In Vitro Functional Characterization Of IGFALS Gene Variants Found In ALS Deficient or Idiopathic Short Stature (ISS) Children

MARTUCCI, L.; SCAGLIA, P.; KARABATAS, L.; REY, R.; DOMENÉ, H.; DOMENÉ, S.; JASPER, H.

Barcelona

Congreso; 54th Annual Meeting of the European Society for Paediatric Endocrinology (ESPE); 2015

Background: ALS deficient (ALS-D) patients present severe IGFI and IGFBP3 deficiencies and variable degree of growth retardation. Heterozygous carriers for IGFALS variants, ALS-D relatives or a subset of ISS children, have levels of IGFI, IGFBP3 and ALS intermediate between ALS-D and wildtype (WT) subjects. This supports that IGFALS gene variants may affect ALS synthesis, secretion and/or function and could be responsible for the observed phenotype. Objective and hypotheses: We aim to study the impact of ten IGFALS gene variants identified in ALSD or ISS children (p.E35Kfs*87, p.E35Gfs*17, p.L213F, p.N276S, p.P287L, p.A330D, p.L409F, p.A475V, p.C540R, and p.R548W) on ALS protein synthesis and secretion and to characterize whether the secreted protein variants retain functional capacity for ternary complex formation in vitro (iv-TCF). Method: IGFALS gene variants were introduced by site-directed mutagenesis into a commercial vector containing the entire human IGFALS cDNA. CHO cells were transiently transfected with WT-IGFALS or eachof the ten variants. Both lysates and conditioned media (CM) were analyzed by WIB. Iv-TCF was performed for WT and mutant ALS variants by Superdex 200 exclusion column chromatography. Results: WIB showed that WT-ALS was found mostly secreted into the CM at 24 hours. For variants p.E35Kfs*87, p.E35Gfs*17, p.N276S, p.L409F and p.C540R, we found absence of protein in both lysates and CM; for variant p.L213F, presence of protein in lysates but absence in CM; and for variants p.P287L, p.A330D, p.A457V, and p.R548W, presence of protein in lysates and CM. All the normally synthesized and secreted variants retained their ability for iv-TCF. Conclusion: Six of the IGFALS gene variants analyzed impaired the biosynthesis, secretion and/or stability of the protein. All secreted variants retained the ability to form iv-TCF. It remains to be explored how these variants affect the IGF system in the context of a whole organism.