A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism

Abstract

Primary aldosteronism is the most common and curable form of secondary arterial hypertension. We performed whole-exome sequencing in patients with early-onset primary aldosteronism and identified a de novo heterozygous c.71G>A/p.Gly24Asp mutation in the CLCN2 gene, encoding the voltage-gated ClC-2 chloride channel1, in a patient diagnosed at 9 years of age. Patch-clamp analysis of glomerulosa cells of mouse adrenal gland slices showed hyperpolarization-activated Cl currents that were abolished in Clcn2−/− mice. The p.Gly24Asp variant, located in a well-conserved ‘inactivation domain’2,3, abolished the voltage- and time-dependent gating of ClC-2 and strongly increased Cl conductance at resting potentials. Expression of ClC-2Asp24 in adrenocortical cells increased expression of aldosterone synthase and aldosterone production. Our data indicate that CLCN2 mutations cause primary aldosteronism. They highlight the important role of chloride in aldosterone biosynthesis and identify ClC-2 as the foremost chloride conductor of resting glomerulosa cells.

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Fig. 1: A CLCN2 variant identified in a patient with early-onset primary aldosteronism.
Fig. 2: ClC-2 expression in mouse adrenal glands and electrophysiological analyses of wild-type and mutant channels.
Fig. 3: Effect of ClC-2WT and mutant ClC-2Asp24 channels on aldosterone production and expression of genes and proteins involved in aldosterone biosynthesis.
Fig. 4: Functional impact of the ClC-2Asp24 variant.
Fig. 5: Proposed model for autonomous aldosterone secretion in adrenal zona glomerulosa cells with the ClC-2Asp24 mutant.

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Acknowledgements

This work was funded through institutional support from INSERM and by the Agence Nationale pour la Recherche (ANR-13-ISV1-0006-01), the Fondation pour la Recherche Médicale (DEQ20140329556), the Programme Hospitalier de Recherche Clinique (PHRC grant AOM 06179), and institutional grants from INSERM. The laboratory of M.-C.Z. is also a partner of the H2020 project ENSAT-HT grant number 633983. T.J.J. was supported by institutional funding from the Leibniz and Helmholtz associations, a grant from the BMBF (E-RARE 01GM1403), and the Prix Louis-Jeantet de Médecine.

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M.-C.Z., F.L.F.-R., G.D., I.J.O., and T.J.J. designed experiments and wrote the manuscript. T.M.S., M.-C.Z., T.S., and F.L.F.-R. performed and analyzed whole-exome sequencing. M.-C.Z., F.L.F.-R., G.D., R.E.Z., and S.B. performed and analyzed the results of in vitro studies on H295R-S2 cells. I.J.O. performed electrophysiological studies for which the data were analyzed by I.J.O. and T.J.J. C.G. characterized adrenal glands from wild-type and Clcn2−/− mice and performed western blots. V.J., X.J., L.A., and H.L. were responsible for patient recruitment, medical care, and clinical data acquisition. All authors revised the manuscript draft. C.G. and R.E.Z. contributed equally to this work.

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Correspondence to Fabio L. Fernandes-Rosa or Thomas J. Jentsch or Maria-Christina Zennaro.

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Supplementary Figure 1 Dependence of ClC-2WT and ClC-2Asp24 currents on external pH.

WT and mutant channels were expressed in Xenopus oocytes and measured by two-electrode voltage-clamp using a pulse protocol that clamped the oocytes in 2-s-long 20-mV steps from +60 to –120 mV. a,b, Representative current traces obtained from WT (a) and G24D mutant (b) ClC-2 at indicated pH values. c,d, Mean ClC-2WT (c) and ClC-2Asp24 (d) currents measured after 2 s as a function of voltage and pH. n = 3–6 oocytes; error bars, s.e.m. (e) Currents at –80 mV (approximately the resting voltage of glomerulosa cells) from ClC-2WT (filled circles) and ClC-2Asp24 (open circles) normalized to respective currents at –120 mV at pH 7.4. Note the large pH dependence of WT currents, which is strongly reduced but not abolished by the Gly24Asp mutation.

Supplementary Figure 2 Effect of ClC-2 downregulation on aldosterone production and expression of genes involved in aldosterone biosynthesis.

a, Basal and stimulated (Ang II or K+) mRNA expression of CLCN2 in H295R-S2 cells infected with scrambled (open bars) or ClC-2 (filled bars) shRNA (one-way ANOVA, P < 0.0001, F = 28.11). b, Basal and stimulated aldosterone production by H295R-S2 cells infected with scrambled or ClC-2 shRNA. ce, Basal and stimulated mRNA expression of CYP11B2 (one-way ANOVA, P < 0.0001, F = 84) (c), STAR (Kruskal–Wallis, P = 0.0022) (d), and CYP21A2 (Kruskal–Wallis, P = 0.0002) (e) in H295R-S2 cells transfected with scrambled or ClC-2 shRNA. Results of mRNA expression are represented as fold induction of cells infected with scrambled shRNA in basal conditions. Values of all experiments are represented as means ± s.e.m. of two independent experiments performed in experimental triplicate for each condition (n = 6 for scrambled shRNA, n = 12 for ClC-2 shRNA). *P < 0.05; ***P < 0.001; (i) P < 0.05 stimulated versus basal condition; (ii) P < 0.01 stimulated versus basal condition; (iii) P < 0.001 stimulated versus basal condition.

Supplementary Figure 3 CLCN2 variants identified in subjects with bilateral adrenal hyperplasia.

a, Sanger sequencing chromatograms showing the CLCN2 wild-type sequence and the CLCN2 variant c.143C>G (p.Pro48Arg) identified in subject K963-1 with bilateral adrenal hyperplasia. b, Sanger sequencing chromatograms showing the CLCN2 wild-type sequence and the CLCN2 variant c.197G>A (p.Arg66Gln) identified in subject K1044-1 with bilateral adrenal hyperplasia. c, Alignment and conservation of residues encoded by ClC-2 orthologs. Residues that are conserved among more than three sequences are highlighted in yellow.

Supplementary Figure 4 Electrophysiological analyses of ClC-2Gln66 and ClC-2Arg48 channels.

ac, Representative chloride current traces measured by two-electrode voltage-clamp from Xenopus oocytes injected with 9.2 ng of human ClC-2WT (a), ClC-2Gln66 (b), or ClC-2Arg48 (c) cRNA. d, Mean ± s.e.m. currents measured after 2 s from experiments in ac plotted as a function of clamp voltage. The number of cells, obtained from two different batches of oocytes, is indicated in parentheses. e, Summary of Cl currents at –80 mV and after 2 s for ac. Statistical analyses for ClC-2Gln66 and ClC-2Arg48 were performed by comparison with ClC-2WT, Mann–Whitney test.

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Fernandes-Rosa, F.L., Daniil, G., Orozco, I.J. et al. A gain-of-function mutation in the CLCN2 chloride channel gene causes primary aldosteronism. Nat Genet 50, 355–361 (2018). https://doi.org/10.1038/s41588-018-0053-8

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