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

in European Journal of Endocrinology
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  • 1 Centro de Investigaciones Endocrinológicas ‘Dr. César Bergadá’ (CEDIE) CONICET – FEI – División de Endocrinología, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina
  • 2 Servicio de Genética, Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina
  • 3 INGEMM, IdiPAZ, Hospital Universitario La Paz, Madrid & CIBERER (U753), ISCIII, Madrid, Spain

Correspondence should be addressed to P A Pennisi; Email: ppennisi@cedie.org.ar

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

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.

Abstract

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.

Introduction

The insulin-like growth factors (IGFs) comprise a family of peptides that play important roles in mammalian growth and development. IGF1, a 70-residue polypeptide, mediates many of the growth promoting actions of growth hormone (GH) and has intrinsic growth, metabolic and mitogenic effects (1). IGF1 is essential for both fetal and postnatal growth and development (2). During fetal growth, however, these processes are largely GH independent (3, 4, 5)

Though the major source of circulating IGF1 is the liver, it is also locally produced in a wide variety of tissues having well-defined endocrine and paracrine/autocrine modes of action (6). IGF1 circulates mainly bound to IGF-binding proteins, (IGFBP) -3 or -5 and acid labile subunit (ALS), forming a large ternary complex that prolongs its half-life and modulates its bioactivity. This complex formation depends on GH secretion during postnatal life (7).

To date, human homozygous IGF1 gene defects have been reported in a very few cases (8, 9, 10, 11, 12). The first case, reported by Woods et al., had a partial deletion of the IGF1 gene resulting in intrauterine growth failure, severe postnatal growth retardation, sensorineural deafness, and mental retardation associated with undetectable circulating IGF1 (8). The second case with a homozygous IGF1 alteration reported had a similar clinical phenotype but extremely high serum IGF1 levels (9). The third case had a homozygous missense IGF1 mutation resulting in a milder phenotype associated with variable serum IGF1 levels (10).

Here we describe a patient with a novel homozygous IGF1 mutation c.322T>C, p.Tyr108His, leading to severe pre- and post-natal growth failure, microcephaly, developmental delay and bilateral sensorineural deafness, associated with normal to mildly elevated circulating IGF1, underscoring the variability of the clinical and biochemical presentations of IGF1 gene defects.

Patient and methods

Clinical and auxological parameters: height and sitting height were determined using a wall mounted stadiometer, head circumference (HC) was assessed with a tape measure, and weight, using a calibrated scale. Height (cm), weight (kg) and sitting height/height ratio were expressed in standard deviation score (SDS) based on Argentinean references (13, 14) . HC was expressed in cm and SDS using Dutch references (15). Body mass index SDS and height velocity SDS were calculated according to US references (16, 17). Parents were informed of the purpose of the study and written consent was obtained. The study was approved by the Ethics Committee of Hospital de Niños Dr. Ricardo Gutierrez, Buenos Aires, Argentina.

Hormonal assays

Samples were obtained from the proband, his parents and a half maternal brother. Plasma GH, IGF1 and IGFBP3 concentrations were measured by a chemiluminescent immunometric assay (IMMULITE® 2000system, Siemens Healthcare Diagnostics Products Ltd.), as previously reported (18). Intra- and inter-assay coefficients of variation were <7.2% for all serum measurements. A second method was used for IGF1 measurements (Cobas e411 analyzer; Roche Diagnostics, ‘ECLIA’ IGF-I, Roche). IGF1 (ng/mL) and IGFBP3 (µg/mL) SDS were calculated based on Argentinean references (19). TSH, free T4 (FT4), cortisol, prolactin, LH, FSH, testosterone and insulin levels were determined by electrochemiluminescence (Cobas e411 analyzer; Roche Diagnostics GmbH), anti-thyroperoxidase antibodies by chemiluminescence assay (IMMULITE-2000 system; Siemens Healthcare Diagnostics Products Ltd.), anti-Müllerian hormone (AMH) by an enzyme-linked immunoassay (EIA AMH/MIS®, Beckman-Coulter Co., Marseilles, France).

Genetic studies

Genomic DNA from the patient and his parents and a maternal half-brother was extracted from peripheral venous blood by CTAB method (20), as previously described (21). Multiplex ligation-dependent probe amplification (MLPA) for subtelomeric regions was carried out using the Human Telomere-5 P070-B3 MLPA kit (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Exon dosage was calculated using Coffalyser Net software (MRC Holland). Infinium CytoSNP-850K SNP array (Illumina) was used to perform aCGH-SNP in the proband’s sample. IGF1 exons, intron/exon boundaries and known regulatory regions were PCR amplified and analyzed by high-resolution melting, as previously described (22). Fragments displaying abnormal melting pattern were sequenced with specific oligonucleotides in an ABI 3500 DNA analyzer. Oligonucleotide sequences are available upon request. Additionally, patient’s and parents’ samples were analyzed by targeted next-generation sequencing with a custom panel (EZ-Capture, Nimblegen, Roche) including a total of 433 genes implicated in the etiology of or associated with, syndromic and non-syndromic short stature (gene list available upon request). Peripheral blood karyotype was performed using high-resolution GTG banding.

In silico molecular modeling

Multiple sequence alignments among IGF1 from different species and among IGF1, IGF2 and Insulin were performed with PRALINE program (http://www.ibi.vu.nl/programs/pralinewww/) using reference sequences obtained from Uniprot. Molecular model figures were prepared based on the crystal structure of the mature hIGF1 (PDB accession code 1GZR) (23) using the molecular graphics program PyMOL (PyMOL Molecular Graphics System, Version 1.8.4.0, Schrödinger, LLC, http://www.pymol.org/) which was also used to model in silico the Tyr108His mutation. Tyr108 is equivalent to Tyr60 in IGF1 mature peptide. We used the mature IGF1 peptide amino acid numbering for the molecular modeling in order to maintain the nomenclature of the original hIGF1 crystal structure publication. Side chain conformation of histidine was selected from the PyMOL backbone-dependent rotamer library considering minimal steric clashes with surrounding residues.

IGF1 extraction by acid chromatography

Serum was exposed to C18 Sep-Pak column (Waters Associates, Milford, MA, USA) for acid chromatography extraction to separate the IGFs from IGFBPs as previously described (24). To dissociate IGFs from IGFBPs, 100 µL of each serum were incubated with 150 µL of 0.5 N HCl for 1 h at 37°C. After incubation samples were seeded in Sep-Pak columns that were prewet with 5 mL acetonitrile and then rinsed with 5 mL water. Samples entered the columns by gravity. Unbound proteins were eluted by rinsing the columns with 10 mL 4% acetic acid and IGFs were eluted with 1 mL of methanol. Solvents were removed from the sample using a SpeedVac vacuum concentrator. Once dried, pellets were resuspended in different volumes of DMEM culture medium to obtain equal concentrations of IGF1 for all samples.

IGF1 receptor (IGF1R) phosphorylation

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37°C in a humidified 5% CO2 environment. Cells were seeded on six-well plates, allowed to grow up to 70% confluence, and starved overnight with serum-free medium. Next morning, cells were incubated for 10 min with equivalent concentrations of extracted IGF1 and direct serum from the patient, his mother, father, brother and age and sex-matched control individuals. Cells stimulated with 20 nM recombinant human (rh)-IGF1 were used as positive control for IGF1R phosphorylation. Cells cultured in serum-free medium without stimulation were used as basal condition. After stimulation, cells were harvested on lysis buffer containing protease inhibitors, and proteins were extracted as previously described (25). Experiments were performed in duplicates.

Western blotting

After extraction, protein concentration was determined using the Bradford reagent. Fifty micrograms of proteins were resolved by SDS-PAGE and transferred to PVDF membranes. Blots were blocked and probed with antibodies recognizing the phosphorylated fraction or total IGF1R protein (Cell Signaling Technology).

Proliferation assay

HEK293 cells (4 × 104) were seeded on 24-well plates in complete medium (CM) (DMEM, 10% fetal bovine serum (FBS)). Twenty-four hours after seeding, cells were harvested by trypsinization and counted after 0.5% trypan blue staining to determine the initial number of cells (Time 0). In the remaining plates, medium was replaced by low serum medium (LSM) (DMEM, 1% FBS) or medium supplemented with serum samples from the patient, relatives or controls containing equivalent amount of IGF1. After 48 h of culture, cells were trypsinized and counted using 0.5% trypan blue staining. Each condition was performed in triplicates.

Results

Patient

The index case was referred to the Endocrine Division of the Hospital de Niños Dr. Ricardo Gutierrez, Buenos Aires, at 3.2 years of age because of short stature. He was born from consanguineous parents at 40 weeks of gestational age (GA), with intrauterine growth retardation (IUGR): birth weight and length were 1910 g (−3.1 SDS) and 38 cm (−6.3 SDS), respectively. At 3.2 years, height was 74 cm (−6.2 SDS), sitting height/height ratio 0.567 (−0.4 SDS), weight 6.1 kg (−5.1 SDS) and HC 41 cm (−6.1 SDS). Physical examination revealed facial dysmorphism including frontal bossing, triangular face, bulbous nose, full lips, and retrognathia (Fig. 1). He also presented bilateral sensorineural deafness, mild language and motor delay and hyperactive behavior. His school performance was poor, revealing learning difficulties. His last clinical evaluation at 7.8 years showed persisting postnatal growth failure: height, 90.2 cm (−6.5 SDS); weight, 9.6 kg (−5.0 SDS); HC, 44.2 cm (−4.9 SDS) with slow growth height velocity (Table 1). Routine blood tests (RBC, WBC, glucose, BUN, creatinine, SGOT, SGPT, cholesterol, triglycerides, celiac antibodies) showed normal results. TSH, FT4, cortisol, prolactin, insulin, gonadotropins, testosterone and AMH levels were within normal ranges. Basal levels of GH and IGF1 were variable (Table 2). IGF1 measurements were confirmed using a second method (data not shown). Our results are in accordance to high intra-subject variability for IGF1 observed by other authors (26). Normal-to-high IGFBP3 values were found as shown in Table 2. His karyotype was normal (46, XY).

Figure 1
Figure 1

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.

Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0563

Table 1

Auxological parameters.

CAWeightHeightBMISH/H ratioHCHVBA
yKgSDS*CmSDS*kg/m2SDSRatioSDSCmSDS§cm/ySDSy
32/126.1−5.174−6.211.1 −6.70.567 −0.441−5.6
40/126.7−4.976.5−6.411.5 −5.642.5−5.13.9 NA
56/127.9−5.682−6.511.7 −5.343.4−4.83.4−4.0
610/128.2−5.285.8−6.711.3 −6.243.8−4.82.5−5.44
79/129.7−5.090.2−6.511.9 −4.50.536 0.044.2−4.94.4−1.94

*SDS were calculated relative to the mean, based on Argentinean reference values (13). SDS were calculated based on data from CDC (16). SDS were calculated using Argentinean references (14). §SDS were calculated based on Dutch references (15). SDS were calculated using U.S. references (17).

BA, bone age; BMI, body mass index; CA, chronological age; HC, head circumference; HV, height velocity; NA, not available; SH/H ratio, sitting height/height ratio.

Table 2

Hormonal measurements.

CA (y)RR
34/1237/1240/1259/1265/12610/12
GH, ng/mL9 29.101.572.970.200.481–8 years: 0.1–5
IGF1, ng/mL 8910847752061641–5 years: 29–118
 SDS+0.31+0.75−1.15−0.71+2.95+2.125–7 years: 39–118
IGFBP3, µg/mL3.864.122.662.35.34.51–5 years: 1.7–4.2
 SDS+1.22+1.55−0.32−1.58+2.31+1.275–8 years: 2–4.4
Insulin, µU/mL2.65.41.71–5 years: 1.5–6.5
5–13 years: 1.8–12

A, chronological age; RR, reference range.

Parental height and HC were 148.8 cm (−2.0 SDS) and 52.2 cm (−1.8 SDS) for his mother, and 155.2 cm (−2.6 SDS) and 52.0 cm (−3.1 SDS) for his father. Mid-parental height was 158.2 (−2.14 SDS). Mother's and father's birth weights were 2500 and 1900 g, respectively. His maternal half-brother’s height and HC were 118.0 cm (−2.0 SDS) and 49.6 cm (−1.8 SDS) at 8 years of age. He was born at 39 weeks of GA with weight 3000 g (−0.7 SDS) and length 44 cm (−3.1 SDS). The mother had had two spontaneous miscarriages the first at 4 months of GA, with IUGR, the second at 3 months of GA. Table 3 shows available auxological and biochemical data for family members.

Table 3

Clinical and biochemical features in family members.

Family memberCAHeightBMISH/H ratioHCIGF1IGFBP3
ycmSDS*kg/m2SDSRatio SDScmSDS§ng/mLRRµg/mLRR
Mother23148.8−2.028.31.30.530−0.252.2−1.8182107–36743.4–7.8
Father20155.2−2.622.4−0.20.522−0.152−3.1163105–34652.9–7.2
Half-brother8.7118−2.115.5−0.20.525−1.049.6−1.815539–1324.42.2–5.6

*SDS were calculated based on Argentinean reference values (13). SDS were calculated based on data from CDC (16). SDS were calculated using Argentinean references (14). §SDS were calculated based on Dutch references (15).

RR, reference range.

Identification of a new missense variant in the A domain of IGF1

SNP array showed multiple chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located, a potential candidate gene for the patient's phenotype. IGF1 coding and known regulatory regions were analyzed by high-resolution melting. Fragments displaying abnormal melting pattern were sequenced. The patient was homozygous and his parents and half-brother heterozygous for a novel IGF1 missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). This variant changes a highly conserved Tyr residue, located in the A domain of IGF1 and has been described to be critical for its interaction with IGF1R (27). The presence of further variants in IGF1R, IGF2, IGF2R, IGFBP3, or IGFALS was excluded by targeted NGS analysis (average coverage: 385.9×, 370.12×, 364.3×, 297.3× and 296.5×, respectively).

In silico analysis

In silico studies consistently predicted the NM_000618.4(IGF1):c.322T>C, p.(Tyr108His) variant as pathogenic using multiple bioinformatics tools (CADD, DANN, GERP, FATHMM, LRT, MutationAssessor, MutationTaster, PROVEAN, FATHMM-MKL and SIFT) vs no benign predictions. The analysis of positional conservation of Tyr108 residue by multiple sequence alignment from ten species showed that Tyr108 is highly conserved among species. Tyr108 is also conserved among IGF2 and insulin proteins (Fig. 2A). The mature structure of hIGF1, reached after processing all transcript IGF1 isoforms, consists mainly of three-helix bundle and a flexible loop (C-region), which extends away from the core of IGF1 (23). The Tyr108 is equivalent to Tyr60 in this mature structure. Tyr60 is a key residue involved in IGF1R binding (27) and is located neighboring the C-neck region and completely buried under the side chains of Arg56, Lys27, and Met59 (Fig. 2B). Despite its position toward the core of the molecule, the hydroxyl group of Tyr60 forms a hydrogen bond with Glu46. When Tyr60 is changed into a smaller histidine, no hydrogen bond is formed between the residue and Glu46 (Fig. 2C). However, residues involved in the interaction with IGFBP3 (28) should not be affected according to the molecular model (Fig. 2D).

Figure 2
Figure 2

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.

Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0563

Impaired ability to induce IGF1R phosphorylation

The effect of the p.(Tyr108His) mutation on the ability to activate IGF1R was tested using HEK293 cells. A short stimulus with serum samples before and after acid chromatography to separate IGF1 from IGFBPs, was performed. As shown in Fig. 3A, stimulation with 20 nM of rhIGF1 provoked a strong activation of IGF1R. Similar effects were obtained after 10 min of stimulation using direct or extracted samples from controls (Fig. 3A, lines 3 and 9). However, a much weaker stimulation was observed when patient’s serum or extracted samples were used. This result suggests an impaired IGF1R activation by mutant IGF1 (Fig. 3A, lines 5 and 11). When samples from heterozygous relatives were tested, the responses were lower than control's but stronger than the proband's.

Figure 3
Figure 3

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.

Citation: European Journal of Endocrinology 181, 5; 10.1530/EJE-19-0563

Impaired ability to stimulate cell proliferation

To assess the ability of p.Tyr108His-IGF1 to stimulate proliferation in HEK293T cells, cells were cultured for 48 h with medium supplemented with serum from the patient and his first-degree relatives and age and sex-matched controls (Fig. 3B and C). While controls were able to duplicate the cell number after 48 h of culture, serum from the patient did not increase the cell count. As in the phosphorylation studies, samples from heterozygous subjects had intermediate proliferation effects.

Discussion

We describe a patient with severe short stature born from consanguineous parents with a novel homozygous substitution of a highly conserved tyrosine residue in the A domain of IGF1. This is, to our knowledge, the sixth IGF1 variant reported in the literature (8, 9, 10, 11, 12), and the third associated with marked pre- and postnatal growth restriction, microcephaly, micro/retrognathia, sensorineural deafness and intellectual deficit (8, 9). In addition to the three previous well documented cases of patients with IGF1 defects that were supported by functional analysis (8, 9, 10), two other IGF1 variants have been reported: one (11) was later shown to be a non-pathogenic variant (29). In the other (12), a homozygous IGF1 mutation is reported in two members of a Saudi Arabian family, of whom some clinical (Table 4) but no functional or in silico data are available. A substitution of Arginine by Tryptophan is reported in a position of the mature IGF1 peptide that is not highly conserved. Using ACMG guidelines, we found that this mutation is classified as a variant of uncertain significance (VUS).

Table 4

Summary of phenotypic and biochemical features in homozygous IGF1 defects.

Patients with homozygous variants in the IGF1 gene
Current patientWoods et al. (8)Walenkamp et al. (9)Netchine et al. (10)Bonapace et al. (11)Shaheen et al. (12)
IGF1 variantsc.322T>C,p.Tyr108HisDeletion exons 4 and 5c.274G>A,p.Val92Met*c.251G>A,p.Arg84Glnc.*263A>Tc.292C>T, p.Arg98Trp
IGF1 domainA DomainTruncated proteinA DomainC DomainUCPASA Domain
Statushomozygoushomozygoushomozygoushomozygoushomozygoushomozygous
SexMaleMaleMaleMaleMaleFemale and Male
Birth weight (SDS)−3.05−3.9−3.9−2.4−4.1F: −3.5; M: NA
Birth length (SDS)−6.3−5.4−4.3−3.7−6.5NA
Age3.215.85511.61.6F: 2.8; M: 5
Weight (SDS)−5.1−6.5NANA−6.2F: −4.1; M: −4.9
Height (SDS)−6.15−6.9−8.5−1.9−6.2F: −3.2; M: −4.3
Parents consanguinity+++++NA
Mother height (SDS)−1.95−1.4+0.3−2.95th centileNA
Father height (SDS)−2.6−1.8−2.4−1.05th centileNA
Head circumference (SDS)−6.05−4.9−8.0−2.5 at 11 months

−4.8 at 9 years
<5th centileF: −4.7; M: −5.4
DeafnessPresentPresentPresentAbsentPresentNA
Mental retardationMildSevereSevereMildMildAbsent
DysmorphiasFrontal bossing, triangular face, bulbous nose, full lips, and retrognathiaMicrognathia, bilateral ptosis, low hairline, bilateral clinodactylyDeep-set eyes, flat occiput, micrognathia, broad end phalanges, convex nailsUnilateral clinodactylyNANA
IGF1 (SDS)−1.15 to +2.95Undetectable+7.3Undetectable to >2§LowNA
IGFBP3 (SDS)−1.58 to +2.31NA
IGFBP3 (mg/l)

3.3 (normal)


1.98 (normal)


7.2 (elevated)


3.6 (normal)
Basal GH levels (ng/ml)Normal-High (9, range 0.2–29)Elevated (18)Elevated (13)Normal (0.7)Elevated (10)NA

*Originally published as Val44Met (V44M); originally published as Arg36Gln (R36Q); UCPAS, upstream core polyadenylation signal; §IGF1 serum concentrations using different assays (with monoclonal/polyclonal antibodies).

NA, not available.

The missense variant presented in this report resulted in a substitution for histidine of a highly conserved tyrosine residue, located in a region of the A domain of IGF1 that has been reported to be critical for its interaction with IGF1R (27). Previous cases with such a severe phenotype presented undetectable (8) or, on the opposite, very high (9) IGF1 levels in circulation. Conversely, our patient had normal to mildly elevated serum IGF1, underscoring the importance of considering the heterogeneity of the presentation of IGF1 deficiency to avoid misled diagnoses in patients with short stature.

The change of the highly conserved Tyr108 residue (Tyr60 in the mature IGF1 peptide) was consistently predicted as pathogenic by multiple bioinformatic tools. In silico analysis predicts that this substitution may lead to conformational changes in the receptor binding domain that in turn could destabilize the contact of IGF1 with its receptor (27). Evidence of a decreased ability to activate the IGF1R was obtained in vitro using serum from our patient, which proved that phosphorylation of the IGF1R was diminished as compared to age-matched controls. Furthermore, Tyr108His-IGF1 also induced cell growth less efficiently than IGF1 from controls. Early work in the 1990s searching for determinants on human IGF1 for its receptors and binding proteins had identified the Tyr60 of the mature IGF1 (i.e. Tyr108 replaced by a histidine in our patient) as a conserved residue with paramount relevance in the maintenance of binding to its receptor (27). Other reports showed that substitution of valine by methionine in position 92 (Met 44 in the mature protein), also in the A domain (9), substantially impaired IGF1R binding by a factor of 90. On the other hand, residues involved in the interaction with IGFBP3 (27, 28, 30) should not be affected according to the molecular model. Recombinant IGF1 peptides with modifications such as p.Arg84Gln and p.Val92Met have normal binding to IGFBP3 (9, 10). Accordingly, in our study, in vitro experiments showed that when IGFBPs were removed from the patient’s serum by acid chromatography, the phosphorylated band of IGF1R was stronger than in the presence of IGFBPs, suggesting that in unextracted serum, IGF1 was mainly bound to its binding proteins. Taken together, our results show that the substitution of tyrosine 108 by histidine in the mature IGF1 peptide renders the molecule highly likely to have a reduced affinity for IGF1R.

Several authors (31, 32, 33) have reported short stature and/or microcephaly in patients carrying heterozygous IGF1 mutations. Walenkamp et al (9) also described the impact of p.Val92Met-IGF1 on a wide pedigree of heterozygous carriers. In the present case, auxological data of the parents and half-brother could suggest that heterozygosity for the mutation leads to mild to moderate short stature and microcephaly. Unfortunately, we did not have the possibility to study any other member of this family to further support this observation.

The first patient with severe IGF1 deficiency was reported in 1996 by Woods et al (8), describing a male born from consanguineous parents with a homozygous deletion of exon 4 and 5 of IGF1 gene resulting a severely truncated IGF1 protein (Table 4). GH levels were elevated, and serum IGF1 was undetectable at baseline and after a 4-day IGF1 generation test with normal levels of IGFBP3 and ALS. Clinical presentation but not biochemical features were very similar to the patient described here. In 2005, Walenkamp et al (9) described a 55-year-old male with a severe IGF1 deficiency phenotype, with final height of 118 cm (Table 4). Molecular analysis revealed a homozygous missense mutation in IGF1 gene predicting the change of valine 92 into methionine. Interestingly, IGF1 levels were at + 7.3 SDS, and functional studies showed an about 90-fold decreased affinity of IGF1 for its receptor, which could explain the high levels of IGF1. Our patient had clinical features that could relate to a very low activity of IGF1, such as marked pre and postnatal growth retardation, microcephaly, sensorineural deafness and mental retardation. However, his IGF1 levels were normal or just above normal range. Therefore, patients with IGF1 molecular defects and a very severe clinical presentation, could have substantially different IGF1, IGFBP3, ALS and GH serum profiles. Thus, serum IGF1 could be elusive in the diagnosis of a child with IUGR, sensorineural deafness and severe postnatal growth failure.

The third patient, reported by Netchine et al. (10), was a boy with pre- and postnatal growth failure, but he had normal hearing and only a mild developmental delay (Table 4). IGF1 levels were variable between undetectable to +2 SDS measured by different assays. IGFBP3 and ALS, in the upper normal range. IGF1 gene showed a homozygous missense mutation that predicted the change of a highly conserved arginine located in C domain of the protein into a glutamine. The affinity of IGF1 for IGF1R was decreased by approximately four-fold, leading to diminished IGF1R phosphorylation. The patient reported in our study showed a more severe phenotype, but with a very similar GH/IGF1 axis serum profile. Therefore, our results suggest that similar serum profiles for GH/IGF axis could be associated with variable clinical features. Furthermore, molecular defects in IGF1R have been reported in patients with similar clinical features, including IUGR and postnatal growth failure, despite variable GH-IGFs axis profiles. Heterozygous mutations in the IGF1R gene lead to incomplete resistance to IGF1, IUGR and postnatal growth failure, microcephaly, modest impairment of mental development and normal hearing, associated with normal or increased levels of IGF1 and IGFBP3 (34, 35).

In summary, many clinical and biochemical features may overlap within molecular defects in different genes belonging to the same signaling path. The report of our patient expands the spectrum of presentation of IGF1 defects and shows the variability these patients may present depending on the IGF/IGF1R pathway activity.

Declaration of interest

I certify that neither I nor my co-authors have a conflict of interest that is relevant to the matter or materials included in this work.

Funding

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas, República Argentina, Grant PUE CEDIE # 131-2017; Instituto Nacional del Cáncer, Ministerio de Salud de la Nación, República Argentina, Grant 2015 (P A P); FIS 12/00649 from ISCIII, Spain and EU FEDER Funding (to A C B) and ENDOSCREEN S2010/BMD-2396, from Comunidad de Madrid, Spain & EU FEDER FUNDING (to P L-B, J N B, and A C B).

Acknowledgements

The authors wish to thank Dr Saul Malozowski for his helpful discussion and revision of the manuscript. They also would like to thank Dr Mariana Del Pino for providing references and calculating SDS for SH/H ratio. Ignacio Bergadá and Patricia A Pennisi: Joint senior authors.

References

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    Netchine I, Azzi S, Le Bouc Y, Savage MO. IGF1 molecular anomalies demonstrate its critical role in fetal, postnatal growth and brain development. Best Practice and Research. Clinical Endocrinology and Metabolism 2011 181190. (https://doi.org/10.1016/j.beem.2010.08.005)

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    Agrogiannis GD, Sifakis S, Patsouris ES, Konstantinidou AE. Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review). Molecular Medicine Reports 2014 579584. (https://doi.org/10.3892/mmr.2014.2258)

    • Search Google Scholar
    • Export Citation
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    Hellström A, Ley D, Hansen-Pupp I, Hallberg B, Löfqvist C, van Marter L, van Weissenbruch M, Ramenghi LA, Beardsall K, Dunger D et al. Insulin-like growth factor 1 has multisystem effects on foetal and preterm infant development. Acta Paediatrica 2016 576586. (https://doi.org/10.1111/apa.13350)

    • Search Google Scholar
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    Livingstone C. Insulin-like growth factor-I (IGF-I) and clinical nutrition. Clinical Science 2013 265280. (https://doi.org/10.1042/CS20120663)

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    Rosenfeld RG, Hwa V. The growth hormone cascade and its role in mammalian growth. Hormone Research 2009 3640. (https://doi.org/10.1159/000192434)

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    Woods KA, Camacho-Hübner C, Savage MO, Clark AJL. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. New England Journal of Medicine 1996 13631367. (https://doi.org/10.1056/NEJM199610313351805)

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    Walenkamp MJE, Karperien M, Pereira AM, Hilhorst-Hofstee Y, van Doorn J, Chen JW, Mohan S, Denley A, Forbes B & van Duyvenvoorde HA Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. Journal of Clinical Endocrinology and Metabolism 2005 28552864. (https://doi.org/10.1210/jc.2004-1254)

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  • 11

    Bonapace G, Concolino D, Formicola S, Strisciuglio P. A novel mutation in a patient with insulin-like growth factor 1 (IGF1) deficiency. Journal of Medical Genetics 2003 913917. (https://doi.org/10.1136/jmg.40.12.913)

    • Search Google Scholar
    • Export Citation
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    Shaheen R, Faqeih E, Ansari S, Abdel-Salam G, Al-Hassnan ZN, Al-Shidi T, Alomar R, Sogaty S, Alkuraya FS. Genomic analysis of primordial dwarfism reveals novel disease genes. Genome Research 2014 291299. (https://doi.org/10.1101/gr.160572.113)

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    Lejarraga H, del Pino M, Fano V, Caino S, Cole TJ. [Growth references for weight and height for Argentinian girls and boys from birth to maturity: incorporation of data from the World Health Organisation from birth to 2 years and calculation of new percentiles and LMS values]. Archivos Argentinos de Pediatria 2009 126133. (https://doi.org/10.1590/S0325-00752009000200006)

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    del Pino M, Orden AB, María A, Arenas VF. Referencias argentinas para la evaluación de proporciones corporales desde el nacimiento hasta los 17 años Argentine references for the assessment of body proportions from birth to 17 years of age. Archivos Argentinos de Pediatria 2017 234240. (https://doi.org/10.5546/aap.2017.234)

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    Fredriks AM, van Buuren S, Burgmeijer RJ, Meulmeester JF, Beuker RJ, Brugman E, Roede MJ, Verloove-Vanhorick SP, Wit JM. Continuing positive secular growth change in the Netherlands 1955–1997. Pediatric Research 2000 316323. (https://doi.org/10.1203/00006450-200003000-00006)

    • Search Google Scholar
    • Export Citation
  • 16

    Kuczmarski RJ, Ogden CL, Guo SS, Grummer-Strawn LM, Flegal KM, Mei Z, Wei R, Curtin LR, Roche AF, Johnson C. CDC growth charts for the United States: methods and development. National Center for Health Statistics. In Vital and Health Statistics. Series 11 (246) 2002 .

    • Search Google Scholar
    • Export Citation
  • 17

    Kelly A, Winer KK, Kalkwarf H, Oberfield SE, Lappe J, Gilsanz V, Zemel BS. Age-based reference ranges for annual height velocity in US children. Journal of Clinical Endocrinology and Metabolism 2014 21042112. (https://doi.org/10.1210/jc.2013-4455)

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    • Export Citation
  • 18

    Ballerini MG, Braslavsky D, Scaglia PA, Keselman A, Rodríguez ME, Martínez A, Freire AV, Domené HM, Jasper HG & Bergadá I Circulating IGF-I, IGFBP-3 and the IGF-I/IGFBP-3 molar ratio concentration and height outcome in prepubertal short children on rhGH treatment over two years of therapy. Hormone Research in Paediatrics 2017 354363. (https://doi.org/10.1159/000479691)

    • Search Google Scholar
    • Export Citation
  • 19

    Ballerini MG, Domené HM, Scaglia P, Martínez A, Keselman A, Jasper HG, Ropelato MG. Association of serum components of the GH-IGFs-IGFBPs system with GHR-exon 3 polymorphism in normal and idiopathic short stature children. Growth Hormone and IGF Research 2013 229236. (https://doi.org/10.1016/j.ghir.2013.08.003)

    • Search Google Scholar
    • Export Citation
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    Scaglia PA, Keselman AC, Braslavsky D, Martucci LC, Karabatas LM, Domené S, Gutiérrez ML, Ballerini MG, Ropelato MG & Spinola-Castro A Characterization of four Latin American families confirms previous findings and reveals novel features of acid-labile subunit deficiency. Clinical Endocrinology 2017 300311. (https://doi.org/10.1111/cen.13361)

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    • Export Citation
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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.

  • View in gallery

    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.

  • View in gallery

    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.

  • 1

    Lund PK. Insulin-like growth factor I: molecular biology and relevance to tissue-specific expression and action. Recent Progress in Hormone Research 1994 125148. (https://doi.org/10.1016/B978-0-12-571149-4.50010-6)

    • Search Google Scholar
    • Export Citation
  • 2

    Netchine I, Azzi S, Le Bouc Y, Savage MO. IGF1 molecular anomalies demonstrate its critical role in fetal, postnatal growth and brain development. Best Practice and Research. Clinical Endocrinology and Metabolism 2011 181190. (https://doi.org/10.1016/j.beem.2010.08.005)

    • Search Google Scholar
    • Export Citation
  • 3

    Agrogiannis GD, Sifakis S, Patsouris ES, Konstantinidou AE. Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review). Molecular Medicine Reports 2014 579584. (https://doi.org/10.3892/mmr.2014.2258)

    • Search Google Scholar
    • Export Citation
  • 4

    Hellström A, Ley D, Hansen-Pupp I, Hallberg B, Löfqvist C, van Marter L, van Weissenbruch M, Ramenghi LA, Beardsall K, Dunger D et al. Insulin-like growth factor 1 has multisystem effects on foetal and preterm infant development. Acta Paediatrica 2016 576586. (https://doi.org/10.1111/apa.13350)

    • Search Google Scholar
    • Export Citation
  • 5

    Ranke MB, Wit JM. Growth hormone – past, present and future. Nature Reviews Endocrinology 2018 285300. (https://doi.org/10.1038/nrendo.2018.22)

    • Search Google Scholar
    • Export Citation
  • 6

    Livingstone C. Insulin-like growth factor-I (IGF-I) and clinical nutrition. Clinical Science 2013 265280. (https://doi.org/10.1042/CS20120663)

    • Search Google Scholar
    • Export Citation
  • 7

    Rosenfeld RG, Hwa V. The growth hormone cascade and its role in mammalian growth. Hormone Research 2009 3640. (https://doi.org/10.1159/000192434)

    • Search Google Scholar
    • Export Citation
  • 8

    Woods KA, Camacho-Hübner C, Savage MO, Clark AJL. Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. New England Journal of Medicine 1996 13631367. (https://doi.org/10.1056/NEJM199610313351805)

    • Search Google Scholar
    • Export Citation
  • 9

    Walenkamp MJE, Karperien M, Pereira AM, Hilhorst-Hofstee Y, van Doorn J, Chen JW, Mohan S, Denley A, Forbes B & van Duyvenvoorde HA Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation. Journal of Clinical Endocrinology and Metabolism 2005 28552864. (https://doi.org/10.1210/jc.2004-1254)

    • Search Google Scholar
    • Export Citation
  • 10

    Netchine I, Azzi S, Houang M, Seurin D, Perin L, Ricort JM, Daubas C, Legay C, Mester J & Herich R Partial primary deficiency of insulin-like growth factor (IGF)-I activity associated with IGF1 mutation demonstrates its critical role in growth and brain development. Journal of Clinical Endocrinology and Metabolism 2009 39133921. (https://doi.org/10.1210/jc.2009-0452)

    • Search Google Scholar
    • Export Citation
  • 11

    Bonapace G, Concolino D, Formicola S, Strisciuglio P. A novel mutation in a patient with insulin-like growth factor 1 (IGF1) deficiency. Journal of Medical Genetics 2003 913917. (https://doi.org/10.1136/jmg.40.12.913)

    • Search Google Scholar
    • Export Citation
  • 12

    Shaheen R, Faqeih E, Ansari S, Abdel-Salam G, Al-Hassnan ZN, Al-Shidi T, Alomar R, Sogaty S, Alkuraya FS. Genomic analysis of primordial dwarfism reveals novel disease genes. Genome Research 2014 291299. (https://doi.org/10.1101/gr.160572.113)

    • Search Google Scholar
    • Export Citation
  • 13

    Lejarraga H, del Pino M, Fano V, Caino S, Cole TJ. [Growth references for weight and height for Argentinian girls and boys from birth to maturity: incorporation of data from the World Health Organisation from birth to 2 years and calculation of new percentiles and LMS values]. Archivos Argentinos de Pediatria 2009 126133. (https://doi.org/10.1590/S0325-00752009000200006)

    • Search Google Scholar
    • Export Citation
  • 14

    del Pino M, Orden AB, María A, Arenas VF. Referencias argentinas para la evaluación de proporciones corporales desde el nacimiento hasta los 17 años Argentine references for the assessment of body proportions from birth to 17 years of age. Archivos Argentinos de Pediatria 2017 234240. (https://doi.org/10.5546/aap.2017.234)

    • Search Google Scholar
    • Export Citation
  • 15

    Fredriks AM, van Buuren S, Burgmeijer RJ, Meulmeester JF, Beuker RJ, Brugman E, Roede MJ, Verloove-Vanhorick SP, Wit JM. Continuing positive secular growth change in the Netherlands 1955–1997. Pediatric Research 2000 316323. (https://doi.org/10.1203/00006450-200003000-00006)

    • Search Google Scholar
    • Export Citation
  • 16

    Kuczmarski RJ, Ogden CL, Guo SS, Grummer-Strawn LM, Flegal KM, Mei Z, Wei R, Curtin LR, Roche AF, Johnson C. CDC growth charts for the United States: methods and development. National Center for Health Statistics. In Vital and Health Statistics. Series 11 (246) 2002 .

    • Search Google Scholar
    • Export Citation
  • 17

    Kelly A, Winer KK, Kalkwarf H, Oberfield SE, Lappe J, Gilsanz V, Zemel BS. Age-based reference ranges for annual height velocity in US children. Journal of Clinical Endocrinology and Metabolism 2014 21042112. (https://doi.org/10.1210/jc.2013-4455)

    • Search Google Scholar
    • Export Citation
  • 18

    Ballerini MG, Braslavsky D, Scaglia PA, Keselman A, Rodríguez ME, Martínez A, Freire AV, Domené HM, Jasper HG & Bergadá I Circulating IGF-I, IGFBP-3 and the IGF-I/IGFBP-3 molar ratio concentration and height outcome in prepubertal short children on rhGH treatment over two years of therapy. Hormone Research in Paediatrics 2017 354363. (https://doi.org/10.1159/000479691)

    • Search Google Scholar
    • Export Citation
  • 19

    Ballerini MG, Domené HM, Scaglia P, Martínez A, Keselman A, Jasper HG, Ropelato MG. Association of serum components of the GH-IGFs-IGFBPs system with GHR-exon 3 polymorphism in normal and idiopathic short stature children. Growth Hormone and IGF Research 2013 229236. (https://doi.org/10.1016/j.ghir.2013.08.003)

    • Search Google Scholar
    • Export Citation
  • 20

    Del Sal G, Manfioletti G, Schneider C. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. BioTechniques 1989 514520.

    • Search Google Scholar
    • Export Citation
  • 21

    Scaglia PA, Keselman AC, Braslavsky D, Martucci LC, Karabatas LM, Domené S, Gutiérrez ML, Ballerini MG, Ropelato MG & Spinola-Castro A Characterization of four Latin American families confirms previous findings and reveals novel features of acid-labile subunit deficiency. Clinical Endocrinology 2017 300311. (https://doi.org/10.1111/cen.13361)

    • Search Google Scholar
    • Export Citation
  • 22

    Arias-Salgado EG, Galvez E, Planas-Cerezales L, Pintado-Berninches L, Vallespin E, Martinez P, Carrillo J, Iarriccio L, Ruiz-Llobet A & Catalá A Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes. Orphanet Journal of Rare Diseases 2019 82. (https://doi.org/10.1186/s13023-019-1046-0)

    • Search Google Scholar
    • Export Citation
  • 23

    Brzozowski AM, Dodson EJ, Dodson GG, Murshudov GN, Verma C, Turkenburg JP, De Bree FM, Dauter Z. Structural origins of the functional divergence of human insulin-like growth factor-I and insulin. Biochemistry 2002 93899397. (https://doi.org/10.1021/bi020084j)

    • Search Google Scholar
    • Export Citation
  • 24

    Mohan S, Nakao Y, Honda Y, Landale E, Leser U, Dony C, Lang K, Baylink DJ. Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells. Journal of Biological Chemistry 1995 2042420431. (https://doi.org/10.1074/jbc.270.35.20424)

    • Search Google Scholar
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  • 25

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