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.

中文翻译：

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GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征中 KDM1A 缺失：一项多中心、回顾性、队列研究

背景

GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征是由肾上腺病变中 GIP 受体的异常表达引起的。这种疾病的双侧性质表明生殖系遗传易感性。我们旨在确定导致 GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征的遗传驱动事件。

方法

我们在法国、加拿大、意大利、希腊、比利时和荷兰的内分泌医院和大学医院进行了一项多中心、回顾性、队列研究。我们收集了因 GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征而接受单侧或双侧肾上腺切除术的患者的血液和肾上腺样本。来自患有原发性双侧大结节性肾上腺增生的患者的肾上腺样本被用作对照组，这些患者因明显或轻度的库欣综合征而接受了肾上腺切除术，但没有食物依赖性皮质醇产生的证据，以及患有 GIP 依赖​​性单侧肾上腺皮质腺瘤的患者的肾上腺样本。我们进行了全基因组、全外显子组和靶向下一代测序，对家族性或散发性疾病患者的血液和肾上腺 DNA 进行拷贝数分析。我们对肾上腺样本进行了 RNA 测序，并对人类肾上腺皮质细胞系 H295R 中已鉴定的遗传缺陷进行了功能分析。

发现

对 17 例 GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征患者进行了研究。患者的中位年龄为 43·3 (95% CI 38·8–47·8) 岁，大多数患者 (15 [88%]) 为女性。我们在所有 17 名患者中鉴定了KDM1A基因中的种系杂合致病性或最可能的致病性变异。我们还在这些患者的肾上腺病变中发现了含有KDM1A基因座的 1 号染色体短 p 臂的复发性缺失。对照组中的 29 名患者均未出现KDM1A种系或体细胞改变。两个KDM1A等位基因的伴随遗传失活导致KDM1A丢失肾上腺病变中的表达。全局基因表达分析显示 GIP 受体上调，log2 倍变化为 7·99（95% CI 7·34–8·66；p=4·4 × 10 ^-125），以及其他几种 G 蛋白偶联物的差异调节与对照样品相比，GIP 依赖​​性原发性双侧大结节增生样品中的受体。通过 CRISPR-Cas9 基因组编辑对 KDM1A 的体外药理抑制和失活导致人肾上腺皮质 H295R 细胞中 GIP 受体转录物和蛋白质的增加。

解释

我们提出 GIP 依赖​​性原发性双侧大结节性肾上腺增生伴库欣综合征是由KDM1A的两次打击失活引起的，这与肿瘤发生的抑癌基因模型一致。应向这些患者及其亲属提供基因检测和咨询。

资金

Agence Nationale de la Recherche、Fondation du Grand défi Pierre Lavoie 和法国国家癌症研究所。