ESPE Abstracts (2014) 82 FC7.2

ESPE2014 Free Communications Growth promoting therapies (6 abstracts)

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.

Article tools

My recent searches

No recent searches.