In vitro impairment of protein synthesis and/or secretion of IGFALS gene variants characterized in ALS deficient or idiopathic short stature children

MARTUCCI, LUCIA; SCAGLIA, PAULA; KARABATAS, LILIANA; REY, RODOLFO; DOMENE, HORACIO; DOMENE, SABINA; JASPER, HECTOR

Playa del Carmen

Congreso; XXIV Reunión anual de la SLEP; 2014

Sociedad Latinoamericana de Endocrinoologia Pediatrica

Background: ALS is essential for the stabilization of IGF-I and IGFBP-3 in ternary complexes in the vascular system. ALS defi- cient (ALS-D) patients, homozygous or compound heterozygous for IGFALS gene mutations present severe IGF-I and IGFBP-3 deficiencies and variable degree of growth retardation. In addition, heterozygous carriers for these mutations, either first degree rela- tives of ALS-D patients or a subset of children with idiopathic short stature (ISS), have levels of IGFBP-3 and ALS intermediate be- tween ALS-D and wildtype (WT) subjects. These findings suggest that IGFALS gene variants affect ALS synthesis, secretion and/or function and are responsible for the phenotype observed (height and biochemical parameters). Aim: To study the impact of ten IGFALS gene variants we iden- tified in ALS-D 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 through cell culture transient transfections. Methods: IGFALS gene variants were introduced by site-di- rected mutagenesis into a commercial vector containing the entire human IGFALS cDNA (pCMV6-XL5-IGFALS). CHO cells (lack- ing ALS expression) were transiently transfected with wildtype IGFALS on each of the 10 variants using lipofectamine and har- vested at 48 hours. Both lysates and conditioned media were ana- lyzed by Western immunoblots using a mouse anti ALS antibody to detect protein localization. Results: Western immunoblots showed that WT-ALS was found mostly secreted into the culture medium at 48 hours. This assay was able to discriminate between variants that affect protein synthesis (absence or diminished levels of protein in both lysates and culture medium: p.E35Kfs*87, p.E35Gfs*17, p.N276S, p.L409F and p.C540R), defects in protein secretion (presence of protein in lysates but absence in culture medium: p.L213F) and neutral variants that do not affect protein synthe- sis and/or secretion (similar levels of protein in lysates and cul- ture medium as WT-ALS: p.P287L, p.A330D, p.A457V and p. R548W). Conclusions: The majority of the IGFALS gene variants ana- lyzed impaired the biosynthesis of the protein and only p.L213F affected its secretion. Whether the normally synthesized and se- creted protein variants retain complete functional capacity for ter- nary complex formation remains to be characterized.