A new p.(Ile66Serfs*93) IGF2 variant is associated with pre- and postnatal growth retardation

in European Journal of Endocrinology
Correspondence should be addressed to D Rockstroh; Email: Denise.Rockstroh@medizin.uni-leipzig.de
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Objective

The IGF/IGF1R axis is involved in the regulation of human growth. Both IGF1 and IGF2 can bind to the IGF1R in order to promote growth via the downstream PI3K/AKT pathway. Pathogenic mutations in IGF1 and IGF1R determine intrauterine growth restriction and affect postnatal body growth. However, to date, there are only few reports of pathogenic IGF2 mutations causing severe prenatal, as well as postnatal growth retardation.

Results

Here we describe a de novo c.195delC IGF2 variant (NM_000612, p.(Ile66Serfs*93)) in a 4-year-old patient with severe pre- and post-natal growth retardation in combination with dystrophy, facial dimorphism, finger deformities, as well as a patent ductus. Cloning and sequencing of a long-range PCR product harboring the deletion and a SNP informative site chr11:2153634 (rs680, NC_000011.9:g.2153634T>C) demonstrated that the variant resided on the paternal allele. This finding is consistent with the known maternal imprinting of IGF2. 3D protein structure prediction and overexpression studies demonstrated that the p.(Ile66Serfs*93) IGF2 gene variation resulted in an altered protein structure that impaired ligand/receptor binding and thus prevents IGF1R activation.

Conclusion

The severity of the phenotype in combination with the dominant mode of transmission provides further evidence for the involvement of IGF2 in growth disorders.

 

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European Society of Endocrinology

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Figures

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    Pedigree, growth curve and clinical characteristics of the patient. (A) The index patient (III.2) bearing the heterozygous p.(Ile66Serfs*93) variation is indicated by a half-filled symbol and marked by an arrow. Height (cm) and standard deviation score (SDS) of the last reported visit is shown below the symbol. Crossed out lines indicate half siblings (n.a., no data available). Growth curve of the index patient (B) indicated a severe growth failure. Bone age is shown by a gray square, while initiation of rhGH therapy is shown by a gray arrow. White circles indicated patients’ height adjusted for week of pregnancy, while black circles indicate unadjusted values. (C) Clinical features of the index patient: short stature (I, II), triangular face (III), low-set ears (IV, V), frontal bossing (V) and a clinodactyly (VI–VIII marked by an arrow).

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    Identification of a novel p.(Ile66Serfs*93) IGF2 variation. (A) Electropherograms of Sanger sequencing analyses. Arrow, position of the c.195delC deletion; *frameshift of the aa sequence. (B) Structure of the IGF2 protein. Normal protein translation generates a 180-aa IGF2 precursor protein. The I66S variation is predicted to results in a frameshift (position 66) in combination with a premature stop codon at position 93. The changed aa sequence in the mutant beginning from position 66 is shown in italics. (C) Schematic illustration of so far identified IGF2 mutations (9, 11, 12). IGF2 encode an inactive 180-aa (Isoform 1, NP_000603) or 236-aa (Isoform 2, NP_001121070) precursor protein that includes a 24-aa signal peptide (SP), a 67-aa core IGF2 and a 89-aa trailer sequence (18). The p.I66S variation is localized in the core-IGF2 sequence of isoform 1 (NM_000612).

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    Illustration of potential conformational changes in p.I66S protein structure. (A) Schematic diagram of IGF2 maturation. IGF2 encodes an inactive 180-aa precursor protein which is post-translationally cleaved into a 67-aa bioactive protein. First, the terminal signal peptide is proteolytically removed creating a 156-aa sequence. In the next step, the trailer sequence is proteolytically cleaved at two separated sites (TQRLRR 104 and PAKSER 68) generating the bioactive protein. (B) Illustration of p.I66S protein structure. Sequence analyses of the cleavage sites indicated a changed aa sequence at basic residues in the mutant: TQRLRR→PSACAG and PAKSER→PPSPRG. (C) Comparison of the WT (IGF2-WT, blue) and mutant (IGF2-I66S, red) protein structure. 3D protein structure was predicted based on the crystal structure of IGF2-WT (pdb: 1IGL) using the iTASSER server and superpositioned with the WT structure. The mostly unstructured C-terminal extension due to the lack of posttranslational processing is illustrated. (D) Upper panel: Schematic presentation of IGF2 plasmids. The position of the c.195delC mutation is marked in red. Lower panel: Cell lysates and supernatants from transfected cells after immunoblotting with the indicated antibodies. (E) Whole-cell lysates from IGF1 stimulated (30 min) cells transfected with an IGF1R plasmid were subjected to immunoblotting using antibodies as indicated. (F) Whole-cell lysates were prepared from IGF1R transfected cells after stimulation (30 min) with IGF2-WT supernatants and immunoblotted for pIGF1R, total IGF1R and β-actin as loading control. Representative blot out of three independent experiments is shown.

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    The p.I66S variant inhibits IGF1R activation. (A) Immunoblots of untreated or rhIGF1 treated (30 min, 10 nM) cell lysates after co-transfection with IGF1R and IGF2 constructs were analyzed using the antibodies as indicated. In addition, supernatants from every approach were analyzed using an IGF2 antibody. (B) Densitometric evaluation of IGF1R activation (fold change of the variant compared to empty vector). pIGF1R levels were normalized to total IGF1R and β-actin protein amounts and results are shown as mean ± s.e.m. Significant P-values are indicated. ***P < 0.001. (C) Cell lysates or supernatant from IGF1R transfected cells after stimulation with IGF2 supernatants (30 min) analyzed with the indicated antibodies. (D) Densitometric evaluation of IGF1R activation (fold change of the variants compared to an empty vector control). *P < 0.05, **P < 0.01. (E) Illustration of IGF2 residues interacting with domains of the IGF1 receptor according to Vashisth et al. (20). L1 residues are shown in red, CR residues in green, L2 residues in blue, and (F1–F2)′ residues are shown in yellow. The IGF2 variant lacks L1, L2 and (F1–F2)′ residues (highlighted in bold). (F) Rescue experiment with the p.I66S variation. Lysates of IGF1R/IGF2 co-transfected HEK293 cells after stimulation with recombinant IGF1 (30 min, 10 nM) in comparison to unstimulated approaches. For each approach a representative Western blot out of three independent experiments is shown.

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