Foussier Loïc, Vitellius Géraldine, Bouligand Jérôme, Amazit Larbi, Bouvattier Claire, Young Jacques, Trabado Séverine, Lombès Marc
INSERM UMR_S U1185, Fac Med Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, France.
Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpitaux Universitaires Paris Sud, AH-HP, CHU Bicêtre, France.
J Endocr Soc. 2019 Mar 6;3(5):865-881. doi: 10.1210/js.2019-00028. eCollection 2019 May 1.
Recent advances in genetic analysis technologies such as next-generation sequencing (NGS) have considerably increased the incidental discovery of genetic abnormalities. Six heterozygous missense mutations of the human glucocorticoid receptor (GR; encoded by the gene) have been identified in the context of genetic screening of endocrine pathologies. GR, a nuclear receptor, hormone-induced transcription factor, is involved in many physiological processes. Nevertheless, the pathogenic significance of incidentally discovered mutations remains obscure. The aim of this work was to characterize these variants by evaluating their functional impact on GR signaling. Six original GR variants, located in exon 2, led to amino acid substitutions of the -terminal domain of GR (F65V, M86V, A229T, A304E, N374S, and R386Q), excluding mainly the activation function tau core 1 domain, the potential site of functional interaction with transcriptional coregulators. Transient cotransfection in HEK293T cells of mutated GR-expressing vectors and a luciferase reporter established dose-response curves for dexamethasone. This excluded any major transactivation abnormality of the mutated GRs (ligand concentration leading to 50% maximal transactivation capacity ≈ 0.2 nM), with maximal transactivation capacity identical to that of the wild-type (WT) GR and without modification of the potentiation of transcriptional coactivator steroid receptor coactivator 2 except in N374S. Moreover, protein expression of mutated GRs and their cytonuclear translocation studied by immunocytochemistry were almost unchanged compared with WT GR. These results underline the silent nature of these missense GR variants and call for cautious interpretation of the discovery of genetic incidentalomas by NGS in the absence of detailed characterization in order to appropriately assess their functional impact on a particular signaling pathway.
诸如新一代测序（NGS）等遗传分析技术的最新进展显著增加了基因异常的偶然发现。在内分泌疾病的基因筛查中，已鉴定出人类糖皮质激素受体（GR；由 基因编码）的六个杂合错义突变。GR是一种核受体、激素诱导的转录因子，参与许多生理过程。然而，偶然发现的突变的致病意义仍不清楚。这项工作的目的是通过评估它们对GR信号传导的功能影响来表征这些变体。位于外显子2的六个原始GR变体导致GR的 -末端结构域发生氨基酸取代（F65V、M86V、A229T、A304E、N374S和R386Q），主要排除了激活功能tau核心1结构域，这是与转录共调节因子进行功能相互作用的潜在位点。在HEK293T细胞中瞬时共转染表达突变GR的载体和荧光素酶报告基因，建立了地塞米松的剂量反应曲线。这排除了突变GR的任何主要反式激活异常（导致50%最大反式激活能力的配体浓度≈0.2 nM），最大反式激活能力与野生型（WT）GR相同，并且除了N374S外，转录共激活因子类固醇受体共激活因子2的增强作用没有改变。此外，通过免疫细胞化学研究的突变GR的蛋白质表达及其细胞核转位与WT GR相比几乎没有变化。这些结果强调了这些错义GR变体的沉默性质，并呼吁在没有详细表征的情况下，对NGS发现的基因偶发瘤进行谨慎解释，以便适当地评估它们对特定信号通路的功能影响。
