SLC30A151_57del lentiviruses to generate the cell lines HAC15B2Luc-Doxy-SLC30A1WT and HAC15B2Luc-Doxy-SLC30A151_57del respectively, followed by antibiotic selection with geneticin (Gaussia luciferase selection marker, 1 mg/mL) and blasticidin (SLC30A1 selection marker, 2 µg/mL).  Cells were plated at a density of 75,000 cells/well in a 48-well dish for 48 hours. After incubation in a low serum medium (DMEM/F-12 containing 0.1% CCS and antibiotics) for 24 hours, expression of SLC30A1WT and SLC30A151_57del in the cells was initiated by addition of 1 µg/mL Doxy (Sigma Aldrich, St. Louis, MO) for the indicated times.