ESPE Abstracts (2014) 82 FC7.2

The rs1024531 GRB10 Promoter Polymorphism is Associated with Response to GH Therapy in Patients with GH Deficiency: Validation by in vitro Functional Analysis

Chiara De Leonibus, Daniel Hanson, Philip Murray, Adam Stevens & Peter Clayton

Manchester Academic Health Sciences Centre, Royal Manchester Children’s Hospital, Manchester, UK

Background: GH response is influenced by genetic polymorphisms, including the rs1024531 polymorphism (A/G) in the promoter region of GRB10, a negative regulator of signaling through the IGF1 receptor. Allele A is associated with borderline lower baseline IGF1 SDS and 1.5-fold higher response to GH compared to allele G in children with GHD (P=0.0006).

Objective: To test functional impact of the rs1024531 polymorphism in an in vitro cell system.

Methods: Each allele, in a 500-bp fragment of GRB10 promoter sequence, was cloned into a secreted alkaline phosphatase (ALP) reporter gene plasmid (pSEAP). The transcriptional activity (TA) of each construct was evaluated by ALP induction [relative light units (RLU)]. Transfection experiments were performed at baseline using the human HEK293 cell line. GH-stimulation was performed in human MCF7 cells known to be GH responsive,2 with maximal dose (200 ng/ml). A GH dose-dependent titration (24 h) was also performed (range: 0, 2, 20, and 200 ng/ml).

Results: At baseline, allele A was associated with greater TA than allele G (0.4 vs 1.4 RLU, P=0.003). Conversely, when GH stimulation was performed, allele G was associated with a relative 4.8-fold ALP induction compared to a 3.1-fold increase for allele A (P<0.001). When the cells were exposed to various GH concentrations, allele G induced significantly higher TA of the reporter construct than allele A at all levels of stimulation (P<0.05). A GH-induced suppression was found for allele A at lower concentrations [2 and 20 ng/ml vs baseline (both P<0.05)], a range comparable to in vivo stimulation.

Conclusion: These cell models provide a platform to test the functional mechanisms that underlie clinical observations for GRB10, a negative growth regulator. Allele A is associated with higher TA at baseline, consistent with lower IGF1 values, but suppressed TA after GH stimulation, consistent with better growth on GH in GHD children.

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