ESPE Abstracts (2014) 82 P-D-1-1-139

Functional Characterization of Three Novel Mutations in the IGF1R Gene

Matias Juanes, Gabriela Guercio, Roxana Marino, Esperanza Berensztein, Marta Ciaccio, Silvia Gil, Marco A Rivarola & Alicia Belgorosky


Hospital de Pediatria Garrahan, Buenos Aires, Argentina


Background: IGF1R gene mutations have been associated with varying degrees of intrauterine and postnatal growth retardation, and microcephaly. We have previously reported three novel variants in the IGF1R gene: de novo p.Arg1256Ser, de novo p.Asn359Tyr and p.Tyr865Cys (ENDO 2013, OR20-2).

Aim: To characterize the functional effects of the novel IGF1R gene allelic variants.

Methods: In silico tools were used to predict the pathogenicity of the variations. Functional effects were evaluated by two in vitro assays: i) IGF1 dependent DNA synthesis in fibroblast cell primary cultures from patients and two control subjects (C1 and C2) were analyzed by 3[H]thymidine incorporation into DNA treated with IGF1 (50 ng/ml) for 16, 20, and 24 h and ii) PI3K/Akt pathway phosphorylation by phospho Akt (Ser473) STAR ELISA Kit (Millipore) assay in fibroblast cultures from patients and two controls (C3 and C4) stimulated with 100 ng/ml of IGF1 for 10 min. Protein concentration for each well was measured by Bradford assay.

Results: In silico prediction models indicated that the substitutions probably affect protein function. Proliferation assay showed that IGF1 significantly induced DNA synthesis in C1 and C2 at 20 h (5.15±0.67 and 6.37±1.00 fold increase over ‘0’ dose respectively, P<0.05 by ANOVA and Bonferroni tests) and also Akt phosphorylation was significantly stimulated in C3 and C4 after 10 min of treatment with IGF1 (P<0.05 by ANOVA and Bonferroni tests). However, no significant increase was observed in any of the three patients in both functional studies.

Conclusion: We characterized three novel heterozygous mutations in the IGF1R gene that inhibit cell proliferation induced by IGF1 and affect IGFR signal transduction in patients’ fibroblast cultures. These findings strongly suggest that these mutations lead to failure of the IGF1R and cause the phenotype of pre and postanatal growth retardation and microcephaly.

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