Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing’s syndrome
Anne-Lise Lecoq, Constantine A. Stratakis, Say Viengchareun, Ronan Chaligné, Lucie Tosca, Vianney Deméocq, Mirella Hage, Annabel Berthon, Fabio R. Faucz, Patrick Hanna, Hadrien-Gaël Boyer, Nicolas Servant, Sylvie Salenave, Gérard Tachdjian, Clovis Adam, Vanessa Benhamo, Eric Clauser, Anne Guiochon-Mantel, Jacques Young, Marc Lombès, Isabelle Bourdeau, Dominique Maiter, Antoine Tabarin, Jérôme Bertherat, Hervé Lefebvre, Wouter de Herder, Estelle Louiset, André Lacroix, Philippe Chanson, Jérôme Bouligand, Peter Kamenický
Anne-Lise Lecoq, Constantine A. Stratakis, Say Viengchareun, Ronan Chaligné, Lucie Tosca, Vianney Deméocq, Mirella Hage, Annabel Berthon, Fabio R. Faucz, Patrick Hanna, Hadrien-Gaël Boyer, Nicolas Servant, Sylvie Salenave, Gérard Tachdjian, Clovis Adam, Vanessa Benhamo, Eric Clauser, Anne Guiochon-Mantel, Jacques Young, Marc Lombès, Isabelle Bourdeau, Dominique Maiter, Antoine Tabarin, Jérôme Bertherat, Hervé Lefebvre, Wouter de Herder, Estelle Louiset, André Lacroix, Philippe Chanson, Jérôme Bouligand, Peter Kamenický
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Research Article Endocrinology

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing’s syndrome

Abstract

GIP-dependent Cushing’s syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing’s syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing’s syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Authors

Anne-Lise Lecoq, Constantine A. Stratakis, Say Viengchareun, Ronan Chaligné, Lucie Tosca, Vianney Deméocq, Mirella Hage, Annabel Berthon, Fabio R. Faucz, Patrick Hanna, Hadrien-Gaël Boyer, Nicolas Servant, Sylvie Salenave, Gérard Tachdjian, Clovis Adam, Vanessa Benhamo, Eric Clauser, Anne Guiochon-Mantel, Jacques Young, Marc Lombès, Isabelle Bourdeau, Dominique Maiter, Antoine Tabarin, Jérôme Bertherat, Hervé Lefebvre, Wouter de Herder, Estelle Louiset, André Lacroix, Philippe Chanson, Jérôme Bouligand, Peter Kamenický

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Figure 1

Adrenal glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in patients with GIP-dependent Cushing’s syndrome or aldosteronoma.

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Adrenal glucose-dependent insulinotropic polypeptide receptor (GIPR) exp...
(A) GIPR expression profile in adrenal tissue samples obtained on reverse transcription quantitative PCR (RT-qPCR) and normalized to the β-actin housekeeping gene. Similar results were obtained when GIPR expression was normalized to 18S. Four samples of normal adrenal tissue (NA1–NA4); non-tumorous adrenal tissue (PT) adjacent to adenoma of patient #1; 5 adrenocortical adenomas; and 10 bilateral adrenal hyperplasias from patients with aberrant adrenal sensitivity to GIP were studied. In one patient with bilateral adrenal hyperplasia (#7), samples from both adrenal lesions (R, right; L, left) were analyzed. Results are expressed as relative GIPR expression compared with mean expression level in the unaffected adrenal tissue samples, arbitrary set at 1, representing 1.60 × 10–4 ± 0.54 × 10–4 amol/amol of β-actin. The expression of GIPR in adrenal lesions of patients with GIP-dependent Cushing’s syndrome or aldosteronoma was increased (570 ± 159–fold, ranging from 23- to 2,152-fold) compared with the mean expression level in the unaffected adrenal tissue (P < 0.0001, Mann-Whitney U test). Experiments were performed twice; one representative experiment is shown here. Three independent determinations were done for each sample. The lines represent the median values; the whiskers represent the minimal and maximal values. (B) Representative examples of adrenal GIPR expression as assessed by RNA FISH in adrenal lesions with or without 19q13 duplication. Monoallelic (a maximum of one RNA FISH signal is visible per nucleus) GIPR expression in interphase nuclei is visible as one green pinpoint in adrenal lesions of patients with GIP-dependent Cushing’s syndrome or aldosteronoma. RNA FISH analysis was performed on all adrenal lesions with aberrant sensitivity to GIP, except for lesions of patient #11 and #12, due to the lack of good quality frozen tissue in these two samples. Normal adrenal tissue did not show any GIPR expression. (C) Percentage of nuclei with monoallelic GIPR expression determined by RNA FISH. At the bottom of the histogram, the number of analyzed nuclei is indicated. Scale bars: 5 μm.