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Endocrine Abstracts (2021) 78 OC6.1 | DOI: 10.1530/endoabs.78.OC6.1

Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University, London, United Kingdom


Background: Growth hormone insensitivity (GHI) is a continuum defined by normal/elevated growth hormone (GH), low IGF-I levels and growth restriction. Non-classical/mild-moderate GHI is poorly characterised and is frequently underdiagnosed. Heterozygous dominant negative (DN) gene variants located in the regions encoding the intracellular/transmembrane domains of the GH receptor cause a ‘non-classical’ GHI phenotype.

Hypothesis/Objective: Detailed characterisation of novel, naturally occurring dominant negative GHR gene variants will improve our understanding of the physiology of human growth and enhance patient care.

Methods: Two novel 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 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 as well as constructs with either NanoLuc® Large BiT (LgBiT) or Small BiT (SmBiT) subunits. 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: 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. Analysis of the conditioned cell media demonstrated increased GH binding protein (GHBP) production. 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 with MUT GHR receptor homo/heterodimers.

Conclusion: The novel truncated GHR variants exert a dominant negative effect with blunted GHR signalling. Increased cell surface expression and GHBP production may also contribute to reduced function of these GHR variants. The creation of NanoLuc®-GHR constructs provide an innovative methodology for characterising the functional role of GHR variants in GH binding and GHBP physiology.

Volume 78

48th Meeting of the British Society for Paediatric Endocrinology and Diabetes

Online, Virtual
24 Nov 2021 - 26 Nov 2021

British Society for Paediatric Endocrinology and Diabetes 

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