ESPE Abstracts (2015) 84 P-2-480

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

Lucia Martucci, Paula Scaglia, Liliana Karabatas, Rodolfo Rey, Horacio Domené, Sabina Domené & Héctor Jasper

Centro de Investigaciones Endocrinológicas “Dr. César Bergadá” (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina

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 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 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 each of 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.

Funding information: This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (PICT-2010 Nro.1916), and SANDOZ International GmbH, Business Unit Biopharmaceuticals.

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