In vitro funcional characterizationof two novel missense IGF1 variants

MARÍA CELIA FERNANDEZ; MARÍA GABRIELA RAMPI; AYELEN MARTIN ; FLORENCIA CLEMENT; PATRICIA PENNISI

Buenos Aires

Congreso; 11th International Meeting of Paediatric Endocrinology (IMPE); 2023

Insulin-like growth factor 1 (IGF1) plays key roles in promotinggrowth, during both pre- and post-natal life and regulating neurologicdevelopment in mammals. IGF1 gene mutations are extremelyrare causes of pre- and post-natal growth retardation. Phenotypecan be heterogenous with varying degrees of neurosensory deafness,cognitive defects, glucose metabolism impairment and short stature.We have previously described two patients with novel homozygousIGF1 missense variants (c.247A>T,pSer83Cys-IGF1 and c.322T>C,p.Tyr108His-IGF1) presenting a wide variability in the clinical presentationand biochemical profiles. In silico modelling predicts thatvariant 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 depositioncompared to WT-IGF1.Methods: Site-directed mutagenesis was performed on the RG21 2527 vector containing the full length IGF1 protein codingsequence as a fusion protein with GFP. Specific primers weredesigned 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 withWT-IGF1-GFP, pS83C-IGF1-GFP or pT108H-IGF1-GFP and stableclones selected. HEK293 cells were used for phosphorylation,viability and apoptosis assays. SaOS-2 cells were used for osteocytesdifferentiation and bone matrix deposition assays. Geneexpression was quantified by rqPCR and IGF1R phosphorylationwas assessed by western blotting (WB).Results: HEK293 cells expressing similar amounts ofp.Ser83Cys-IGF1 and p.Tyr108His-IGF1 showed a significantdecrease in IGF1R autocrine phosphorylation compared toWT-IGF1 expressing cells. Also, cells carriyng the variants had apronounced 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 similarto HEK293 parental cells stimulated with rhIGF1. Apoptosisassay on day 3 of culture showed a significant increase in cell deathfor HEK293 pSer83Cys-IGF1 clone compared to HEK293 cellsexpressing WT-IGF1. SaOS-2 cell differentiation from osteoblast toosteocytes resulted accelerated for cells expressing WT-IGF1 anddelayed in time for p.Tyr108His-IGF1 variant. SaOS-2 cells expressingpSer83Cys-IGF1 behaved like parental cells.