Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause

A. E D Teo, S. Garg, L. Haris Shaikh, J. Zhou, F. E. Karet Frankl, M. Gurnell, L. Happerfield, A. Marker, M. Bienz, Azizan Elena Aisha, M. J. Brown

Research output: Contribution to journalArticle

Abstract

Background: Primary aldosteronism is a curable cause of hypertension if the hypertension is due to an aldosterone-producing adenoma (APA) [1,2]. The discovery of somatic mutations defining pathogenetic subtypes of this adenoma has increased recognition of common but small zona glomerulosa-like APAs [3-6]. In this subtype, we have sought APAs with distinctive presentation or outcome, in which analysis of genotype-phenotype associations could help explain the low success rate and variability of adrenalectomy for curing hypertension. Methods: Ten zona glomerulosa-like APAs from three women with primary aldosteronism were analysed by exome sequencing and microarray analysis. Nine had a mutation of ATP1A1 or CACNA1D, and one had a distinctive genotype and transcriptome. Targeted sequencing of further zona glomerulosa-like APAs led to the finding of two with a similar genotype, and to the recognition that all three women presented in early pregnancy or menopause. Quantitative PCR and immunohistochemistry were performed for genes upregulated in the index case. Similar analyses and a TCF-LEF assay for Wnt signalling were performed on cells transfected with the mutant gene. Findings: All three APAs had novel mutations in exon 3 of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway. The mutations prevented phosphorylation of β-catenin, resulting in an increase in total form of β-catenin and active to total ratio, as quantified by immunoblotting. Transfection of mutant constructs into HEK cells caused near doubling of Wnt signalling in a luciferase reporter assay. Microarray of the index case found a greater than 100-fold increase in expression of gonadal receptors LHCGR and GNRHR, confirmed by qPCR (both genes) and immunohistochemistry (LHCGR) in all three APAs. Increased GATA4 expression was further evidence of adrenocortical cell de-differentiation towards their common gonad-adrenal precursor cell-type. Transfection of LHCGR-negative APA cells with GFP-mutant-CTNNB1 switched on LHCGR expression in GFP-positive cells. Interpretation: The role of the Wnt system in adrenal physiology and tumour development is well recognised [7,8]. De-differentiation of zona glomerulosa-cells after Wnt activation by the CTNNB1 mutation seems to cause aberrant gonadal receptor expression, which is also previously described [9,10]. Our findings connect these observations, and explain the unmasking of small APAs by increases in luteinising hormone and human chorionic gonadotropin hormone in early pregnancy or menopause. Strikingly, all three women, including the treatment-resistant older woman, were clinically cured by adrenalectomy. This contrasts with the failure of adrenalectomy to completely cure hypertension in the majority of patients. This failure is attributable to decades of exposure to aldosterone excess, and the secondary consequences of fibrosis and remodelling in the cardiovascular system [11,12]. CTNNB1 mutations have now been reported in 10% of APAs without a previously reported mutation [13]. Elucidation of APA subtypes could, with preoperative genotyping, permit recognition of patients in whom recent onset of primary aldosteronism is likely to be completely resolved by surgery. Funding NIHR Cambridge Biomedical Research Centre (Cardiovascular and Metabolic); Wellcome Trust; Agency for Science, Technology and Research (A*STAR) Singapore; British Heart Foundation; Cambridge Overseas Trust Scholarship; NIHR Senior Investigator; Medical Research Council; Tunku Abdul Rahman Centenary Fund (Cambridge); Austin Doyle Award (Servier Australia). Contributors A.E.D.T., E.A.B.A. and M.J.B. designed the study, with the help of M.B. A.E.D.T. performed functional analyses of the mutant gene and ex-vivo studies of the adenomas (including qPCR, immunohistochemistry, and immunofluorescence), with the help of S.G. S.G. carried out sequencing and provided the constructs for transfection. L.H.S. performed the TCF-LEF assay. M.J.B., E.A.B.A., and J.Z. designed the microarray. E.A.B.A. carried out exome sequencing. F.E.K.F. and M.G. provided clinical data and were involved in patient care. L.H. and A.M. conducted the histological analyses and facilitated tissue collection. A.E.D.T. and M.J.B. analysed and interpreted the data. All authors approved the final submitted version. Disclosure of interest The authors declare that they have no competing interest.

Original languageEnglish
Pages (from-to)161-162
Number of pages2
JournalAnnales d'Endocrinologie
Volume77
Issue number2
DOIs
Publication statusPublished - 1 Jun 2016

Fingerprint

Menopause
Adenoma
Cell Differentiation
Zona Glomerulosa
Pregnancy
Mutation
Aldosterone
Catenins
Hyperaldosteronism
Adrenalectomy
Hypertension
Exome
Transfection
Immunohistochemistry
Genes
Biomedical Research
Genotype
Singapore
Disclosure
Gonads

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Teo, A. E. D., Garg, S., Haris Shaikh, L., Zhou, J., Karet Frankl, F. E., Gurnell, M., ... Brown, M. J. (2016). Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause. Annales d'Endocrinologie, 77(2), 161-162. https://doi.org/10.1016/j.ando.2016.04.022

Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause. / Teo, A. E D; Garg, S.; Haris Shaikh, L.; Zhou, J.; Karet Frankl, F. E.; Gurnell, M.; Happerfield, L.; Marker, A.; Bienz, M.; Elena Aisha, Azizan; Brown, M. J.

In: Annales d'Endocrinologie, Vol. 77, No. 2, 01.06.2016, p. 161-162.

Research output: Contribution to journalArticle

Teo, AED, Garg, S, Haris Shaikh, L, Zhou, J, Karet Frankl, FE, Gurnell, M, Happerfield, L, Marker, A, Bienz, M, Elena Aisha, A & Brown, MJ 2016, 'Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause', Annales d'Endocrinologie, vol. 77, no. 2, pp. 161-162. https://doi.org/10.1016/j.ando.2016.04.022
Teo, A. E D ; Garg, S. ; Haris Shaikh, L. ; Zhou, J. ; Karet Frankl, F. E. ; Gurnell, M. ; Happerfield, L. ; Marker, A. ; Bienz, M. ; Elena Aisha, Azizan ; Brown, M. J. / Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause. In: Annales d'Endocrinologie. 2016 ; Vol. 77, No. 2. pp. 161-162.
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abstract = "Background: Primary aldosteronism is a curable cause of hypertension if the hypertension is due to an aldosterone-producing adenoma (APA) [1,2]. The discovery of somatic mutations defining pathogenetic subtypes of this adenoma has increased recognition of common but small zona glomerulosa-like APAs [3-6]. In this subtype, we have sought APAs with distinctive presentation or outcome, in which analysis of genotype-phenotype associations could help explain the low success rate and variability of adrenalectomy for curing hypertension. Methods: Ten zona glomerulosa-like APAs from three women with primary aldosteronism were analysed by exome sequencing and microarray analysis. Nine had a mutation of ATP1A1 or CACNA1D, and one had a distinctive genotype and transcriptome. Targeted sequencing of further zona glomerulosa-like APAs led to the finding of two with a similar genotype, and to the recognition that all three women presented in early pregnancy or menopause. Quantitative PCR and immunohistochemistry were performed for genes upregulated in the index case. Similar analyses and a TCF-LEF assay for Wnt signalling were performed on cells transfected with the mutant gene. Findings: All three APAs had novel mutations in exon 3 of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway. The mutations prevented phosphorylation of β-catenin, resulting in an increase in total form of β-catenin and active to total ratio, as quantified by immunoblotting. Transfection of mutant constructs into HEK cells caused near doubling of Wnt signalling in a luciferase reporter assay. Microarray of the index case found a greater than 100-fold increase in expression of gonadal receptors LHCGR and GNRHR, confirmed by qPCR (both genes) and immunohistochemistry (LHCGR) in all three APAs. Increased GATA4 expression was further evidence of adrenocortical cell de-differentiation towards their common gonad-adrenal precursor cell-type. Transfection of LHCGR-negative APA cells with GFP-mutant-CTNNB1 switched on LHCGR expression in GFP-positive cells. Interpretation: The role of the Wnt system in adrenal physiology and tumour development is well recognised [7,8]. De-differentiation of zona glomerulosa-cells after Wnt activation by the CTNNB1 mutation seems to cause aberrant gonadal receptor expression, which is also previously described [9,10]. Our findings connect these observations, and explain the unmasking of small APAs by increases in luteinising hormone and human chorionic gonadotropin hormone in early pregnancy or menopause. Strikingly, all three women, including the treatment-resistant older woman, were clinically cured by adrenalectomy. This contrasts with the failure of adrenalectomy to completely cure hypertension in the majority of patients. This failure is attributable to decades of exposure to aldosterone excess, and the secondary consequences of fibrosis and remodelling in the cardiovascular system [11,12]. CTNNB1 mutations have now been reported in 10{\%} of APAs without a previously reported mutation [13]. Elucidation of APA subtypes could, with preoperative genotyping, permit recognition of patients in whom recent onset of primary aldosteronism is likely to be completely resolved by surgery. Funding NIHR Cambridge Biomedical Research Centre (Cardiovascular and Metabolic); Wellcome Trust; Agency for Science, Technology and Research (A*STAR) Singapore; British Heart Foundation; Cambridge Overseas Trust Scholarship; NIHR Senior Investigator; Medical Research Council; Tunku Abdul Rahman Centenary Fund (Cambridge); Austin Doyle Award (Servier Australia). Contributors A.E.D.T., E.A.B.A. and M.J.B. designed the study, with the help of M.B. A.E.D.T. performed functional analyses of the mutant gene and ex-vivo studies of the adenomas (including qPCR, immunohistochemistry, and immunofluorescence), with the help of S.G. S.G. carried out sequencing and provided the constructs for transfection. L.H.S. performed the TCF-LEF assay. M.J.B., E.A.B.A., and J.Z. designed the microarray. E.A.B.A. carried out exome sequencing. F.E.K.F. and M.G. provided clinical data and were involved in patient care. L.H. and A.M. conducted the histological analyses and facilitated tissue collection. A.E.D.T. and M.J.B. analysed and interpreted the data. All authors approved the final submitted version. Disclosure of interest The authors declare that they have no competing interest.",
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T1 - Effect of somatic CTNNB1 mutations on adrenocortical cell de-differentiation in adenoma presenting in early pregnancy or menopause

AU - Teo, A. E D

AU - Garg, S.

AU - Haris Shaikh, L.

AU - Zhou, J.

AU - Karet Frankl, F. E.

AU - Gurnell, M.

AU - Happerfield, L.

AU - Marker, A.

AU - Bienz, M.

AU - Elena Aisha, Azizan

AU - Brown, M. J.

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N2 - Background: Primary aldosteronism is a curable cause of hypertension if the hypertension is due to an aldosterone-producing adenoma (APA) [1,2]. The discovery of somatic mutations defining pathogenetic subtypes of this adenoma has increased recognition of common but small zona glomerulosa-like APAs [3-6]. In this subtype, we have sought APAs with distinctive presentation or outcome, in which analysis of genotype-phenotype associations could help explain the low success rate and variability of adrenalectomy for curing hypertension. Methods: Ten zona glomerulosa-like APAs from three women with primary aldosteronism were analysed by exome sequencing and microarray analysis. Nine had a mutation of ATP1A1 or CACNA1D, and one had a distinctive genotype and transcriptome. Targeted sequencing of further zona glomerulosa-like APAs led to the finding of two with a similar genotype, and to the recognition that all three women presented in early pregnancy or menopause. Quantitative PCR and immunohistochemistry were performed for genes upregulated in the index case. Similar analyses and a TCF-LEF assay for Wnt signalling were performed on cells transfected with the mutant gene. Findings: All three APAs had novel mutations in exon 3 of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway. The mutations prevented phosphorylation of β-catenin, resulting in an increase in total form of β-catenin and active to total ratio, as quantified by immunoblotting. Transfection of mutant constructs into HEK cells caused near doubling of Wnt signalling in a luciferase reporter assay. Microarray of the index case found a greater than 100-fold increase in expression of gonadal receptors LHCGR and GNRHR, confirmed by qPCR (both genes) and immunohistochemistry (LHCGR) in all three APAs. Increased GATA4 expression was further evidence of adrenocortical cell de-differentiation towards their common gonad-adrenal precursor cell-type. Transfection of LHCGR-negative APA cells with GFP-mutant-CTNNB1 switched on LHCGR expression in GFP-positive cells. Interpretation: The role of the Wnt system in adrenal physiology and tumour development is well recognised [7,8]. De-differentiation of zona glomerulosa-cells after Wnt activation by the CTNNB1 mutation seems to cause aberrant gonadal receptor expression, which is also previously described [9,10]. Our findings connect these observations, and explain the unmasking of small APAs by increases in luteinising hormone and human chorionic gonadotropin hormone in early pregnancy or menopause. Strikingly, all three women, including the treatment-resistant older woman, were clinically cured by adrenalectomy. This contrasts with the failure of adrenalectomy to completely cure hypertension in the majority of patients. This failure is attributable to decades of exposure to aldosterone excess, and the secondary consequences of fibrosis and remodelling in the cardiovascular system [11,12]. CTNNB1 mutations have now been reported in 10% of APAs without a previously reported mutation [13]. Elucidation of APA subtypes could, with preoperative genotyping, permit recognition of patients in whom recent onset of primary aldosteronism is likely to be completely resolved by surgery. Funding NIHR Cambridge Biomedical Research Centre (Cardiovascular and Metabolic); Wellcome Trust; Agency for Science, Technology and Research (A*STAR) Singapore; British Heart Foundation; Cambridge Overseas Trust Scholarship; NIHR Senior Investigator; Medical Research Council; Tunku Abdul Rahman Centenary Fund (Cambridge); Austin Doyle Award (Servier Australia). Contributors A.E.D.T., E.A.B.A. and M.J.B. designed the study, with the help of M.B. A.E.D.T. performed functional analyses of the mutant gene and ex-vivo studies of the adenomas (including qPCR, immunohistochemistry, and immunofluorescence), with the help of S.G. S.G. carried out sequencing and provided the constructs for transfection. L.H.S. performed the TCF-LEF assay. M.J.B., E.A.B.A., and J.Z. designed the microarray. E.A.B.A. carried out exome sequencing. F.E.K.F. and M.G. provided clinical data and were involved in patient care. L.H. and A.M. conducted the histological analyses and facilitated tissue collection. A.E.D.T. and M.J.B. analysed and interpreted the data. All authors approved the final submitted version. Disclosure of interest The authors declare that they have no competing interest.

AB - Background: Primary aldosteronism is a curable cause of hypertension if the hypertension is due to an aldosterone-producing adenoma (APA) [1,2]. The discovery of somatic mutations defining pathogenetic subtypes of this adenoma has increased recognition of common but small zona glomerulosa-like APAs [3-6]. In this subtype, we have sought APAs with distinctive presentation or outcome, in which analysis of genotype-phenotype associations could help explain the low success rate and variability of adrenalectomy for curing hypertension. Methods: Ten zona glomerulosa-like APAs from three women with primary aldosteronism were analysed by exome sequencing and microarray analysis. Nine had a mutation of ATP1A1 or CACNA1D, and one had a distinctive genotype and transcriptome. Targeted sequencing of further zona glomerulosa-like APAs led to the finding of two with a similar genotype, and to the recognition that all three women presented in early pregnancy or menopause. Quantitative PCR and immunohistochemistry were performed for genes upregulated in the index case. Similar analyses and a TCF-LEF assay for Wnt signalling were performed on cells transfected with the mutant gene. Findings: All three APAs had novel mutations in exon 3 of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway. The mutations prevented phosphorylation of β-catenin, resulting in an increase in total form of β-catenin and active to total ratio, as quantified by immunoblotting. Transfection of mutant constructs into HEK cells caused near doubling of Wnt signalling in a luciferase reporter assay. Microarray of the index case found a greater than 100-fold increase in expression of gonadal receptors LHCGR and GNRHR, confirmed by qPCR (both genes) and immunohistochemistry (LHCGR) in all three APAs. Increased GATA4 expression was further evidence of adrenocortical cell de-differentiation towards their common gonad-adrenal precursor cell-type. Transfection of LHCGR-negative APA cells with GFP-mutant-CTNNB1 switched on LHCGR expression in GFP-positive cells. Interpretation: The role of the Wnt system in adrenal physiology and tumour development is well recognised [7,8]. De-differentiation of zona glomerulosa-cells after Wnt activation by the CTNNB1 mutation seems to cause aberrant gonadal receptor expression, which is also previously described [9,10]. Our findings connect these observations, and explain the unmasking of small APAs by increases in luteinising hormone and human chorionic gonadotropin hormone in early pregnancy or menopause. Strikingly, all three women, including the treatment-resistant older woman, were clinically cured by adrenalectomy. This contrasts with the failure of adrenalectomy to completely cure hypertension in the majority of patients. This failure is attributable to decades of exposure to aldosterone excess, and the secondary consequences of fibrosis and remodelling in the cardiovascular system [11,12]. CTNNB1 mutations have now been reported in 10% of APAs without a previously reported mutation [13]. Elucidation of APA subtypes could, with preoperative genotyping, permit recognition of patients in whom recent onset of primary aldosteronism is likely to be completely resolved by surgery. Funding NIHR Cambridge Biomedical Research Centre (Cardiovascular and Metabolic); Wellcome Trust; Agency for Science, Technology and Research (A*STAR) Singapore; British Heart Foundation; Cambridge Overseas Trust Scholarship; NIHR Senior Investigator; Medical Research Council; Tunku Abdul Rahman Centenary Fund (Cambridge); Austin Doyle Award (Servier Australia). Contributors A.E.D.T., E.A.B.A. and M.J.B. designed the study, with the help of M.B. A.E.D.T. performed functional analyses of the mutant gene and ex-vivo studies of the adenomas (including qPCR, immunohistochemistry, and immunofluorescence), with the help of S.G. S.G. carried out sequencing and provided the constructs for transfection. L.H.S. performed the TCF-LEF assay. M.J.B., E.A.B.A., and J.Z. designed the microarray. E.A.B.A. carried out exome sequencing. F.E.K.F. and M.G. provided clinical data and were involved in patient care. L.H. and A.M. conducted the histological analyses and facilitated tissue collection. A.E.D.T. and M.J.B. analysed and interpreted the data. All authors approved the final submitted version. Disclosure of interest The authors declare that they have no competing interest.

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