Abstract
Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.
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Data availability
Our institutional review board approval allows for the NGS analysis of somatic variants on archival tissue and sharing of these data upon request within a scientific cooperation. The tissue-related NGS data that support the findings of the present study will, therefore, be available from the authors upon reasonable request. Cell line-related RNA-seq data are available at the Gene Expression Omnibus (accession no. GSE236437). Source data are provided with this paper.
Code availability
FastQC is available at https://www.bioinformatics.babraham.ac.uk/projects/fastqc. Read adapters trimming with the bbduk tool from bbtools is available at https://sourceforge.net/projects/bbmap. The pheatmap Pretty Heatmaps, R package v.1.0.12 is available at https://CRAN.R-project.org/package=pheatmap. EnhancedVolcano: publication-ready volcano plots with enhanced coloring and labeling, R package v.1.12.0 is available at https://github.com/kevinblighe/EnhancedVolcano.
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Acknowledgements
We thank M. Vinco and F. Keyoumarsi at the University of Michigan for assistance in slide preparation. We also thank E. Klayman, M. Cieslik, F. Su, R. Wang, X. Cao and A. M. Chinnaiyan at the University of Michigan for support with WES and C.-J. Liu at the University of Michigan for technical support with the targeted NGS. We would also like to acknowledge F. Gürtler and E. Wacker at the University of Regensburg for their technical assistance with the electrophysiological studies. This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; grant nos. R01DK106618 and R01DK043140 to W.E.R.), National Center for Advancing Translational Sciences/Michigan Institute for Clinical and Health Research (grant no. UL1TR002240 to J.R.), the National Heart, Lung, and Blood Institute (grant nos. 1R01HL130106 to T.E. and R01HL155834 to A.F.T.), the Doris Duke Charitable Foundation (grant no. 2019087 to A.F.T.), the American Heart Association (grant nos. 17SDG33660447 to K.N. and 20CDA35320016 to J.R.), the Ministry of Health, Labour, and Welfare, Japan (grant no. 20FC1020 to H.S.) and Deutsche Forschungsgemeinschaft (DFG; German Research Foundation, grant nos. WA 1275/6 to R.W., BA 4436/2-1 to S.B., CRC/TRR 205 ‘The Adrenal Gland’, and RE 752/31-1 and WI 5359/2-1 ‘Project 444776998’ to M.R. and T.A.W.). M.R. also received funding from the Else Kröner-Fresenius-Stiftung (grant nos. 2012_A103, 2015_A228 and 2019_A104 ‘Else-Kröner Hyperaldosteronismus-German Conn Registry’) and the European Research Council (grant no. 694913). The lentiviral preparation was performed at the University of Michigan Vector Core and was supported by a grant from the NIDDK to the University of Michigan Center for Gastrointestinal Research (grant no. 5P30DK034933). Confocal microscopy performed at the University of Regensburg was funded by DFG (project no. 471535567).
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J.R., S.B., K.N. and W.E.R. designed the experiments. J.R., S.B., K.N. and W.E.R. wrote the paper. P.V. and C.K.-S. performed and analyzed WES. A.R.B. performed IHC studies and tissue DNA/RNA extraction. A.M.U. analyzed targeted NGS and RNA-seq data from tissue samples. J.R., A.R.B. and W.E.R. performed and analyzed the results of in vitro studies on HAC15-B2Luc cells. A.M.L. analyzed RNA-seq data in the engineered HAC15-B2Luc cells. S.B., C.K., A.P. and R.W. performed electrophysiological studies, calcium measurements, IF staining and flame photometry measurements of cells. A.F.T. and J.R. performed LC/MS–MS studies and analyzed the results. A.F.T., T.E., Y.Y., F.S., H.S., T.J.G., T.A.W. and M.R. assisted with patient recruitment, medical care, specimen collection and clinical data acquisition. All authors reviewed and revised the paper draft.
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Rege, J., Bandulik, S., Nanba, K. et al. Somatic SLC30A1 mutations altering zinc transporter ZnT1 cause aldosterone-producing adenomas and primary aldosteronism. Nat Genet 55, 1623–1631 (2023). https://doi.org/10.1038/s41588-023-01498-5
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DOI: https://doi.org/10.1038/s41588-023-01498-5