ESPE Abstracts (2016) 86 P-P1-596

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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