ESPE Abstracts

Background: Growth hormone insensitivity (GHI) encompasses normal/elevated growth hormone (GH), low IGF-I levels and growth restriction. Non-classical/mild-moderate GHI is an emerging entity which is poorly characterised, and, in many subjects, the underlying cause is unclear. Heterozygous dominant negative (DN) variants located in the intracellular/transmembrane domain of the GH receptor (GHR) cause a ‘non-classical’ GHI phenotype.

Hypothesis/Objective: Detailed characterisation of novel, naturally occurring GHR variants will improve our understanding of the physiology of human growth and lead to improved diagnosis/enhanced patient care.

Methods: Two novel heterozygous GHR variants were identified in 2 GHI patients by our short stature whole genome panel. In vitro splicing assays were performed using an exon trap vector. Gibson assembly created GHR wild type (WT) and variant (MUT 1 & 2) constructs. Additional WT and MUT constructs with either NanoLuc® Large BiT (LgBiT; 17.6kDa) or Small BiT (SmBiT; 11 amino acids) subunits were generated. These constructs were transfected into HEK293T cells and western blotting (WB) was performed using anti-STAT5b, anti-pSTAT5, anti-GHBP and anti-NanoLuc® Luciferase antibodies (anti-beta-actin/GAPDH as controls). NanoBiT complementation assays allowed quantitative assessment of WT and MUT GHR homo/hetero dimerization.

Results: Heterozygous GHR variants (c.876-15T>G (MUT 1) and c.902T>G (MUT 2) in intron 8/exon 9, respectively) were identified in 2 GHI subjects. Segregation studies confirmed MUT 1 was maternally inherited (mother’s height SDS -2.4) and MUT 2 arose de-novo. In-vitro splicing assays confirmed both GHR variants activate the same alternative splice acceptor site resulting in abnormal splicing and exclusion of 26 base pairs of GHR exon 9. Wildtype (WT) and MUT constructs were co-transfected to mimic the heterozygous state of patients. WB analysis confirmed the production of truncated MUT variants and reduced GH-induced STAT5B phosphorylation. WB analysis of the supernatant of cells transfected with the NanoLuc®-GHR constructs, showed increased GH binding protein (GHBP) production in MUT: MUT and WT: MUT compared to WT: WT GHR. Novel NanoBiT complementation assays showed increased luminescence readings of MUT: MUT and WT: MUT GHR homo/heterodimers compared to WT: WT homodimers suggesting increased cell surface expression of MUT: MUT and WT: MUT GHR receptor dimers.

Conclusion: Heterozygous defects in the intracellular domain of GHR should be considered in cases with a non-classical GHI phenotype. Our novel truncated GHR variants exert a dominant negative effect with blunted GHR signalling. The creation of NanoLuc®-GHR constructs provide a novel, innovative methodology for characterising the functional role of GHR variants.