IMPE Abstracts

Insulin-like growth factor 1 (IGF1) plays key roles in promoting growth, during both pre- and post-natal life and regulating neurologic development in mammals. IGF1 gene mutations are extremely rare causes of pre- and post-natal growth retardation. Phenotype can be heterogenous with varying degrees of neurosensory deafness, cognitive defects, glucose metabolism impairment and short stature. We have previously described two patients with novel homozygous IGF1missense variants (c.247A>T, pSer83Cys-IGF1 and c.322T>C, p.Tyr108His-IGF1) presenting a wide variability in the clinical presentation and biochemical profiles. In silico modelling predicts that variant pSer83Cys-IGF1 could interfere with IR signalling.

Aim: To evaluate in vitro the effect of the IGF1 variants in bioactivity, proliferation and cellular differentiation and matrix deposition compared to WT-IGF1.

Methods: Site-directed mutagenesis was performed on the RG 21 2527 vector containing the full length IGF1 protein coding sequence as a fusion protein with GFP. Specific primers were designed to perform the punctual change of A>T at position 247 (c.247A>T, p.Ser83Cys) or of T>C at position 322 (c.322T>C, p.Tyr108His). HEK293 and SaOS-2 cells were transfected with WT-IGF1-GFP, pS83C-IGF1-GFP or pT108H-IGF1-GFP and stable clones selected. HEK293 cells were used for phosphorylation, viability and apoptosis assays. SaOS-2 cells were used for osteocytes differentiation and bone matrix deposition assays. Gene expression was quantified by rqPCR and IGF1R phosphorylation was assessed by western blotting (WB).

Results: HEK293 cells expressing similar amounts of p.Ser83Cys-IGF1 and p.Tyr108His-IGF1 showed a significant decrease in IGF1R autocrine phosphorylation compared to WT-IGF1 expressing cells. Also, cells carriyng the variants had a pronounced decreased in viability by day 3 of culture. Interestingly, on day 6 of culture viability of HEK293 cells expressing p.Tyr108His-IGF1 became different from pSer83Cys-IGF1 reaching values similar to HEK293 parental cells stimulated with rhIGF1. Apoptosis assay on day 3 of culture showed a significant increase in cell death for HEK293 pSer83Cys-IGF1 clone compared to HEK293 cells expressing WT-IGF1. SaOS-2 cell differentiation from osteoblast to osteocytes resulted accelerated for cells expressing WT-IGF1 and delayed in time for p.Tyr108His-IGF1 variant. SaOS-2 cells expressing pSer83Cys-IGF1 behaved like parental cells.

Conclusion: In vitro studies of c.247A>T, pSer83Cys-IGF1 and c.322T>C, p.Tyr108His-IGF1 demonstrated that both variants impaired cell viability and increased cell apoptosis when expressed in HEK293 cells, as well as delayed bone cell differentiation and matrix deposition in SaOS-2 cells, with a more pronounced phenotype for the pSer83Cys-IGF1. Whether this effect is related to activation/interference of the IR by pSer83Cys-IGF1 or not remains to be elucidated.