Characterization of an activating R1353H insulin-like growth factor 1 receptor variant in a male with extreme tall height

in European Journal of Endocrinology
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Objective

The insulin-like growth factor1 receptor (IGF1R) is important in growth and development, and inactivating IGF1R mutations cause short stature and relatively high levels of serum IGF-I. We identified an unclassified IGF1RR1353H variant in a male with extreme tall height, very low levels of serum IGF-I and delayed and prolonged growth spurt. The index case’s mother and three sons all carried the variant, but so far only the eldest son (age 18 years) presented with tall height. We hypothesized that the variant could constitute an activating mutation.

Design

The IGF1RR1353H variant was investigated in Igf1r/ mouse embryonic fibroblasts (R-cells) by cell cycle, colony formation and transcriptome analyses.

Results

The IGF1RR1353H (R-1353) exhibited significantly increased cell proliferation, G1-S progression and colony formation in soft agar. RNA sequencing identified 195 differentially expressed genes between R-WT and R-1353 (adjusted P < 1E-100). Most genes were upregulated in R-1353, including the gene encoding the androgen receptor (AR). Gene expression profiling showed the most significant enrichment in extracellular matrix organization (P = 2.76E-7), collagen biosynthesis (P = 1.21E-5) and cell adhesion (P = 7.38E-5). Retrospective biochemical analysis of the index case revealed decreased testosterone and sex hormone-binding globulin levels, whereas LH and FSH were within normal ranges. This profile suggests an increased sensitivity to androgen, which is compatible with the enhanced expression of Ar in R-1353 cells.

Conclusions

Our findings suggest that R1353H constitutes an activating IGF1R variant. The possible deregulation of collagen turnover and increased androgen sensitivity implicates an association to tall phenotype in male carriers.

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  • Supplementary Figure 1
  • Supplementary Figure 2

 

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European Society of Endocrinology

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    Family pedigree of the index case with the identified IGF1R R1353H variant. The index case developed an extreme tall stature (+3.6 SDS) and low-serum IGF-I levels (−3.0 SDS). The variant was observed in the index case’s three sons and mother. Variant carriers are marked by black shading. Squares indicate males, and circles indicate females. The arrow indicates the index case/proband (II:1). A full colour version of this figure is available at https://doi.org/10.1530/EJE-18-0176.

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    Characterization of R− cells stably transfected with WT and R1353H-mutated IGF1R. (A) Relative IGF1R transcription levels against Gapdh levels (Y axis) in R− clones were analyzed by qRT-PCR. Two cell lines with equal expression of WT-2C4 and R1353H-mutated (1353-2B1) IGF1R (both indicated with arrows) were selected for further investigation and named R-WT and R-1353 respectively. Another pair of cell lines, R-WT-2D5 and R-1353-3A2 (indicated by dotted arrows), with similar IGF1R expression levels were selected for validation experiments. No IGF1R expression was detected in R-puro where empty virus was used for mock transduction. (B) IGF-IR protein expression in R-puro, R-WT and R-1353 cells were assayed by western blotting using anti-IGF-IRβ. GAPDH was blotted as loading control. (C) Western blots of IGF-1R in subcellular fractionations from R-puro, R-WT and R-1353 cells. The receptor was detected at equivalent levels in the membrane and nucleus fractions from R-WT and R-1353 cells and was undetectable in R-puro cells. N, K-ATPase and histone 3 were blotted as fraction specific markers for membrane and nucleus proteins respectively. (D) R-puro, R-WT and R-1353 cells were serum-starved for 36 h followed with or without 10 min of IGF-I stimulation. Immunoprecipitated IGF-IR was used in western blot analysis with an anti-phospho-tyrosine antibody to investigate IGF-IR phosphorylation, re-blot with IGF-IRβ was performed to confirm equal input. Separate western blot experiments were performed to investigate pAkt and Erk (pErk) in total cell lysates. GAPDH was used as loading control. (E) R-WT-2D5 and R-1353-3A2 were serum-starved for 36 h followed with or without 10 min of IGF-I stimulation. Immunoprecipitated IGF-IR was used in western blot analysis with an anti-phospho-tyrosine antibody to investigate IGF-IR phosphorylation, re-blot with IGF-IRβ was performed to confirm equal input. (F) The kinetics of the IGF-1R WT and the R1353H variant were assessed by measuring the IGF-1R autophosphorylation using a phospho-IGF1R (Tyr1135/1136) antibody after IGF-I stimulation. R-WT and R-1353 were stimulated by 50 mg/μL IGF1 for 0, 5, 15, 30 and 45 min, and the total IGF1Rβ level was determined as loading control. All immunoprecipitation and immunoblotting experiments were successfully repeated at least three times. The figures show representative pictures from one of the experiments. A full colour version of this figure is available at https://doi.org/10.1530/EJE-18-0176.

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    Proliferation and colony formation of R-puro, R-WT and R-1353 cells. (A) R-puro, R-WT and R-1353 cells were seeded in 96-well plates and cultured under basal condition. Cell proliferation was monitored with XTT proliferation assay kit every 24 h. The results are from five replications. (B) 1000 R-puro, R-WT or R-1353 cells were seeded in six-well plates with 1.0% soft agar and cultured for 2 weeks. The colony numbers in each well were determined using microscopy counting. The results are means from five replications.

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    Cell cycle progression of R-puro, R-WT and R-1353 cells after stimulation with 50 ng/mL IGF-I for 0, 10, 16 and 24 h. (A) Cell cycle progression (G1-S) was analyzed by FACS using BrdU/7-AAD staining. G1 (yellow), S (red) and G2 (purple) phases were gated respectively to investigate the cell cycle progression. (B) Percentage changes of S phase cells after 0, 10, 16 and 24 h of 50 ng/mL IGF-I stimulation are shown for R-puro, R-WT and R-1353 cells after normalization against unstimulated samples. Data represents three replications.

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    Gene expression differences between R-1353 and R-WT cells. (A) Heatmap of DE genes between R-1353 and R-WT. RNA sequencing was performed for six replicates of R-WT and R-1353. A total of 195 DE genes were identified by DESeq2 at adjusted p<1E-100 and is depicted in the heatmap. Unsupervised Euclidean clustering separated genes into four expression clusters (Group 1–4). (B) Relative AR mRNA expression as determined by qRT-PCR in R-WT and R-1353 cells, showing 70-fold (74.0 ± 13.14) higher expression in the R-1353 cells. (C) Relative AR mRNA expression as determined by qRT-PCR in R-WT-2D5 and R-1353-3A2 cells, showing 55.6-fold (55.6 ± 17.03) higher expression in R-1353-3A2 cells. (D) Relative AR mRNA expression as determined by qRT-PCR in R-WT/WT and R-1353/WT cells, showing 51.1-fold (51.1 ± 7.14) higher expression in R-1353/WT cells. AR, androgen receptor; DE, differentially expressed.

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