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Divisions of Pediatrics and Perinatology (Y.K., S.K., T.K., K.H., J.N.) and Molecular Medicine and Therapeutics (I.H.) and Department of Neurobiology (H.N.), Faculty of Medicine and Gene Research Center (E.N.), Tottori University, Yonago 683-8504, Japan; Mitsubishi Kagaku BCL, Inc. (Y.O.), Tokyo 174-8555, Japan; Department of Animal Sciences and Applied Biological Chemistry (T.F., S.-I. T.), The University of Tokyo, Tokyo 113-8657, Japan; and Department of Pediatrics (F.Y.), West China Second University Hospital, Sichuan University, Chengdu 610041, China
Address all correspondence and requests for reprints to: Susumu Kanzaki, M.D., Ph.D., Division of Pediatrics and Perinatology, Tottori University, 36-1, Nishi-machi, Yonago 683-8504, Japan. E-mail: smkanzak{at}grape.med.tottori-u.ac.jp.
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Abstract |
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Objective: We hypothesized that mutations of IGF-IR gene might predispose to short stature in children born with intrauterine growth retardation (IUGR).
Patients: Twenty-four children with unexplained IUGR (birth weight < –1.5 SD) and short stature (<–2.0 SD) were screened for abnormalities of the IGF-IR gene.
Methods: Direct DNA sequencing was used to identify IGF-IR gene mutations. Unprocessed IGF-IR proreceptor in fibroblasts was detected by immunoblot analysis. Functions of mutated IGF-IR in fibroblasts were evaluated by IGF-I binding, and IGF-I-stimulated DNA synthesis and ß-subunit autophosphorylation.
Results: We found the following results: 1) a heterozygous mutation (R709Q) changing the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Gln-Arg was identified in a 6-yr-old Japanese girl (case 1) and her mother who also had IUGR with short stature (case 2); 2) fibroblasts from case 2 contained more IGF-IR proreceptor protein (189 ± 26% of normal) and less mature ß-subunit protein (63 ± 12%); 3) [125I]IGF-I binding to fibroblasts from case 2 was reduced, compared with normal control (0.61 ± 0.16 x 106 vs. 1.14 ± 0.12 x 106 sites per cell; P < 0.05); and 4) both IGF-I-stimulated [3H]thymidine incorporation and IGF-IR ß-subunit autophosphorylation were low in fibroblasts from case 2, compared with those of control (P < 0.05).
Conclusions: These findings strongly suggest that this mutation leads to failure of processing of the IGF-IR proreceptor to mature IGF-IR and causes short stature and IUGR.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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2/ß2 heterotetramer structurally related to the insulin receptor (IR). IGF-I is known to mediate the growth-promoting effects of GH in postnatal life. Mouse knockout models have clearly demonstrated the critical importance of IGF-I and IGF-IR for embryonic growth; mice homozygous null for Igf-i or Igf-ir having birth weights 60 and 45% of those of wild-type mice, respectively (3, 4). Intrauterine growth retardation (IUGR) is a common condition in which fetal events constrain the birth size. Although 15–20% of children born with IUGR are at risk of being short as adults (5, 6), the mechanism of postnatal growth retardation is not fully understood. Ducos et al. (7) and Cutfield et al. (8) reported that higher plasma concentrations of IGF-I, IGF-II, and IGF binding protein (IGFBP)-3 were observed in short children with IUGR. These studies also showed that the affinity of erythrocyte IGF-IR to IGF-I was reduced in 17 short children born with IUGR. These data suggest that short children with IUGR might have at least partial resistance to IGF-I. Therefore, it is hypothesized that mutations of the IGF-IR gene might predispose to short stature in children born with IUGR.
In regard to IR, which is structurally related to IGF-IR, more than 40 gene mutations are reported to cause insulin-resistant diabetes mellitus (9). In contrast, little has been reported on studies of the IGF-IR gene in short-stature children born with IUGR, although there are sporadic reports of deletions encompassing IGF-IR in IUGR patients (10, 11). Recently IGF-IR mutations have been reported in only two patients with short stature born with IUGR (12).
In this report, we analyzed the nucleotide sequence of the IGF-IR gene in 24 patients with IUGR and short stature. A novel missense mutation at the cleavage site of IGF-IR (R709Q) was found in this study. We speculate that this mutant IGF-IR, which could not be cleaved to a mature receptor, leads to IGF-I resistance.
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Subjects and Methods |
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Table 1
shows the characteristics of the subjects. We studied 24 Japanese patients between 3 and 24 yr of age (eight boys and 16 girls) with short stature (below mean –2.0 SD) associated with IUGR. IUGR was defined as a birth weight less than mean –1.5 SD for gestational age according to the commonly used definition of IUGR in Japan. Subjects having chromosomal anomalies or intrauterine infections were excluded. Their mean birth weight was –2.2 SD (range –4.0 SD to –1.5 SD), and their mean birth height was –2.1 SD (range –5.4 SD to –0.8 SD). Four patients showed low peak plasma GH levels (<10 ng/ml) after two conventional stimulation tests and were treated with recombinant human GH. The control subjects were nine Japanese subjects (four boys and five girls) with normal stature born with IUGR and 20 Japanese subjects (eight men and 12 women) with normal stature not born with IUGR. The mean birth weight and height for normal controls born with IUGR were –2.5 SD (range –4.3 to –1.7 SD) and –1.9 SD (range –2.9 to –0.3 SD), respectively. Those for normal controls not born with IUGR were –0.2 SD (range –1.0 to 2.4 SD) and –0.9 SD (range –2.2 to –0.2 SD), respectively. Their mean height was –0.7 SD (range –1.7 SD to 1.6 SD).
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Materials
Recombinant human IGF-I was donated by Fujisawa Pharmaceutical Co. (Osaka, Japan). [125I]IGF-I was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK).
DNA sequence analysis of IGF-IR gene
Genomic DNA was isolated from peripheral blood lymphocytes and amplified by PCR using 27 pairs of primers that flank the coding regions of the 21 exons of the IGF-IR gene. PCR primers were designed based on published sequences (Fig. 1 and Table 2) (13). PCR for the exons except exon 1 was performed using an Ampli Taq Gold (PE Applied Biosystems, Foster City, CA) with 100 ng genomic DNA as follows: 1 cycle of 94 C for 4 min, followed by 30 cycles of 94 C for 30 sec, 55 or 58 C for 30 sec, and 72 C for 90 sec, with a final cycle of 72 C for 5 min. PCR for exon 1 was performed using LA Taq with GC buffer (Takara Shuzo, Kyoto, Japan) instead of Ampli Taq Gold. Each PCR product was sequenced using an automated DNA sequencer [Dye terminator mix (ABI PRISM 3100 genetic analyzer, Applied Biosystems, Tokyo, Japan)]. DNA sequences were compared with the published IGF-IR sequence (accession no. IGF-IR gene NT035325 and NM000875).
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We identified the R709Q mutation in exon 11 of the IGF-IR gene in one patient. For identification of the R709Q substitution, a 338-bp region of the IGF-IR gene covering this mutation in exon 11 was amplified by PCR using the primers for exon 11 (Fig. 1
) in this patient, her parents, and an unrelated individual (normal control). The PCR product was digested with AciI (New England Biolabs, Inc., Beverly, MA). Cleaved PCR products were fractionated on 2–3% agarose gels, stained with 10 µg/ml ethidium bromide, and visualized.
Case reports
Case 1 (proband).
The patient was a 6-yr-old Japanese girl. She was the third child of her parents. The height of her family members is shown in Fig. 2. Her father and brothers were not born with IUGR and had normal stature. The pregnancy was not complicated. She was born at 40 wk gestation, with a birth weight of 2686 g (–1.5 SD) and birth height of 48 cm (–1.0 SD). She was healthy except for IUGR at birth. At the age of 2 yr, she was noticed to have growth failure. Her growth curve is shown in Fig. 2A. Her height was 102 cm (–2.1 SD) and weight 13 kg (–2.2 SD) at 6 yr of age. Her bone age was 3.9 yr when she was 6 yr old. She was diagnosed as having mental retardation at age 6 yr, and intelligence test (Wechsler Intelligence Scale for Children III) showed an intelligence quotient of 60. Serum levels of IGF-I and IGFBP-3 at age 5 yr were 208 ng/ml (1.5 SD) and 2.22 µg/ml (–1.0 SD), respectively. Serum-free T4, free T3, and TSH levels were within the normal range for her age.
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). Fibroblast culture
Fibroblasts were obtained from the left axillary fold in case 2, the mother of the proband (case 1), bearing the same IGF-IR gene mutation and phenotype, and an unrelated normal individual aged 8 yr, with written informed consent. Fibroblasts were maintained in DMEM (Invitrogen Co., Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Nichirei Co., Tokyo, Japan) at 37 C in a humidified atmosphere with 5% CO2. Cells were subcultured and then used for experiments 2–3 d after reaching confluence.
Binding assay of IGF-I to IGF-IR in fibroblasts
For the IGF-I-binding study, we used fibroblasts from case 2 and an unrelated individual. The fibroblasts (2 x 104/well) were plated in 24-well plates and cultured in DMEM containing 10% FBS at 37 C for more than 12 h in a humidified atmosphere with 5% CO2 until they became quiescent. The fibroblasts were washed three times with Hanks’ balanced salt solution (HBSS) and starved for 16 h in serum-free medium (DMEM containing 0.1% BSA). Then they were washed twice with ice-cooled PBS and incubated for 7 h at 4 C in 0.5 ml PBS with 0.1% BSA containing 20,000–40,000 cpm [125I]IGF-I and various amounts of unlabeled IGF-I, as previously described (14, 15, 16). After washing three times with ice-cooled PBS, the residual radioactivity was recovered in 0.5 ml of 0.1 N NaOH. Radioactivity recovered in NaOH was defined as cell surface radioactivity. Nonspecific binding in the presence of a 100-fold excess of unlabeled IGF-I was always less than 5% of the total radioactivity recovered. Bound IGF-I count (molecules per cell) was obtained as the mean count of three replicate wells. The number of binding sites and dissociation constant (Kd) values were calculated according to Scatchard plot analysis (17).
Immunoblot analysis of IGF-IR using anti-IGF-IR ß-subunit antibody
Semiconfluent fibroblasts in 60-mm dishes from case 2 and an unrelated individual were harvested by a cell scraper and collected by centrifugation. The pellets were suspended in Laemmli’s sample buffer [10 mM Tris-HCl (pH 8.0), 5% glycerol, 3% sodium dodecyl sulfate, and 0.017% bromophenol blue] containing 2% ß-mercaptoethanol. Protein concentrations were determined using a BCA protein assay kit (Bio-Rad Laboratories, Hercules, CA). Then 10 µg protein was separated on a 7.5% gradient SDS-PAGE (Bio-Rad) and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in TBS-T [10 mM Tris-HCl (pH 8.0), 150 nM NaCl, and 0.1% Tween 20] overnight at 4 C. They were then incubated with either a 1:500 dilution of rabbit polyclonal anti-IGF-IR ß-antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or a 1:5000 dilution of mouse monoclonal antiactin antibody (EMD Biosciences Inc., Darmstadt, Germany) in TBS-T containing 5% skim milk for 45 min at room temperature. After washing three times with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences Corp., Piscataway, NJ) for 30 min at room temperature. After washing three times with TBS-T, the membranes were incubated with enhanced chemiluminescence reagents (Amersham Biosciences), and the chemiluminescence on the blots was detected. Densitometric analysis was performed using National Institutes of Health Image software (version 1.63).
Autophosphorylation assay of IGF-IR ß-subunit
Fibroblasts from case 2 and an unrelated individual were plated in 100-mm dishes (5 x 105 cells/well) and cultured in DMEM containing 10% FBS and maintained at 37 C in a humidified atmosphere with 5% CO2 until they became subconfluent. Fibroblasts were washed with HBSS and starved for 16 h in serum-free medium (DMEM containing 0.1% BSA). Then fibroblasts were stimulated with 0, 1, 10, 100, or 250 ng/ml IGF-I for 5 min. IGF-I-treated fibroblasts were lysed at 0 C in 0.3 ml lysis buffer solution [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM NaF, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 500 mM Na3VO4, 10 mg/ml leupeptin, 5 mg/ml pepstatin, 20 mg/ml phenylmethylsulfonyl fluoride, 100 kIU/ml aprotinin, and 10 mg/ml p-nitrophenyl phosphate]. The lysates were centrifuged at 3000 x g for 10 min at 4 C. The supernatants (200 µg protein) were incubated with anti-IGF-IR ß-antibody for 2 h at 4 C. Then 20 µl protein A-Sepharose (50% vol/vol; Amersham Biosciences) was added to the lysate, followed by incubation for 2 h at 4 C. At 4 C, the immunoprecipitates were collected by centrifugation, washed three times with 1 ml lysis buffer solution, and boiled for 5 min in a mixture of 60 µl lysis buffer and 30 µl of 3x Laemimli’s sample buffer. Each sample was subjected to SDS-PAGE, and proteins were transferred onto polyvinylidene fluoride membranes. Immunoblotting for phosphorylation was carried out as described above, except using 10 mM Tris-HCl (pH 7.2), 50 mM NaCl, 1 mM EDTA, 0.025% NaN3, and 3% BSA as blocking buffer, and anti-phosphotyrosine mouse monoclonal antibodies PY20 (Transduction Laboratories Inc., Lexington, KY).
DNA synthesis assay of fibroblasts
Fibroblasts from case 2 and an unrelated individual were incubated in 24-well plates at a concentration of 2.5 x 104 cells/well with DMEM containing 10% FBS. Subconfluent cells were washed three times with HBSS and starved for 29 h in DMEM containing 0.1% BSA. Quiescent cells were washed twice with HBSS and stimulated with various concentrations of IGF-I (0, 1, 5, 10, 50, 100 ng/ml). [Methyl-3H]thymidine (60 Ci/mmol; Amersham Biosciences) was added after 0, 4, 8, 12, 16, and 20 h of IGF-I treatment to give a final concentration of 1 µCi/ml. Cells were incubated with [methyl-3H]thymidine for 4 h. Ice-cold 1 M ascorbic acid was added to the wells, and the plates were left overnight. Cells were washed twice with PBS and fixed by incubation with 1 ml of 10% trichloric acid for 2 h at 4 C. The cells were washed with 10% trichloric acid twice. Cell lysates were solubilized with 0.1 M NaOH and 0.2% sodium dodecyl sulfate for over 2 h at 37 C, and the solubilized radioactivity was counted.
Statistical analysis
Conventional methods were used for comparisons (Student’s t test) and correlation and regression analysis. All experiments were performed at least three times, and means of triplicates are presented.
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Results |
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Table 2 shows the results of sequence analysis of the IGF-IR gene in patients with IUGR and short stature. We identified a missense mutation (R709Q) in one of 24 probands, five silent variants in the coding region of IGF-IR, three intron variants, and a deletion (4 bp) in 5'-untranslated region (UTR) of the IGF-IR gene. Three intron variants were also found in subjects with normal stature born with IUGR and control subjects (data not shown). The 4-bp deletion (CTTT) of 5'UTR, 45 bp upstream of the transcription initiation site of exon 1, was a homozygous mutation.
Missense mutation (R709Q) of IGF-IR gene
The missense mutation (R709Q), identified in a 6-yr-old girl with IUGR and short stature (case 1), was a heterozygous mutation that would change the cleavage site of the proreceptor of IGF-IR (proIGF-IR) from Arg-Lys-Arg-Arg to Arg-Lys-Glu-Arg due to a C to T point mutation of the antisense sequence (Fig. 3). This mutation resulted in loss of a restriction site for AciI and yielded a 103-bp band instead of 61- and 43-bp bands from PCR products of exon 11, which also included the adjacent upstream 22 bp of intron 10 and the adjacent downstream 27 bp of intron 11 (Fig. 4A). As shown in Fig. 4B, both the proband (case 1) and her mother (case 2), who was also diagnosed with IUGR and short stature, possessed the same mutation. However, her brothers and father, who were not born with IUGR and had normal stature, did not have this mutation.
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The identified heterozygous mutation changes the amino acid sequence of the cleavage site of the IGF-IR precursor. Because this mutation was presumed to cause failure of processing of proIGF-IR to mature IGF-IR, we measured the amount of unprocessed proIGF-IR and mature IGF-IR in fibroblasts from case 2 by immunoblotting using anti-IGF-IR ß-subunit antibody. As shown in Fig. 5A, fibroblasts from case 2 had more 200-kDa proIGF-IR and less 97-kDa processed ß-subunit, compared with fibroblasts from control subjects. By densitometric analysis, the protein level of the 200-kDa proIGF-IR in case 2 was 189 ± 26% of that of the normal control, whereas that of the 97-kDa ß-subunit of case 2 was decreased to 63 ± 12% of that of the normal control (Fig. 5B).
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The results of IGF-I binding to fibroblasts are shown in Fig. 6. To assess the possible interference of IGFBPs in the binding assay, an IGF-I displacement study for IGF-IR in fibroblasts of case 2 and normal subjects was performed. Labeled IGF-I in both case 2 and the control subject was similarly displaced by cold IGF-I (EC50 = 1.06 and 1.11 nM, respectively), thus showing that IGFBPs did not interfere significantly in the binding of IGF-I to its receptor (Fig. 6A). The maximum number of IGF-I binding sites in fibroblasts from case 2 was significantly lower than that of control subject (0.61 ± 0.16 x 106 sites/cell vs. 1.14 ± 0.12 x 106 sites/cell: P < 0.05) (Fig. 6B). However, we could not find significant difference in both the high-affinity Kd and low-affinity Kd value between case 2 (high-affinity Kd: 2.67 M, low-affinity Kd: 30.9 M) and the normal control subject (high-affinity Kd: 2.28 M, low-affinity Kd: 23.7 M).
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To clarify the function of mutated IGF-IR, we evaluated autophosphorylation of the IGF-IR ß-subunit in response to IGF-I in fibroblasts from case 2 and a normal control subject. Immunoprecipitates of IGF-I-stimulated cell lysates with anti-IGF-IR ß-antibody were subjected to Western blotting analysis using antiphosphotyrosine antibodies (Fig. 7). IGF-I treatment for 5 min increased tyrosine phosphorylation of a 97-kDa protein, which represents the processed IGF-IR ß-subunit, in both case 2 and the normal control in a dose-dependent manner. However, autophosphorylation of the IGF-IR ß-subunit stimulated by 100 and 250 ng/ml IGF-I was significantly lower in case 2 than in the normal control subject [IGF-I (100 ng/ml): 102 ± 32 vs. 242 ± 49%, P < 0.05; IGF-I (250 ng/ml): 139 ± 40 to 340 ± 120%; P < 0.05]. Phosphorylation was normalized for IGF-IR protein and expressed as a percentage of that of unstimulated lysate.
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To elucidate the function of mutated IGF-IR in IGF-I-stimulated cell proliferation, we measured IGF-I-dependent DNA synthesis in fibroblasts from case 2 and a control subject. Figure 8A shows [3H]thymidine incorporation into DNA in fibroblasts treated with IGF-I (50 ng/ml) for various times. IGF-I treatment increased [3H]thymidine incorporation of control fibroblasts, and the highest incorporation was observed after 20 h of incubation. However, there was no significant increase in [3H]thymidine incorporation in IGF-I-treated fibroblasts from case 2. Figure 8B shows the effect of IGF-I at different concentrations on [3H]thymidine incorporation into DNA in fibroblasts after 16–20 h of IGF-I treatment. IGF-I significantly induced DNA synthesis in fibroblasts from the control subject dose dependently. On the contrary, IGF-I treatment at all concentrations failed to increase [3H]thymidine incorporation in fibroblasts from case 2.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGF-IR consists of an 2/ß2 heterotetramer structurally related to IR. The -subunit bears the IGF binding site, whereas the ß-subunit possesses intrinsic tyrosine kinase activity. IGF-IR, like IR, is synthesized as a high-molecular-weight IGF-IR precursor protein, which is proteolytically processed into mature - and ß-subunits, and these subunits then link to each other by disulfide bonds. The cleavage site of proIGF-IR, containing the Arg-Lys-Arg-Arg peptide, belongs to a family of cleavage sites comprising pairs of basic amino acids that are involved in the posttranslational processing of many proteins (18, 19, 20). As shown in Fig. 9, the Arg-X (Lys)-Arg-Arg sequence is conserved in both IGF-IR and IR of various species (21, 22, 23, 24, 25). The conserved amino acid sequence of the cleavage site in these receptors strongly suggests that this amino acid sequence is essential for processing into mature - and ß-subunits.
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Several previous studies have revealed that anomalies of the cleavage site of the proreceptor of IR (proIR) affect the receptor function. Yoshimasa et al. (26) and Kobayashi and colleagues (27, 28, 29, 30, 31) found a homozygous point mutation in the IR gene, which changed Arg-Lys-Glu-Arg to Arg-Lys-Glu-Ser at the cleavage site of proIR, in patients with insulin-resistant diabetes mellitus . The 210-kDa unprocessed proIR instead of IR was observed on the surface of cells transfected with this mutated IR gene. It has been speculated that a structural change in the cleavage region is responsible for severe insulin resistance (29, 31). Williams et al. (32) synthesized an IR mutant lacking the cleavage site from the cDNA encoding the human proIR and transfected it into Chinese hamster ovary cells, with expression on the cell surface as uncleaved proIR. This uncleaved proIR on the cell surface exhibited markedly decreased affinity for insulin (32).
Lehmann et al. (33) reported that LoVo-C5 cells lacking furin, which cleaves proIGF-IR into - and ß-subunits, expressed a major high-molecular-mass proIGF-IR (200 kDa) instead of mature IGF-IR on the cell surface. [125I]IGF-I binding study using these LoVo-C5 cells showed that the affinity for IGF-I of each IGF-IR on these cells was not decreased, but the number of IGF-I binding sites on the cells was significantly lower. Therefore, these LoVo-C5 cells expressing proIGF-IR could not induce intracellular signaling such as ß-subunit tyrosine autophosphorylation (33). These reports strongly suggest that mutation at the cleavage site of proIGF-IR, as observed in our patients, may cause failure of intracellular signaling transduction and abolish IGF-IR functions such as cell growth, transformation, and development.
In our study using fibroblasts, the maximum number of IGF-I binding sites in fibroblasts from our patient (case 2) (0.61 ± 0.16 x 106 sites/cell) was half of that in fibroblasts from the control subject (1.14 ± 0.12 x 106 sites/cell). However, we could not find any difference in the affinity of each IGF-I binding site on fibroblasts between case 2 and the normal control. As for IGF-IR function, IGF-I treatment resulted in an apparently lower response of DNA synthesis in fibroblasts from case 2 (mut/wt) in vitro. These data suggest that the mutation in the cleavage site of proIGF-IR in our patients may have induced failure of IGF function due to a decreased number of IGF-I binding sites.
The most important question in our cases is whether a heterozygous mutation at the cleavage site of IGF-IR gene could cause manifestations of IGF-IR dysfunction such as short stature. In the prestigious work of Liu et al. (3) and Baker et al. (4), mice carrying heterozygous mutations of Igf-1r were phenotypically normal. However, Holzenberger et al. reported that heterozygous Igf-1r gene knockout mice grew along the –2 SD growth curve of normal mice (34). In the recent report by Abuzzahab et al. (12), a patient having the heterozygous point mutation CGA to TGA (Arg59stop) in exon 2 of the IGF-IR gene manifested IUGR with short stature. The growth pattern of that patient closely resembled that of our mutated IGF-IR proband. Moreover, Okubo et al. (35) reported that a patient bearing only a single copy of IGF-IR was born with IUGR and showed postnatal growth failure and recurrent hypoglycemia. These data support our speculation that heterozygous mutation of the IGF-IR gene can cause IUGR with short stature.
The mechanism by which a heterozygous IGF-IR gene mutation is able to cause IUGR with short stature remains unclear. Okubo et al. (35) recently reported that skin fibroblasts from subjects with only one copy of IGF-IR grew slowly, whereas fibroblasts from subjects with three copies of IGF-IR showed accelerated growth, compared with controls. They concluded that IGF-IR gene copy number is functionally and clinically important in humans. This suggests that IGF-IR dysfunction due to a heterozygous IGF-IR gene mutation is attributable to a haploinsufficiency effect. However, our study revealed that both IGF-I-induced IGF-IR ß-subunit phosphorylation and thymidine incorporation by IGF-I in fibroblasts from case 2 were extremely low, considering the number of IGF-I binding sites on the fibroblasts, about half of normal control. This may suggest that the anomaly in the cleavage site of proIGF-IR in our patients behaves as a dominant-negative inhibitor. One possibility is that unprocessed IGF-IR binds IGF-I, even with low affinity, and prevents the ligand from reaching the normal processed IGF-IR. de Lacerda et al. (36) reported a severely growth-retarded girl with ring chromosome 15 and deletion of a single allele of the IGF-IR gene. This girl showed IGF-I insensitivity in vivo, whereas the responsiveness of her fibroblasts to IGF-I was similar to that of control fibroblasts. Therefore, the fibroblast response may not be representative of that of other tissues, such as the growth plate. In vitro studies using transfected cells with our IGF-IR mutation are warranted to clarify the exact mechanism of IGF-IR dysfunction.
Another question raised by our study is the mental retardation in our proband case (case 1). Both IGF-I and -II are known to be important factors for brain development. In Igf-Ir gene knockout mice, Liu and colleagues (3, 4) observed central nervous system abnormalities as well as generalized organ hypoplasia. In humans, Woods et al. (37) reported a patient with deletion of the IGF-I gene who showed impaired mental development in addition to IUGR and postnatal growth failure. Abuzzahab et al. (12) also reported psychiatric disorder in patients with the mutated IGF-IR gene. These findings suggest that the mental retardation in case 1 may have been associated with the heterozygous IGF-IR mutation. However, her mother bearing the same IGF-IR gene mutation (case 2) had normal intellectual development. Thus, we cannot yet conclude an association between mental retardation in our case and the heterozygous IGF-IR mutation.
We identified one patient (case 1) with a biologically significant IGF-IR gene mutation from 24 patients with unexplained IUGR and short stature. Recently Abuzzahab et al. (12) reported two children with IUGR and short stature due to IGF-IR gene mutation. One patient, who had a compound heterozygous missense mutation (R108Q and K115N), was found among 42 children with unexplained IUGR and persistent short stature. Another patient with a heterozygous nonsense mutation (Arg59stop) was found among 50 children with short stature (<–2.5 SD) and high serum IGF-I levels (>2 SD). These findings suggest that the actual prevalence of IGF-IR mutations in IUGR with short stature might be considerably higher than previously thought. This conclusion is also supported by the fact that heterozygous IGF-IR gene mutation can manifest as IUGR with short stature, as in our cases.
In conclusion, we report a family with IUGR and short stature bearing a missense mutation at the cleavage site of IGF-IR. This IGF-IR mutation resulted in the failure of processing of proIGF-IR to mature IGF-IR and caused IGF-IR dysfunction. The results of this study provide new important information on the mechanism of short stature in children born with IUGR.
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Footnotes |
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First Published Online May 31, 2005
Abbreviations: FBS, Fetal bovine serum; HBSS, Hanks’ balanced salt solution; IGFBP, IGF binding protein; IGF-IR, IGF receptor type 1; IR, insulin receptor; IUGR, intrauterine growth retardation; Kd, affinity constant; proIGF-IR, proreceptor of IGF-IR; proIR, proreceptor of IR; TBS-T, Tris-buffered saline and Tween 20; UTR, untranslated region.
Received October 3, 2004.
Accepted May 19, 2005.
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References |
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Ser735 point mutation at the cleavage site. Metabolism 41:820–826[CrossRef][Medline]
T point mutation at the cleavage site of insulin receptors to COS7 cells. Biochem Biophys Res Commun 167:1073–1078[CrossRef][Medline]
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M. J. E. Walenkamp, H. J. van der Kamp, A. M. Pereira, S. G. Kant, H. A. van Duyvenvoorde, M. F. Kruithof, M. H. Breuning, J. A. Romijn, M. Karperien, and J. M. Wit A Variable Degree of Intrauterine and Postnatal Growth Retardation in a Family with a Missense Mutation in the Insulin-Like Growth Factor I Receptor J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3062 - 3070. [Abstract] [Full Text] [PDF]
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K. Raile, J. Klammt, A. Schneider, A. Keller, S. Laue, R. Smith, R. Pfaffle, J. Kratzsch, E. Keller, and W. Kiess Clinical and Functional Characteristics of the Human Arg59Ter Insulin-Like Growth Factor I Receptor (IGF1R) Mutation: Implications for a Gene Dosage Effect of the Human IGF1R J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2264 - 2271. [Abstract] [Full Text] [PDF]
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 P. J. Schlueter, T. Royer, M. H. Farah, B. Laser, S. J. Chan, D. F. Steiner, and C. Duan Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors FASEB J, June 1, 2006; 20(8): 1230 - 1232. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
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