Abstract

Primary aldosteronism, a common cause of severe hypertension1, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II)2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism.

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Acknowledgements

We thank our patients and their families for their invaluable contributions, the Yale Center for Genome Analysis for next-generation sequencing, the Center for Advanced Imaging (CAi) at Heinrich Heine University for providing a confocal microscope, S. Weidtkamp-Peters and S. Hänsch for technical assistance, J. Zhang for helpful discussions, E. Seidel, N. Erlenhardt and N. Klöcker for providing immunoprecipitation protocols and helpful discussions, C. Gomez-Sanchez (University of Mississippi) for providing plasmids, W. Rainey (University of Michigan) for providing HAC15 cells and M. Haase (Heinrich Heine University Düsseldorf) for providing H295R cells. Computational support and infrastructure were in part provided by the Centre for Information and Media Technology (Düsseldorf). This study was supported in part by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen (Rückkehrprogramm and Junges Kolleg) and the Deutsche Forschungsgemeinschaft (SCHO 1386/2-1) (all to U.I.S.) and the NIH Center for Mendelian Genomics (5U54HG006504), NIH P01DK17433 and the Howard Hughes Medical Institute (all to R.P.L.).

Author information

Author notes

  1. These authors contributed equally: Gabriel Stölting and Julia Schewe.

Affiliations

  1. Department of Nephrology, Medical School, Heinrich Heine University Düsseldorf, Düsseldorf, Germany

    • Ute I. Scholl
    • , Julia Schewe
    • , Anne Thiel
    •  & Lars C. Rump
  2. Department of Nephrology and Medical Intensive Care, Charité–Universitätsmedizin Berlin, Berlin Institute of Health, Berlin, Germany

    • Ute I. Scholl
  3. Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Jülich, Germany

    • Gabriel Stölting
    • , Hua Tan
    • , Verena Untiet
    •  & Christoph Fahlke
  4. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    • Carol Nelson-Williams
    • , Alfred A. Vichot
    • , Sheng Chih Jin
    • , Erin Loring
    • , Jungmin Choi
    •  & Richard P. Lifton
  5. Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA

    • Carol Nelson-Williams
    • , Alfred A. Vichot
    • , Erin Loring
    • , Jungmin Choi
    •  & Richard P. Lifton
  6. Yale Center for Mendelian Genomics, New Haven, CT, USA

    • Erin Loring
    •  & Richard P. Lifton
  7. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Republic of Korea

    • Taekyeong Yoo
    •  & Murim Choi
  8. Endocrine Hypertension Research Center, University of Queensland Diamantina Institute, Greenslopes and Princess Alexandra Hospitals, Brisbane, Queensland, Australia

    • Shengxin Xu
    • , Aihua Wu
    • , Richard Gordon
    •  & Michael Stowasser
  9. Proteomics Platform, Max Delbrück Center for Molecular Medicine in the Helmholtz Society and Core Unit of Proteomics, Berlin Institute of Health, Berlin, Germany

    • Marieluise Kirchner
    •  & Philipp Mertins
  10. Nephrology, Le Bonheur Children’s Hospital, Memphis, TN, USA

    • Ali Mirza Onder
  11. Cooper Clinic, PA, Fort Smith, AR, USA

    • Cory Gamble
  12. Peyton Manning Children’s Hospital at St. Vincent, Indianapolis, IN, USA

    • Daniel McKenney
  13. Division of Metabolism, Endocrinology and Diabetes, University of Michigan Medical School, Ann Arbor, MI, USA

    • Robert W. Lash
  14. Division of Nephrology and Hypertension, Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, USA

    • Deborah P. Jones
  15. Olin Teague Veterans Administration Hospital, Temple, TX, USA

    • Gary Chune
  16. Division of Endocrinology, Nemours Children’s Specialty Care, Jacksonville, FL, USA

    • Priscila Gagliardi
  17. Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, NY, USA

    • Richard P. Lifton

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Contributions

U.I.S., G.S., C.F. and R.P.L. designed the study. M.S. and R.G. recruited and characterized family 3. A.M.O., C.G., D.M., R.W.L., D.P.J., G.C., P.G., E.L., C.N.-W. and R.P.L. ascertained and recruited probands with early-onset primary aldosteronism. A.A.V., E.L. and U.I.S. recruited additional members of selected families. C.N.-W., S.X. and A.W. prepared DNA samples. U.I.S., A.A.V., S.C.J., T.Y., M.C. and R.P.L. analyzed exome sequencing results. U.I.S. identified the disease-associated gene. C.N.-W., A.T. and A.A.V. performed and analyzed the results of targeted DNA sequencing. J.S. performed immunohistochemistry, immunoprecipitation and real-time PCR. J.S. and A.T. made constructs and generated stable cell lines. J.S. and J.C. prepared samples for and analyzed the results of RNA sequencing. J.S. and U.I.S. performed and analyzed the results of splicing assays and confocal microscopy. G.S., H.T., V.U. and C.F. performed and analyzed the results of FLIM and electrophysiology. M.K. and P.M. performed and analyzed the results of mass spectrometry. L.C.R. read and revised the manuscript. U.I.S. wrote the initial draft of the manuscript, with contributions and/or revisions from all authors.

Competing interests

Heinrich Heine University Düsseldorf has filed a patent application: EP17209972, Diagnosis and Therapy of Primary Aldosteronism.

Corresponding author

Correspondence to Ute I. Scholl.

Integrated supplementary information

  1. Supplementary Figure 1 Sanger sequences.

    a, Sanger sequences of kindreds with CLCN2 variants. M/+ denotes the indicated novel CLCN2 variant in the heterozygous state, and +/+ denotes homozygous wild-type sequence. Mutant bases are indicated by a red frame, and encoded amino acid sequences are shown above. b, Splicing assay for the p.Lys362del mutation. Exons 9–11 of CLCN2 were cloned, and HEK293T cells were transfected with WT DNA and DNA carrying the variant found in kindred 1492. RNA was isolated and cDNA was transcribed. Shown are Sanger sequences of PCRs covering the exon 10/11 splice site affected by the variant. A new donor site on exon 10 is used, resulting in deletion of the last 3 bp of exon 10 (red rectangle in WT sequence, red line in mutant sequence). These results were consistent with in silico prediction.

  2. Supplementary Figure 2 Immunohistochemistry.

    a, Immunohistochemistry as in Fig. 2 was performed on the adrenal gland of a second human subject (one of two technical replicates shown). Peptide control, antibody was preincubated with immunogenic peptide. Scale bar, 100 µm. C, capsule; G, glomerulosa; F, fasciculata. b, Immunofluorescent staining of mouse adrenal gland, with DAB2 as a marker of the zona glomerulosa. Scale bar, 10 µm.

  3. Supplementary Figure 3 Gating analysis, time constants of activation/deactivation and current density–voltage plots of WT and mutant ClC-2 channels.

    a, Whole-cell patch–clamp recordings of representative ClC-2MUT and voltage protocol (150 mM Cl outside, 75 mM Cl inside; see the Methods for solutions) for the indicated mutations are shown. b, Time constants for WT and mutant ClC-2 channels (see the Methods for details). Mean values ± 95% confidence intervals are shown (WT, p.Arg172Gln and p.Ser865Arg are reproduced from Fig. 3; WT, n = 11; p.Met22Lys, n = 12; p.Tyr26Asn, n = 13; p.Arg172Gln, n = 13; p.Lys362del, n = 12; p.Ser865Arg, n = 11). c, Plot of steady-state current densities. Values are shown as means ± 95% confidence intervals; WT, n = 11; p.Met22Lys, n = 12; p.Tyr26Asn, n = 13; p.Arg172Gln, n = 13; p.Lys362del, n = 12; p.Ser865Arg, n = 11. Median values at –80 mV and the results of Kruskal–Wallis one-way ANOVA (H = 14.823, d.f. = 5) followed by Dunn’s method when appropriate are as follows: WT: –5.75; p.Met22Lys: –10.28, n.s. vs. WT; p.Tyr26Asn: –13.66, P < 0.05 vs. WT; p.Arg172Gln: –9.13, n.s. vs. WT; p.Lys362del: –13.69, P < 0.05 vs. WT; p.Ser865Arg, –12.58, P < 0.05 vs. WT. df, Total (d), protopore (e) and common gate (f) open probabilities were determined as described (see the Methods for details; WT, p.Arg172Gln and p.Ser865Arg total and common open probabilities are reproduced from Fig. 3; WT, n = 11; p.Met22Lys, n = 12; p.Tyr26Asn, n = 13; p.Arg172Gln, n =13; p.Lys362del, n = 12; p.Ser865Arg, n = 12). Data points were fit using a Boltzmann function (bold line), and 95% confidence intervals were determined using bootstrap sampling. The overall shift in activation of mutant ClC-2 channels apart from p.Ser865Arg mostly results from a higher common gate open probability, whereas the fast gate open probability of p.Ser865Arg is shifted to more positive potentials. g, Individual data and stable cell line clones used in the analysis shown in Fig. 3 and this figure.

  4. Supplementary Figure 4 Mass spectrometry and confocal microscopy.

    a,b, LC–MS/MS-based identification of the phosphorylation site of ClC-2 at Ser865. ClC-2 was identified with 43 peptides and sequence coverage of 52.8% Shown are annotated MS/MS spectra of the unmodified (a) and phosphorylated (b) peptide DSATSSSDTETTEVHALWGPHSR corresponding to amino acids 859–881 of ClC-2. The x axis shows m/z, the left y axis shows relative abundance and the right y axis shows absolute signal intensity. Unmodified peptide: 16 MS/MS counts, posterior error probability (PEP) 1.21 × 10–35, score 160.47; Ser865-phosphorylated peptide: 11 MS/MS counts, PEP 1.61 × 10–36, score 166.49. The phosphorylation site at Ser865 was fully localized, with a localization probability of 0.813811 (mass error of 0.28027 p.p.m.). One of two independent preparations (Methods) is shown; similar results were obtained for another two independent preparations using a different solvent. c,d, Live cell confocal microscopy of YFP-tagged WT and mutant ClC-2 and the surface membrane marker CFP-mem in H295R cells. c, The respective variant is noted on the left. Left, confocal image using the YFP channel; middle, CFP channel; right, overlay. Scale bars, 20 µm. Two independent clones of each plasmid were assessed, and representative images are shown. d, Correlation R between YFP and CFP fluorescence for each construct. WT, 0.29 ± 0.03 (n = 71); p.Met22Lys, 0.15 ± 0.02 (n = 81; P = 0.0001 vs. WT); p.Tyr26Asn, 0.25 ± 0.03 (n = 53; P > 0.9999 vs. WT); p.Arg172Gln, 0.24 ± 0.03 (n = 56; P = 0.5862 vs. WT); p.Lys362del, 0.29 ± 0.03 (n = 65; P > 0.9999 vs. WT); p.Ser865Arg, 0.30 ± 0.02 (n = 59; P > 0.9999 vs. WT); all mean ± s.e.m., Kruskal–Wallis test; Dunn’s multiple-comparisons test; Kruskal–Wallis statistic 28.47, d.f. = 5. Box, interquartile range; whiskers, 1.5 times the interquartile range; line, median; ***P < 0.001; n.s., not significant.

  5. Supplementary Figure 5 CYP11B2 expression in H295R cells after transfection of non-functional CLCN2.

    Relative expression levels of CYP11B2 determined by real-time PCR in H295R cells transfected with empty vector control, wild-type CLCN2 (blue) or two constructs with C-terminal deletions that affect ion channel function (yellow, corresponding deletions noted below the graph)30. Values were normalized to empty vector control. CYP11B2 expression is significantly lower after transfection of non-functional channels than after transfection of the wild-type channel (mean ± s.e.m.; WT, 16.35 ± 2.72; p.Arg751Ter, 1.46 ± 0.18, P = 0.0089 vs. WT; p.His573_Leu636del, 2.38 ± 0.50, P = 0.0077 vs. WT; **P < 0.01). n = 5, one-way ANOVA for all constructs, Dunnett’s multiple-comparisons test, F = 31.52, d.f. = 4. Box, interquartile range; whiskers, 1.5 times the interquartile range; line, median.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Tables 1–7 and 10, and Supplementary Note

  2. Life Sciences Reporting Summary

  3. Supplementary Table 8

    Details of statistical analysis shown in Figs. 3 and 4, and Supplementary Fig. 3

  4. Supplementary Table 9

    Whole-transcriptome analysis of CLCN2-transfected H295R cells

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