Loss of KDM1A in GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome: a multicentre, retrospective, cohort study

Summary

Background

GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome is caused by aberrant expression of the GIP receptor in adrenal lesions. The bilateral nature of this disease suggests germline genetic predisposition. We aimed to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome.

Methods

We conducted a multicentre, retrospective, cohort study at endocrine hospitals and university hospitals in France, Canada, Italy, Greece, Belgium, and the Netherlands. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Adrenal samples from patients with primary bilateral macronodular adrenal hyperplasia who had undergone an adrenalectomy for overt or mild Cushing's syndrome without evidence of food-dependent cortisol production and those with GIP-dependent unilateral adrenocortical adenomas were used as control groups. We performed whole genome, whole exome, and targeted next generation sequencing, and copy number analyses of blood and adrenal DNA from patients with familial or sporadic disease. We performed RNA sequencing on adrenal samples and functional analyses of the identified genetic defect in the human adrenocortical cell line H295R.

Findings

17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome were studied. The median age of patients was 43·3 (95% CI 38·8–47·8) years and most patients (15 [88%]) were women. We identified germline heterozygous pathogenic or most likely pathogenic variants in the KDM1A gene in all 17 patients. We also identified a recurrent deletion in the short p arm of chromosome 1 harboring the KDM1A locus in adrenal lesions of these patients. None of the 29 patients in the control groups had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in adrenal lesions. Global gene expression analysis showed GIP receptor upregulation with a log2 fold change of 7·99 (95% CI 7·34–8·66; p=4·4 × 10⁻¹²⁵), and differential regulation of several other G protein-coupled receptors in GIP-dependent primary bilateral macronodular hyperplasia samples compared with control samples. In vitro pharmacological inhibition and inactivation of KDM1A by CRISPR-Cas9 genome editing resulted in an increase of GIP receptor transcripts and protein in human adrenocortical H295R cells.

Interpretation

We propose that GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome results from a two-hit inactivation of KDM1A, consistent with the tumour suppressor gene model of tumorigenesis. Genetic testing and counselling should be offered to these patients and their relatives.

Funding

Agence Nationale de la Recherche, Fondation du Grand défi Pierre Lavoie, and the French National Cancer Institute.

Introduction

Primary bilateral macronodular adrenal hyperplasia is a rare cause of pituitary adrenocorticotropic hormone-independent cortisol excess with Cushing's syndrome.¹ Germline and secondary somatic inactivating mutations in the ARMC5 gene are the most frequent underlying genetic cause in around a third of patients with primary bilateral macronodular adrenal hyperplasia.2, 3

In a large proportion of patients with primary bilateral macronodular adrenal hyperplasia and less frequently in those with unilateral adenoma, cortisol excess is also driven by aberrant (ectopic or excessive) expression of several G protein-coupled receptors in adrenal lesions.⁴ Ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor in primary bilateral macronodular adrenal hyperplasia is associated with abnormal circadian cortisol rhythm with low fasting morning plasma cortisol concentrations, which increase after food intake.5, 6 The postprandial rise of cortisol secretion is induced by GIP, an incretin produced by intestinal K cells following oral lipid, carbohydrate, or protein intake. Activation of the ectopic GIP receptor, functionally coupled to cAMP signalling, triggers adrenal cell proliferation and excessive steroid production. The molecular events leading to ectopic GIP receptor expression in the adrenocortical tissue are not well understood. In 2017, we reported somatic 19q13·32 microduplications containing the GIP receptor locus rearranged with other chromosomal regions in cortisol-secreting adenomas from two patients with GIP-dependent Cushing's syndrome.⁷ However, the molecular pathogenesis of ectopic GIP receptor expression in patients with primary bilateral macronodular adrenal hyperplasia remains unknown.

Research in context

Evidence before this study

No systematic literature search was done. Relevant articles were cited on the basis of the authors' knowledge of the scientific literature available on PubMed from database inception to June 26, 2021. Since the initial description of sporadic cases of glucose-dependent insulinotropic polypeptide (GIP)-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome nearly 30 years ago, the underlying mechanism leading to ectopic or aberrant GIP receptor expression in the adrenal tissue remained unknown. Several other aberrant G-protein coupled receptors and ligands, such as corticotropin, were found to regulate development and steroidogenesis in primary bilateral macronodular adrenal hyperplasia. The bilateral character of the disease suggested a genetic predisposition, and germline and biallelic pathogenic variants of ARMC5 were identified in approximately 25% of patients with primary bilateral macronodular adrenal hyperplasia. However, no cases of GIP-dependent primary bilateral macronodular adrenal hyperplasia were found to harbour ARMC5 mutations. By contrast, in two patients with GIP-dependent unilateral cortisol-secreting adenoma, GIP receptor overexpression resulted from somatic 19q13·32 microduplications containing the GIP receptor locus, rearranged with other chromosomal regions.

Added value of this study

In this multicentre, retrospective, cohort study, we performed sequencing of germline and adrenal DNA derived from 17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. We found that familial and sporadic GIP-dependent primary bilateral macronodular adrenal hyperplasia is a genetic disease caused by germline inactivating pathogenic variants of the lysine demethylase 1A (KDM1A) with loss of heterozygosity of the second KDM1A locus in adrenal lesions. This stepwise inactivation of KDM1A is suggestive of a tumour suppressor gene model of tumourigenesis. RNA sequencing revealed the global effect of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes, including those encoding for GIP receptor and several other G protein-coupled receptors that might be involved in adrenal tumourigenesis and complex regulation of steroidogenesis. Functional in vitro studies in human adrenocortical and pancreatic β cells further showed the link between KDM1A and ectopic and physiological GIP receptor expression.

Implications of all the available evidence

Uncovering a common genetic mechanism of GIP-dependent primary bilateral macronodular adrenal hyperplasia represents a substantial advancement in the field of adrenal Cushing's syndrome. This finding will enable genetic testing and counselling of patients and earlier detection of the disease, which is important because KDM1A pathogenic variants predispose to myelomas or monoclonal gammopathy of undetermined significance. Further, this novel role of KDM1A as an epigenetic regulator of GIP receptor expression and that of several other G protein-coupled receptors can have pharmacological implications. Targeting KDM1A by inhibitors could possibly be applied beyond the field of adrenal hyperplasia—eg, in the field of endocrine and metabolic diseases—and warrants further investigation.

The bilateral nature of this primary adrenal disease suggests germline genetic predisposition; however, no familial forms of GIP-dependent Cushing's syndrome have been reported to date. The aim of this study was to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia and Cushing's syndrome.