Background: Response to GH treatment has been associated with single nucleotide polymorphisms (SNPs) within the promoter region of growth-related genes including GRB10 (rs1024531 (A/G, allele A increased response)), IGFBP3 (rs3110697 (G/A, G increased response)), CYP19A1 (rs10459592 (T/C, T increased response)) and SOS1 (rs2888586 (G/C, C increased response)). SOS1 is a positive regulator of GH signalling (MAPK pathway); the aromatase CYP19A1 promotes oestrogen synthesis to impact on GH secretion and growth, while IGFBP-3 is the main carrier of IGF1. Conversely, GRB10 has a negative impact on growth, inhibiting GH and IGF1 signalling.
Objectives: To test the impact of promoter SNPs in IGFBP3, GRB10, CYP19A1 and SOS1 on transcriptional activity (TA) in an in vitro cell system.
Methods: Each allele within a 500 bp fragment of the promoter sequence was cloned into a secreted alkaline phosphatase (ALP) reporter gene plasmid (pSEAP, Clontech) and transfected into human MCF-7 cells, known to be GH responsive. TA of each construct was evaluated by ALP induction after 24 h of GH stimulation with a dose-dependent titration (range: 0, 2, 20 and 200 ng/ml).
Results: When the cells were stimulated with GH, the alleles associated clinically with better GH response had higher TA at all GH concentrations for IGFBP3 (allele G), CYP19A1 (allele T) and SOS1 (allele C) (all P<0.05). Conversely for GRB10, the allele associated with better GH response (allele A) had a significantly lower TA at all GH stimulations (P<0.05).
Conclusions: We have shown that SNPs associated with response to GH therapy can alter TA after GH stimulation. The differential TA of the SNPs after GH stimulation relates directly to the effect of the gene on growth promotion or inhibition. The observation of induction over a range of GH concentrations supports the in vivo relevance of these findings.
Funding: This work was supported by ESPE Research Fellowship, sponsored by Novo Nordisk A/S.
01 - 03 Oct 2015
European Society for Paediatric Endocrinology