LAUSR

OBJECTIVE Growth hormone insensitivity (GHI) encompasses growth restriction, normal/elevated growth hormone (GH), and low insulin-like growth factor I (IGF1). 'Non-classical' GHI is poorly characterised 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 two GHI cases. METHODS AND DESIGN 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 characterised the novel GHR variants. RESULTS Novel heterozygous GHR variants were identified in two 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 signalling. NanoBiT complementation and flow cytometry showed increased cell surface expression of variant GHR homo/heterodimers compared to wild-type (WT) homodimers and increased rhGH 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. CONCLUSION We identified and characterised two 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.