Analysis of the insulin‐like growth factor 1 receptor gene in children born small for gestational age: in vitro characterization of a novel mutation (p.A rg511T rp)
Andrea C. LealLuciana Ribeiro MontenegroRenata de Freitas SaitoT.C. RibeiroDébora CoutinhoBerenice B. MendonçaIvo J.P. ArnholdAlexander A.L. Jorge
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Insulin-like growth factor 1 insensitivity caused by IGF1R mutations has been previously identified as one of the causes of growth impairment in children born small for gestational age (SGA).To analyse the IGF1R in children born SGA.From an initial cohort of 54 sequential children born SGA, without catch-up growth, 25 children were selected for this IGF1R study due to the presence of serum IGF-1 values above the mean for their age and sex.The proximal IGF1R promoter region, the entire coding region and the exon-intron boundaries were directly sequenced, and multiplex ligation-dependent probe amplification analysis was performed. Fibroblast cultures were developed from one patient with a mutation for the in vitro characterization of IGF-1 insensitivity.The copy number variation analysis did not identify deletions involving the IGF1R gene. We identified two children carrying heterozygous nucleotide substitutions in IGF1R: c.16G>A/p.Gly6Arg and c.1531C>T/p.Arg511Trp. The first variant (p.Gly6Arg) was identified in control subjects (0·3%) and in a relative with normal growth; thus, it was considered to be a rare benign allelic variation. The second variant (p.Arg511Trp) was not found in 306 alleles from control subjects, and it segregated with the growth impairment phenotype in the patient's family. Fibroblasts obtained from this patient had a significantly reduced proliferative response and AKT phosphorylation after IGF-1 stimulation compared with control fibroblasts.The identification of an inactivating IGF1R mutation in the present cohort should encourage further studies of larger series to establish the precise frequency of this molecular defect in children with growth impairment of a prenatal onset.Cite
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Insulin-like growth factor 1 (IGF-1) and its receptor (insulin-like growth factor 1 receptor, IGF1R) can regulate the extracellular matrix synthesis and play a crucial role in maintaining the normal functions of the intervertebral disc (IVD). The objective of this study was to investigate whether there would be accelerated IVD degeneration (IVDD) in IGF1R+/- mice. Three IGF1R+/- male mice and three wild-type male mice were sacrificed respectively at 6, 12, and 18 weeks after birth. Six lumbar disc samples were harvested from each mouse, with a total of 54 disc samples taken from each genotype. Histomorphological analysis for the IVD was performed to assess the degenerative extent according to the classification system proposed by Boos et al. Quantitative real-time PCR and semi-quantitative histologic scoring (HScore) for immunohistochemical staining were used to evaluate the expression level of type-II collagen, aggrecan and matrix metallopeptidase 13 (MMP-13). Histomorphological analysis for the discs revealed significantly less amounts of proteoglycan and type-II collagen, and significantly higher total degenerative score in IGF1R+/- mice than in wild-type mice. Real-time PCR showed that the mRNA expressions of type-II collagen and aggrecan in the discs were significantly lower, while MMP-13 was significantly higher in IGF1R+/- mice than in wild-type mice. The results of HScore analysis were similar to those obtained from the quantitative real-time PCR. Taken together, our study indicates that reduced expression of IGF1R would lead to accelerated degeneration of IVD. IGF1R+/- mice could be regarded as a good animal model to study IVD degeneration (IVDD), and studies on the IVD of IGF1R+/- mice could provide further insight into the pathogenesis of IVDD.
Degeneration (medical)
Intervertebral Disc
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The aim of this review was to describe all of the mutations in the growth hormone receptor (GHR) and insulin-like growth factor-1 receptor (IGF1R) genes that have been discovered so far, and their possible impact on final body height, as well as their relationship with catch-up growth in children born small for gestational age (SGA). Mutations in the GHR gene were found to cause a body height below −2 SD, from the mean for sex and age, whereas the mutations in the IGF1R gene were associated with low body height and intrauterine growth restriction (IUGR), and with being born SGA. After birth, when the child’s growth is not restricted by the intrauterine environment, the infant may develop its developmental potential and experience catch-up growth, which makes it possible to catch up with peers born appropriate for gestational age (AGA). Despite this, catch-up growth does not apply to all, but only to about 85% of SGA children, and its mechanism is unknown. It is possible that SGA children who did not experience catch-up growth are carriers of mutations in the GHR and/or IGF1R genes
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Abstract Introduction : Growth failure is a common consequence in small for gestational age (SGA) children. Patients and Methods : The growth patterns and serum insulin like growth factor 1 (IGF1) concentrations before and after the 1st year under growth hormone treatment of 32 short stature SGA born children have been evaluated. In addition, we investigated the insulin like growth factor 1 receptor (IGF1R) exon 2 as a hotspot for IGF1R genetic alterations. It is of note that no dysmorphic features were observed in this group of children. Results : The tests for pituitary reserve were within normal ranges for all 32 patients. Growth hormone (GH) treatment (0.037 mg/kg/day) was initiated at the mean age of 9.32±3.19 years. Growth velocity increased yearly from −1.80 SDS after the first year to −0.03 SDS in the sixth year of treatment. Their IGF1 serum concentrations before treatment were age and sex appropriate, while during treatment a significant increase was observed fitting in the upper third of the normal range: before the treatment IGF1 SDS was 0.84±1.78 after 1st year the concentrations increased to IGF1 SDS 0.94±2.23. No genetic alterations were found in the IGF1R exon 2 by PCR analysis. Conclusions : Herein we present 32 short stature SGA children with no dysmorphic features treated with GH. They all had increased growth velocity and entered the normal growth range on their growth charts. No side-effects were observed. GH treatment in children with no genetic alterations on the IGF1R exon 2 is safe and efficient in treating SGA children with short stature.
Growth hormone treatment
Growth velocity
Dwarfism
Somatomedin
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Abstract Objective . Our previous research demonstrated that, in contrast to normal chondrocytes, human osteoarthritic (OA) chondrocytes were hyporesponsive to stimulation by insulin‐like growth factor 1 (IGF‐1). The aim of the present investigation was to examine whether this finding was due to an alteration in the level of IGF receptors (IGFRs) and/or IGF binding proteins (IGFBP). Methods . A quantitative reverse transcriptase polymerase chain reaction technique (RT‐PCR) was used to measure the type 1 IGFR messenger RNA (mRNA) level, and Northern blotting was used to measure type 2 IGFR and IGFBP mRNA levels. Western immunoblotting was used to identify and measure IGFBP levels. Results . There were similar levels of type 1 IGFR mRNA in normal and OA chondrocytes. The level of type 2 IGFR mRNA, in which an increased amount of which can interfere with the biologic effects of IGF‐1, was lower in OA chondrocytes compared with normal chondrocytes. Articular chondrocytes produced IGFBP‐2, IGFBP‐3, and IGFBP‐4, and OA chondrocytes secreted and expressed higher amounts than did normal chondrocytes. There was also an increased level of IGFBP‐3 in the OA chondrocyte lysates. IGFBPs 1, 5, and 6 were not detectable. Conclusion . OA chondrocytes synthesize and express a larger amount of 3 IGFBPs. This observation, along with a lack of detectable change in type 1 IGFR mRNA level, suggests that the hyporesponsiveness of OA chondrocytes to IGF‐1 might implicate the involvement of IGFBPs in this pathologic process.
Somatomedin
Type II collagen
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Abstract The present study was undertaken to investigate the effect of insulin‐like growth factor‐1 on proteoglycan synthesis and the autocrine/paracrine mechanisms involving insulin‐like growth factor‐1 in the bovine coccygeal intervertebral disc. Insulin‐like growth factor‐1 stimulated proteoglycan synthesis in cultured cells of the nucleus pulposus of bovine intervertebral discs in a dose‐dependent manner, and the effect was inhibited by an anti‐insulin‐like growth factor‐1 monoclonal antibody. In situ hybridization histochemistry revealed the expression of insulin‐like growth factor‐1 mRNA in the cultured cells, and its production in these cells was demonstrated by radioimmunoassay. Insulin‐like growth factor‐1 receptor in the cultured cells was also demonstrated immunohistochemically. Scatchard analysis using an [ 125 I]insulin‐like growth factor‐1 binding assay showed that the cells cultured in monolayer had a single type of insulin‐like growth factor‐1 receptor, whose affinity and number were estimated to be 7.38 × 10 8 / M and 9.27 × 10 4 /cell, respectively. These results suggest that insulin‐like growth factor‐1 stimulates proteoglycan synthesis in cells of the nucleus pulposus and that these cells in culture have an insulin‐like growth factor‐1 autocrine/paracrine mechanism. The expressions of insulin‐like growth factor‐1 mRNA and insulin‐like growth factor‐1 receptor in disc tissue were greater in cells of the nucleus pulposus of fetal bovine intervertebral discs than in those of the adult discs. These findings suggest that the action of autocrine/paracrine insulin‐like growth factor‐1 is more active in cells of the young nucleus pulposus than in cells of mature subjects.
Somatomedin
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The insulin‐like growth factor (IGF) system plays an important role in postnatal somatic and skeletal muscle growth in pigs. There is little information on the occurrence and distribution of components of the IGF system in postnatal porcine skeletal muscle. IGF‐I, IGF receptor 1 (IGF1R) and the IGF‐binding proteins IGFBP‐1 and ‐3 in longissimus dorsi and triceps brachii were localized in muscle biopsies from 12 commercially crossbred pigs aged from 28 to 199 days as well as from the sire generation, by immunohistochemistry. Plasma IGF‐I concentrations were also determined using radioimmunoassays. Unlike other species, IGF‐I was localized in porcine skeletal muscle fibres. Staining intensity correlated with the highest plasma IGF‐I levels and phases of intensive muscle growth from the 11th to 22nd week. The pattern of IGF1R immunostaining, which was strong, correlated with that of IGF‐I. IGF1R was also localized in endomysial tissues. IGFBP‐1 was not detected within muscle fibres, but was found in the endomysium and vessel walls, while IGFBP‐3 was localized with IGF‐1 and its receptor. Higher magnification revealed that IGF1R, IGFBP‐3 and probably IGF‐I appeared in the tubular system. Inhibitory as well as stimulating controls of IGFBP‐1 and ‐3 on IGF functions are discussed, which may maintain a balance between autocrine growth promoting activities of IGF‐I and IGF1R.
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Objective To investigate the effects of insulin-1ike growth factor 1(IGF1) and IGF1 receptor(IGF1R) on proliferation of HO8910PM cells of ovarian cancer. Methods The effects of IGF1 of different concentration on HO8910PM cells were observed by CCK-8 assay.Transfection of IGF1R siRNA by lipofectamine 2000 to silence IGF1R gene expression in HO8910PM,the silence effects were evaluated by real-time PCR and Western blotting.Forty-eight hours after transfection,IGF1 was added on HO8910PM cells to observe the effects on proliferation by CCK-8 assay. ResultsIncubation with IGF1 for 48 h significantly stimulated the proliferation response of HO8910PM cells(P0.05).IGF1R mRNA and protein expression levels were significantly decreased by transfecting IGF1R siRNA for 48 h(P0.05).After transfection of IGF1R siRNA for 48 h and incubation with IGF1,the stimulation of proliferation response was significantly down-regulated(P0.05). Conclusion IGF1 stimulates the proliferation response of HO8910PM cells by IGF1R pathway.IGF1R siRNA may effectively down-regulate the effects.
Lipofectamine
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