Andrews Afiya, Cottrell Emily, Maharaj Avinaash, Ladha Tasneem, Williams Jack, Schilbach Katharina, Kaisinger Lena R, Perry John R B, Metherell Louise A, McCormick Peter J, Storr Helen L
Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University London, London, United Kingdom.
Endocrine Laboratory, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany.
Eur J Endocrinol. 2023 Apr 4;188(4):353-365. doi: 10.1093/ejendo/lvad039.
Growth hormone insensitivity (GHI) encompasses growth restriction, normal/elevated growth hormone (GH), and low insulin-like growth factor I (IGF1). "Nonclassical" GHI is poorly characterized and is rarely caused by heterozygous dominant-negative (DN) variants located in the intracellular or transmembrane domains of the GH receptor (GHR). We sought to determine the molecular mechanisms underpinning the growth restriction in 2 GHI cases.
A custom-made genetic investigative pipeline was exploited to identify the genetic cause of growth restriction in patients with GHI. Nanoluc binary technology (NanoBiT), in vitro splicing assays, western blotting, and flow cytometry, characterized the novel GHR variants.
Novel heterozygous GHR variants were identified in 2 unrelated patients with GHI. In vitro splicing assays indicated both variants activated the same alternative splice acceptor site resulting in aberrant splicing and exclusion of 26 base pairs of GHR exon 9. The GHR variants produced truncated receptors and impaired GH-induced GHR signaling. NanoBiT complementation and flow cytometry showed increased cell surface expression of variant GHR homo/heterodimers compared to wild-type (WT) homodimers and increased recombinant human GH binding to variant GHR homo/heterodimers and GH binding protein (GHBP) cleaved from the variant GHRs. The findings demonstrated increased variant GHR dimers and GHBP with resultant GH sequestration.
We identified and characterized 2 novel, naturally occurring truncated GHR gene variants. Intriguingly, these DN GHR variants act via the same cryptic splice acceptor site, highlighting impairing GH binding to excess GHBP as a potential therapeutic approach.
生长激素不敏感（GHI）包括生长受限、正常/升高的生长激素（GH）以及低胰岛素样生长因子I（IGF1）。“非经典”GHI的特征尚不明确，很少由位于生长激素受体（GHR）细胞内或跨膜结构域的杂合显性负性（DN）变体引起。我们试图确定2例GHI患者生长受限的分子机制。
利用定制的基因研究流程来确定GHI患者生长受限的遗传原因。采用纳米荧光素酶二元技术（NanoBiT）、体外剪接试验、蛋白质印迹法和流式细胞术对新型GHR变体进行表征。
在2例无关的GHI患者中鉴定出新型杂合GHR变体。体外剪接试验表明，这两种变体均激活了相同的可变剪接受体位点，导致异常剪接并排除了GHR外显子9的26个碱基对。GHR变体产生截短的受体，并损害GH诱导的GHR信号传导。NanoBiT互补和流式细胞术显示，与野生型（WT）同二聚体相比，变体GHR同/异二聚体的细胞表面表达增加，重组人生长激素与变体GHR同/异二聚体和从变体GHR切割而来的生长激素结合蛋白（GHBP）的结合增加。这些发现表明变体GHR二聚体和GHBP增加，导致GH被隔离。
我们鉴定并表征了2种新型的、天然存在的截短型GHR基因变体。有趣的是，这些DN GHR变体通过相同的隐蔽剪接受体位点起作用，突出了损害GH与过量GHBP的结合作为一种潜在的治疗方法。
