Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.
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Source data for Figs. 2a–f and 3a–c,e,g are provided with the paper. The raw RNA-seq dataset analyzed to generate Fig. 4a,b, Supplementary Table 3 and Supplementary Fig. 4 is available upon request from the Science for Life Laboratory Data Centre through the link https://doi.org/10.17044/NBIS/G000007. Regulations by the service provider may make access technically restricted to PIs at Swedish organizations. The microarray datasets analyzed to generate Fig. 4a,b are deposited in the Gene Expression Omnibus database (GSE64957) or are available from the corresponding author on reasonable request. The WES raw data of the 41 APAs and controls investigated for recurrent pathogenic somatic mutation are available from the Sequence Read Archive under accession nos. PRJNA732946 and PRJNA729738. All other raw data that support the findings of this study are available from the corresponding author upon reasonable request.
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The CTNNB1 plasmid was a kind gift of M. Bienz, Medical Research Council Laboratory of Molecular Biology, Cambridge. The 11C-metomidate positron emission tomography (PET) CT The project was funded in part by the British Heart Foundation through a Clinical Research Training Fellowship (no. FS/19/50/34566) and PhD Studentship (no. FS/14/75/31134), by the National Institute of Health Research (NIHR) through Senior Investigator award no. NF-SI-0512-10052 (all to M.J.B.) and by NIHR Efficacy and Mechanisms Evaluation Project (no. 14/145/09, to W.M.D., M.G. and M.J.B.) and Barts and the London Charity project (no. MGU0360), to W.M.D. and M.J.B. The project was further funded through institutional support from INSERM, Agence Nationale de la Recherche (no. ANR-15-CE14-0017-03) and Fondation pour la Recherche Médicale (no. EQU201903007864) to M.-C.Z. and the NIHR Advanced Fellowship (no. NIHR3000098) to H.L.S. E.A.B.A. is a Royal Society-Newton Advanced Research Fellow (no. NA170257/FF-2018-033). R.V.T. is supported by a Wellcome Trust Investigator Award (no. 106995/Z/15/Z) and the NIHR Oxford Biomedical Research Centre (BRC) Programme. C.P.C. is supported by the NIHR BRC at Barts and The London School of Medicine and Dentistry. M.G., A.M., and R.S. are supported by the NIHR Cambridge BRC (no. IS-BRC-1215-20014). The research of J.L.K., Z.T. and R.F. was supported by the National Medical Research Council and BRC of Singapore. Research in London and Cambridge, UK, was further supported by the NIHR Barts Cardiovascular BRC (no. IS-BRC-1215-20022) and the Cambridge BRC and BRC-funded Tissue Bank. The research utilized Queen Mary University of London’s Apocrita HPC facility, supported by QMUL Research-IT (https://doi.org/10.5281/zenodo.438045). Assistance from the Endocrine Unit Laboratory of the National University of Malaysia (UKM) Medical Centre, and from L. K. Chin and S. Khadijah (UKM) is acknowledged.
The authors declare no competing interests.
Peer review information Nature Genetics thanks Lee Weinstein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended Data Fig. 1 High LHCGR expression in GNA11 and CTNNB1 double mutant co-transfected primary human adrenal cells.
a, APA 351 T cells transfected with CTNNB1 (untagged plasmid) and GNA11 (GFP-tagged plasmid) wild-type or Q209P (red boxed cell). LHCGR and CTNNB1 expression was visualized as in Fig. 3f using the primary antibody rabbit anti-LHCGR #NLS1436 (1:200; Novus Biologicals, UK) and the primary antibody mouse anti-CTNNB1 #610154 (1:100; BD transduction Lab, USA), respectively. Scale bars, 50 μm. b, Immunofluorescence of LHCGR in APA 351 T cells was quantified using corrected total cell fluorescence (CTCF). LHCGR expression was increased in cells expressing high CTNNB1 and GNA11 Q209P (the exact number, n, of cells quantified from two independent experiment are as indicated below the x-axis; the P-values indicated are according to Kolmogorov–Smirnov statistical test). High CTNNB1 was determined as CTCF > 10,000. Data are presented as mean values + /- s.e.m.
Extended Data Fig. 2 GNA11 somatic mutations were found in the adjacent adrenals to double-mutant APA of patient 6.
a, From six different regions (R1-5, at the edges of the adrenal cortex, R6 and APA, within the circled areas) in the formalin fixed paraffin embedded (FFPE) adjacent adrenal gland, genomic DNA samples of patient 6 were genotyped for CTNNB1 and GNA11 mutations. Immunohistochemistry of KCNJ5 and CYP11B2 were used for region selection. Scale bar, 10 mm and 50 μm as indicated. b, Sanger sequencing identified weak chromatogram peaks of CTNNB1 G34R and GNA11 Q209P somatic mutations in region 6 of the adjacent adrenal gland. c, Next generation sequencing confirmed the CTNNB1 G34R and GNA11 Q209P mutations in region 6 of the adjacent adrenal gland. d, qPCR of R1-6 and APA showed a 337-fold higher of TMEM132E, 38-fold higher of CYP11B2, 14-fold higher of DKK1 and 10-fold higher of LHCGR expression in region 6 compared to region 5. Regions 1-5 have similar expression of the above genes. The APA had the highest expression of CYP11B2, TMEM132E, DKK1, LHCGR and lowest expression of CYP11B1 and LGR5 compared to regions 1–6.
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Zhou, J., Azizan, E.A.B., Cabrera, C.P. et al. Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause. Nat Genet 53, 1360–1372 (2021). https://doi.org/10.1038/s41588-021-00906-y