ESPE Abstracts (2016) 86 P-P1-596

Functional in vitro Characterization of Two Novel Germinal STAT3 Mutations Associated with Short Stature, Immunodeficiency and Autoimmune Disease

Mariana Gutierreza, Paula Scagliaa, Ana Keselmana, Lucia Martuccia, Liliana Karabatasa, Sabina Domenea, Miguel Blancob, Nora Sanguinettia, Liliana Bezrodnikc, Daniela Di Giovannic, Soledad Caldirolac, Maria Esnaola Azcoitic, Nana-Hawa Jonesd, Vivian Hwad, Santiago Revalee, Martin Vazqueze, Hector Jaspera, Ashish Kumarf & Horacio Domenea

aCentro de Investigaciones Endocrinológicas ‘Dr César Bergadá’ (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; bEndocrinología, Hospital Universitario Austral, Buenos Aires, Argentina; cInmunología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; dDivision of Endocrinology, Cincinnati Center for Growth Disorders, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA; eInstituto de Agrobiotecnología de Rosario (INDEAR), CONICET, Rosario, Argentina; fDivision of BM transplantation and Immunodeficiency, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA

Background: We have recently reported the molecular diagnosis of two patients with severe growth failure associated with a spectrum of early-onset autoimmune disease and immunodeficiency. Heterozygous de novo mutations, c.1847_1849delAAG (p.E616del) and c.1276T>C (p.C426R), in the STAT3 gene were found. Functional in vitro studies of these variants are presented.

Objective and hypotheses: We aimed to study the impact of p.E616del and p.C426R mutations on STAT3 activity under basal and GH- or IL-6-stimulated conditions. We hypothesised that both variants are activating, since inactivating STAT3 mutations are associated with hyper-IgE syndrome without growth failure.

Method: STAT3 gene variants were generated by site-directed mutagenesis and transfected into HEK293T cells. The effects of IL-6 (20 ng/ml) and GH (200 ng/ml) on expression, phosphorylation and transcriptional activity of WT and mutants STAT3 were studied using a luciferase reporter system and by Western Immunoblot.

Results: Under basal conditions, variants p.C426R and p.E616del, presented increased reporter activity compared to WT-STAT3 (P<0.01). However, STAT3 mutants were not constitutively phosphorylated. GH stimulation of STAT3 variants induced the luciferase reporter gene 2- and 4-fold for p.E616del and p.C426R, respectively (P<0.01). Nonetheless, only variant p.C426R showed increased transcriptional activity under IL-6-stimulation compared to WT (P<0.05). WT-STAT3 and the two variants were phosphorylated in response to GH and IL-6, but phosphorylation kinetics was different for each mutant: p.C426R exhibited delayed dephosphorylation only under GH treatment, while p.E616del, only under IL-6-stimulation.

Conclusions: i) p.E616del and p.C426R STAT3 variants are gain-of-function mutations since they both presented increased basal transcriptional activity; ii) While GH was able to induce the STAT3 responsive reporter vectors for both variants studied, IL-6 does not lead to enhanced transcriptional activity for p.E616del mutant; iii) Further studies are necessary to disclose the underlying molecular mechanisms that mediate the effect of these STAT3 variants on IL-6 and GH action.

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