A homozygous mutation in the highly conserved Tyr60 of the mature IGF1 peptide broadens the spectrum of IGF1 deficiency

in European Journal of Endocrinology
Correspondence should be addressed to P A Pennisi; Email: ppennisi@cedie.org.ar

*(A C Keselman and A Martin contributed equally to this work)

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Background

IGF1 is a key factor in fetal and postnatal growth. To date, only three homozygous IGF1 gene defects leading to complete or partial loss of IGF1 activity have been reported in three short patients born small for gestational age. We describe the fourth patient with severe short stature presenting a novel homozygous IGF1 gene mutation.

Results

We report a boy born from consanguineous parents at 40 weeks of gestational age with intrauterine growth restriction and severe postnatal growth failure. Physical examination revealed proportionate short stature, microcephaly, facial dysmorphism, bilateral sensorineural deafness and mild global developmental delay. Basal growth hormone (GH) fluctuated from 0.2 to 29 ng/mL, while IGF1 levels ranged from −1.15 to 2.95 SDS. IGFBP3 was normal-high. SNP array delimited chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located. IGF1 screening by HRM revealed a homozygous missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). The change of the highly conserved Tyr60 in the mature IGF1 peptide was consistently predicted as pathogenic by multiple bioinformatic tools. Tyr60 has been described to be critical for IGF1 interaction with type 1 IGF receptor (IGF1R). In vitro, HEK293T cells showed a marked reduction of IGF1R phosphorylation after stimulation with serum from the patient as compared to sera from age-matched controls. Mutant IGF1 was also less efficient in inducing cell growth.

Conclusion

The present report broadens the spectrum of clinical and biochemical presentation of homozygous IGF1 defects and underscores the variability these patients may present depending on the IGF/IGF1R pathway activity.

 

     European Society of Endocrinology

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Figures

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    Growth curves, patient and pedigree. Weight (A), height (B), and head circumference (C) curves between the ages of 3 and 7 years. (D) Patient at the age of 7 years. Parents gave their informed consent for the publication of pictures. (E) Pedigree. Patient’s parents and half-brother were heterozygous for IGF1 mutation. Heights are indicated as the number of SDS values relative to the mean, based on Argentinean reference values. A full colour version of this figure is available at https://doi.org/10.1530/EJE-19-0563.

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    Multiple sequence alignments among different species (A) and among IGF2 and insulin proteins (B) were done with PRALINE software. The color scheme indicates the least conserved alignment position (dark blue), to the most conserved alignment position (red). (C, D, E and F) Cartoon representations of the mature hIGF1 structure (PDB 1GZR). (C) Main structure of IGF1 consisting of three helices (H1, H2 and H3) and a C-loop. Tyr60 (stick, magenta) is located close to the C-neck (dotted lines); N and C-t are the N- and C-termini. (D) The structure has been rotated to show residues Arg56, Lys27, and Met59 (sticks, blue) protecting Tyr60. The only exposed portion of Tyr60 is the hydroxyl group that forms a hydrogen bond (dotted yellow lines) with Glu46. (E) The image has been expanded in Tyr60 area. Substitution of Tyr60 by His cause the loss of the hydrogen bond with Glu46. (F) Key residues involved in IGFBP3 interaction (Glu3, Glu9, Asp12, Phe16, Val17, Gly19, Asp20, Arg21, and Glu58) are shown in orange in cartoon representation. They are located on the surface of IGF1 far from the change Tyr60His. A full colour version of this figure is available at https://doi.org/10.1530/EJE-19-0563.

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    Activation of IGF1R and induction of cell growth by mutant Tyr108His-IGF1. (A) HEK293T cells are stimulated for 10 min with rhIGF1 or serum from the patient, his relatives and controls before and after IGFBPs extraction. Whole lysates (50 µg) were used for Western blotting, and membranes probed for phospho and total IGF1R. The result shown represents a typical experiment. Samples from the patient activate IGF1R less than samples from control and heterozygous individuals when applied directly on cultured cells as well as using extracts after acid chromatography for IGFBPs removal. (B and C) Cell count after 48-h culture with samples from the patient, controls and relatives. There was no increase in the number of cells at the end of the experiment when cultured with serum form the patient. Number of cells increased after rhIGF1 stimulation, as well as after incubation with serum from age-matched controls and heterozygous brother, one-way ANOVA, followed by Dunnett’s multiple comparison test, **P < 0.01 vs time 0; *P < 0.05 vs time 0 (B). Samples from the father and the mother failed to increase the number of cells after 48 h incubation, compared to serum from age-matched controls; one-way ANOVA, followed by Dunnett’s multiple comparison test, **P < 0.01 vs time 0 (C). B, brother; F, father; M, mother; P, proband.

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