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Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant aldosterone-producing adenomas presenting in puberty, pregnancy or menopause

Nature Genetics volume 53, pages 1360–1372 (2021)Cite this article

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Abstract

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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Fig. 1: Clinical and cellular schemata showing the critical roles of GNA11/Q, and their p.Gln209 residue, in the production of aldosterone.
Fig. 2: Mutations of GNA11/Q Q209 increase aldosterone production in human adrenocortical cells.
Fig. 3: High LHCGR expression in GNA11/Q and CTNNB1 double-mutant adrenal cells.
Fig. 4: Gene expression profiles in GNA11/Q and CTNNB1 double-mutant adrenal cells.
Fig. 5: Expression of aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) in GNA11/Q and CTNNB1 double-mutant APAs.
Fig. 6: GNA11 somatic mutations were found in adrenals adjacent to double-mutant APAs.

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Data availability

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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Acknowledgements

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.

Author information

Author notes

  1. These authors contributed equally: Junhua Zhou, Elena A. B. Azizan, Claudia P. Cabrera, Fabio L. Fernandes-Rosa, Sheerazed Boulkroun.

  2. These authors jointly supervised this work: Helen L. Storr, Maria-Christina Zennaro, Morris J. Brown.

Authors and Affiliations

  1. Endocrine Hypertension, Department of Clinical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK

    Junhua Zhou, Elena A. B. Azizan, Giulia Argentesi, Xilin Wu, Sam O’Toole, Emily Goodchild, Sumedha Garg & Morris J. Brown

  2. NIHR Barts Cardiovascular Biomedical Research Centre, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

    Junhua Zhou, Claudia P. Cabrera, Giulia Argentesi, Xilin Wu, Sam O’Toole, Emily Goodchild, Sumedha Garg & Morris J. Brown

  3. Department of Medicine, The National University of Malaysia (UKM) Medical Centre, Kuala Lumpur, Malaysia

    Elena A. B. Azizan

  4. Centre for Translational Bioinformatics, William Harvey Research Institute, Queen Mary University of London, London, UK

    Claudia P. Cabrera

  5. Université de Paris, PARCC, Inserm, Paris, France

    Fabio L. Fernandes-Rosa, Sheerazed Boulkroun, Laurence Amar & Maria-Christina Zennaro

  6. Centre for Endocrinology, William Harvey Research Institute, Queen Mary University of London, London, UK

    Emily Cottrell, Lou A. Metherell & Helen L. Storr

  7. Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Unité Hypertension Artérielle, Paris, France

    Laurence Amar

  8. Department of Histopathology, Addenbrooke’s Hospital, Cambridge, UK

    Alison Marker

  9. Metabolic Research Laboratories, Institute of Metabolic Science, Cambridge Biomedical Campus, Cambridge, UK

    Russell Senanayake, Sumedha Garg & Mark Gurnell

  10. Department of Surgical Sciences, Uppsala University, Uppsala, Sweden

    Tobias Åkerström, Samuel Backman, Per Hellman & Peyman Björklund

  11. Cellular Pathology Department, Royal London Hospital, London, UK

    Suzanne Jordan & Veronica A. Kinsler

  12. Genetics and Genomic Medicine, University College London Great Ormond Street Institute of Child Health, London, UK

    Satyamaanasa Polubothu

  13. Centre for Cancer Biomarkers and Biotherapeutics, Barts Cancer Institute, Queen Mary University of London, London, UK

    Daniel M. Berney

  14. Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford, UK

    Anna Gluck, Kate E. Lines & Rajesh V. Thakker

  15. Department of Endocrinology and Diabetes, Cork University Hospital, Cork, Ireland

    Antoinette Tuthill

  16. Clinical Biochemistry, Cork University Hospital, Cork, Ireland

    Caroline Joyce

  17. Centre for Inherited Cardiovascular Diseases, Great Ormond Street Hospital and University College London Institute of Cardiovascular Science, London, UK

    Juan Pablo Kaski

  18. Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

    Fiona E. Karet Frankl

  19. Dept of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Ada E. D. Teo

  20. Department of Surgery, St Bartholomew’s Hospital, London, UK

    Laila Parvanta

  21. Department of Endocrinology, St Bartholomew’s Hospital, London, UK

    William M. Drake

  22. Barts and London Genome Centre, School of Medicine and Dentistry, Blizard Institute, Queen Mary University of London, London, UK

    Eva Wozniak & Charles A. Mein

  23. Cardiovascular Disease Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    David Klinzing, Jyn Ling Kuan, Zenia Tiang & Roger S. Y. Foo

  24. Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore, Singapore

    Zenia Tiang

  25. G.V. (Sonny) Montgomery VA Medical Center and Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA

    Celso E. Gomez Sanchez

  26. Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Service de Génétique, Paris, France

    Maria-Christina Zennaro

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  1. Junhua Zhou
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  2. Elena A. B. Azizan
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  4. Fabio L. Fernandes-Rosa
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Contributions

C.P.C., E.A.B.A. and M.J.B. discovered the mutations in GNA11 and GNAQ, replicated by J.Z. and F.L.F.-R. J.Z., E.A.B.A., C.P.C., F.L.F.-R., S. Boulkroun, H.L.S., M.-C.Z. and M.J.B. conceived and designed the subsequent experiments/analyses. C.J., A.T., H.L.S., E.C., G.A., X.W., E.G., L.A., S. Backman, P.H., P.B., T.A., R.S., D.M.B., J.P.K., W.M.D., L.P. and F.E.K.F. contributed to cohort ascertainment, phenotypic characterization and recruitment. S. Backman, C.P.C., S.P., Z.T., L.A.M., T.A. and S.G. contributed to WES/RNA-seq production, validation, analysis and reanalysis. J.Z., F.L.F.-R., S. Boulkroun, X.W., A.E.D.T., E.A.B.A., E.C., S.G., G.A. and T.A. performed targeted sequencing and RT–qPCR analyses. J.Z. performed LCM and genotyping of adrenal zones and biopsy punches. S.J., S. Boulkroun, A.M. and J.Z. performed, and F.L.F.-R. and E.A.B.A. analyzed, IHC staining. C.E.G.S. developed antisera for use in IHC. J.Z., S.G., A.G., K.E.L. and R.V.T. contributed to plasmid construction for GNA11 and GNAQ. J.Z., E.A.B.A. and G.A. performed the functional experiments on transfected H295R and primary human adrenal cells. J.Z. and S.O. undertook confocal analyses. J.Z., E.A.B.A., F.L.F.-R., C.A.M., R.F., E.W., D.K., J.L.K., Z.T. and C.P.C. performed ddPCR, WES and NGS for genotyping of adjacent adrenal regions. C.P.C., J.Z., E.A.B.A. and M.J.B. contributed to statistical analyses. E.A.B.A. and M.J.B. drafted the manuscript, for which J.Z., E.A.B.A., C.P.C., F.L.F.-R., S. Boulkroun, T.A., A.M. and M.J.B. contributed figures. C.P.C., F.L.F.-R., S. Boulkroun, M.G., V.A.K. and M.-C.Z. critically reviewed the text. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Elena A. B. Azizan, Maria-Christina Zennaro or Morris J. Brown.

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Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

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

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