The IGF system is a crucial regulator of normal growth and development, however dysregulation of the system on multiple levels is associated with the incidence of a wide variety of malignancies including the breast, thyroid, lung, and colon, making the IGF system an important anti-cancer therapeutic target. Due to its role in mediating cellular proliferation, protection from apoptosis, and metastasis, traditional focus has been set on examining the role of the type 1 IGF receptor [IGF1R] in cancer. However there is mounting evidence to suggest the insulin receptor [IR] may also be involved in the potentiation and pathogenesis of cancers. The observation that IGF-II is overexpressed, compared to normal tissues, by cancers suggests signaling via target receptors by this ligand has important implications on cancer pathogenesis. Indeed, both the IGF1R and IR have been demonstrated to be up-regulated in a variety of malignancies. In regards to IR isoform, the IGF-II binding IR-A is preferentially expressed by a number of cancer cell types. Together with the observation that an autocrine proliferative loop exists between IGF-II and the IR-A in malignant thyrocytes and cultured breast cancer cells, suggests signaling via the IR-A may play a role in cancer cell growth and survival. However, very few studies on the IR-A have been conducted in cells co-expressing the IGF1R. This is mainly due to the difficulties associated with discrimination between signaling arising from IGF1R homodimers, IR-A homodimers, and IGF1R/IR-A hybrid receptors. It is not known how the IR-A interacts, and functions in conjunction with the other receptors of the IGF system to signal biologically relevant outcomes, especially in terms of anti-cancer therapeutics that aim to block and down-regulate the IGF1R. Current anti-cancer therapies targeting the IGF system have concentrated on blocking IGF signaling via the IGF1R, due mostly to the functional properties of the receptor, but also in part due to the metabolic consequences associated with blockade and inhibition of the IR. This individual targeting of the IGF1R potentially leaves a pathway by which IGF-II secreted by the tumour can circumvent current IGF1R based therapies. Consequently, this thesis investigated whether the IR-A could compensate for the targeted loss of the IGF1R and how the IR-A interacts with the IGF1R in cells co-expressing these two receptors. In addition, the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation was assessed. The main experimental techniques used throughout this body of work included; assessment of protein expression and activation by Western blot, siRNA mediated gene silencing, and measures of cell proliferation, survival, and migration. The key areas of investigation included: 1. Investigation of the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation 2. Identification of an appropriate cell line model in which to investigate the interactions between the IR-A and IGF1R 3. Optimisation of siRNA mediated knock-down of the IR-A and IGF1R in SW480 colorectal adenocarcinoma cells 4. Determination of the biological role of the IR-A in SW480 cells co-expressing the IGF1R The key findings from this work included: 1. The IR-A could not compensate for IGF1R depletion in SW480 cells 2. Dual silencing of the IR-A and IGF1R indicated signaling via the IGF1R was dominant to signaling via the IR-A in SW480 cells 3. Signaling via IR-A/IGF1R hybrid receptors may not be as potent as signaling via IGF1R homodimers 4. IGF-I at physiological concentrations can stimulate biological responses via both isoforms of the IR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337339 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

The IGF system is a crucial regulator of normal growth and development, however dysregulation of the system on multiple levels is associated with the incidence of a wide variety of malignancies including the breast, thyroid, lung, and colon, making the IGF system an important anti-cancer therapeutic target. Due to its role in mediating cellular proliferation, protection from apoptosis, and metastasis, traditional focus has been set on examining the role of the type 1 IGF receptor [IGF1R] in cancer. However there is mounting evidence to suggest the insulin receptor [IR] may also be involved in the potentiation and pathogenesis of cancers. The observation that IGF-II is overexpressed, compared to normal tissues, by cancers suggests signaling via target receptors by this ligand has important implications on cancer pathogenesis. Indeed, both the IGF1R and IR have been demonstrated to be up-regulated in a variety of malignancies. In regards to IR isoform, the IGF-II binding IR-A is preferentially expressed by a number of cancer cell types. Together with the observation that an autocrine proliferative loop exists between IGF-II and the IR-A in malignant thyrocytes and cultured breast cancer cells, suggests signaling via the IR-A may play a role in cancer cell growth and survival. However, very few studies on the IR-A have been conducted in cells co-expressing the IGF1R. This is mainly due to the difficulties associated with discrimination between signaling arising from IGF1R homodimers, IR-A homodimers, and IGF1R/IR-A hybrid receptors. It is not known how the IR-A interacts, and functions in conjunction with the other receptors of the IGF system to signal biologically relevant outcomes, especially in terms of anti-cancer therapeutics that aim to block and down-regulate the IGF1R. Current anti-cancer therapies targeting the IGF system have concentrated on blocking IGF signaling via the IGF1R, due mostly to the functional properties of the receptor, but also in part due to the metabolic consequences associated with blockade and inhibition of the IR. This individual targeting of the IGF1R potentially leaves a pathway by which IGF-II secreted by the tumour can circumvent current IGF1R based therapies. Consequently, this thesis investigated whether the IR-A could compensate for the targeted loss of the IGF1R and how the IR-A interacts with the IGF1R in cells co-expressing these two receptors. In addition, the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation was assessed. The main experimental techniques used throughout this body of work included; assessment of protein expression and activation by Western blot, siRNA mediated gene silencing, and measures of cell proliferation, survival, and migration. The key areas of investigation included: 1. Investigation of the individual ability of the IR isoforms to signal biological outcomes in response to IGF stimulation 2. Identification of an appropriate cell line model in which to investigate the interactions between the IR-A and IGF1R 3. Optimisation of siRNA mediated knock-down of the IR-A and IGF1R in SW480 colorectal adenocarcinoma cells 4. Determination of the biological role of the IR-A in SW480 cells co-expressing the IGF1R The key findings from this work included: 1. The IR-A could not compensate for IGF1R depletion in SW480 cells 2. Dual silencing of the IR-A and IGF1R indicated signaling via the IGF1R was dominant to signaling via the IR-A in SW480 cells 3. Signaling via IR-A/IGF1R hybrid receptors may not be as potent as signaling via IGF1R homodimers 4. IGF-I at physiological concentrations can stimulate biological responses via both isoforms of the IR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337339 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Gene-environment (GE) interactions involving the IGF pathway may affect childhood obesity. Detecting such interactions using longitudinal family studies requires accounting for individual and familial correlations. Simulations were performed to study three methods to test for GE interactions in longitudinal family data using repeated outcomes (linear mixed model) or individual outcome averages as summary statistics (twin model, partition based score I test). Interactions between the IGF pathway genes (IGF-1, IGFALS) and environmental factors (physical activity, daycare attendance and sleep duration) were tested using the Quebec Newborn Twin Study data. The twin model yielded the best performance. Results from the QNTS analysis showed suggestive association for an IGF-1 variant at position 102791894 of chromosome 12 interacting with physical activity. However, this association was not statistically significant after multiple testing correction. More robust methods and studies are needed to better understand the IGF pathway’s role in childhood obesity.

The insulin-like growth factor (IGF) system plays an important role in a number of disease states, such as cancer, and has also been implicated in wound and burn healing processes. Two IGF receptors, the type-1 IGF and type-2 IGF receptors, as well as six insulin-like growth factor binding proteins (IGFBP-1 to 6), have well established roles in mediating IGF activity. Earlier studies in this laboratory demonstrated that IGF-II binds to the extracellular matrix (ECM) protein vitronectin (VN), and although IGF-I does not bind directly to VN it can bind indirectly via specific IGFBPs. Therefore the aim of the research described in this thesis was to determine whether binary and ternary complexes of IGF-I/II, IGFBPs and VN affect human keratinocyte cell function. The strategy of pre-binding these complexes to the culture dishes was adopted in this study in an attempt to more accurately reflect the extracellular environment in vivo. These studies demonstrated that the binary complex of IGF-II and VN and the ternary complexes comprised of IGF-I, IGFBP-2, or 3, or 4, or 5 and VN significantly stimulated HaCaT de novo cell protein synthesis in the human keratinocyte cell line. Interestingly, these latter experiments demonstrated that although large increases in protein synthesis were observed using the ternary complexes, IGF-I/IGFBP complexes alone were responsible for the significant increases in protein synthesis and these responses are mediated via the MAPK signaling pathway. In addition, both the dimeric and trimeric complexes significantly enhanced cell migration through 12 μm TranswellsTM. Unlike the protein synthesis assays, VN was critically important in these migratory responses and highlighting the important role that integrins play in cell migration. Cell attachment assays on the other hand demonstrated that the interactions of IGFs with IGFBPs and VN did not affect cell attachment. The data encompassed within this thesis represent the first studies to provide a functional role for the interaction between IGFs, IGFBPs and VN in human keratinocytes. Taken together these results suggest that IGF/IGFBP/VN complexes may hold great potential in situations where enhanced keratinocyte cell migration and proliferation is required, such as in wound healing and skin engineering applications.

Background: Multiple lines of evidence implicate the insulin-like growth factor(IGF) group of proteins in human type 2 diabetes. The actions of IGF-I and IGF-IIare modulated through their interaction with IGF binding proteins. A holisticapproach to study the IGF system is preferable to analyses of individual proteininteractions as the inter-relationships between these proteins are complex. Inparticular, the associations of IGF-II and its associated binding proteins withcardiovascular risk have been inadequately studied. This study aimed to study indetail the genotype and phenotype interactions of the IGF system with longitudinalcardiovascular risk factor trends and phenotypic outcomes in type 2 diabetes.Methods: 1000 subjects of predominantly Caucasian origin from the SalfordDiabetes Cohort were studied. Measurements of IGF proteins (IGF-I, IGF-II,IGFBP-1, IGFBP-2 and IGFBP-3) were performed in 554 of these patients. 991Caucasian subjects were successfully genotyped for 76 single nucleotidepolymorphisms (SNPs) related to ten genes in the IGF system. In this project weanalysed associations of the studied SNPs with the measured IGF proteins as well aslongitudinal risk factor trends. In addition, the baseline concentrations of themeasured proteins were studied for associations with cardiovascular risk factortrends and vascular outcomes.Results: This project demonstrates for the first time that high serum IGF-IIconcentration at baseline predicts longitudinal increases in high-density lipoproteincholesterol. High baseline IGF-II was also observed to predict longitudinal weightloss. High baseline concentration of IGFBP-2 (which has a preferential associationof IGF-II over IGF-I) was associated with a number of favourable longitudinalcardiovascular risk trends like increased HDL cholesterol and decreased diastolicblood pressure. However high IGFBP-2 was also associated with deterioration inrenal function and increased all-cause and cardiovascular mortality. The IGF2 geneand the genes encoding IGFBP-2 and IGFBP-5 (proteins with IGF-II bindingaffinity) were also associated with longitudinal trends in renal function, bloodpressure and cholesterol concentration.Discussion: This study is the most detailed exploration to date of the genotype andphenotype interactions of the IGF system in a Caucasian population with type 2diabetes. Results from this study strongly hint that changes in IGF-II bioavailabilitymay influence inter-individual variations in cardiovascular risk. The precisebiological role of IGF-II merits clarification in future expression studies in renal,adipose and vascular tissues. Replication of significant results in an independentdiabetes cohort and measurement of other IGF binding proteins will be performed inthe next stage of this study.

<p>In this thesis, the importance of the growth hormone (GH)/insulin-like growth factor (IGF) system and sex steroids for male bone metabolism has been investigated, and the effects of continuous low dose GH replacement in GH deficient (GHD) adults. In a population-based sample of men, positive correlations were found between bone mineral density (BMD) and IGF-I, IGF-II, IGF binding protein (IGFBP)-3 and the testosterone/sex hormone binding globulin (SHBG) ratio. Serum IGFBP-3 and testosterone levels and weight accounted for 34% to 48% of the variation in BMD at different sites. Compared to healthy age matched controls, men with idiopathic osteoporosis had lower estradiol/SHBG ratio and higher SHBG levels. There were no differences between the groups in serum levels of IGF-I, IGFBP-3, 24 hour cumulated GH secretion or peak GH secretion. In the patients, there was a positive correlation between the estradiol/SHBG ratio and BMD in femoral neck. Treatment of patients and controls with GH 0.8 mg/day for one week resulted in similar increases in serum markers for bone turnover in both groups. Several positive correlations between indices of GH secretion and markers for bone turnover were found in the patients. Men with idiopathic osteoporosis were treated with GH, continuously (0.4 mg/day) or intermittently (0.8 mg/day for two weeks every third month), for two years followed by one year of follow-up. After two years, the BMD and bone mineral content in lumbar spine and total body and serum osteocalcin levels were increased in both groups. This increase was sustained one year post treatment. Treatment of GHD adults with a low fixed dose of GH (0.17 mg/day) for three months, resulted in increases in serum IGF-I and IGFBP-3 levels and lean body mass, and a reduction in fat mass and total and low-density lipoprotein cholesterol levels. These beneficial effects were accomplished without serious side effects. These findings indicate that: i) the sex hormone and GH/IGF systems are important in male bone metabolism, ii) a combination of subtle disturbances in these two systems could contribute to the development of male idiopathic osteoporosis, iii) GH treatment could be considered as a treatment option in this condition.</p>

<p>Ormarsdóttir, S. 2001. Osteoporosis in Chronic Liver Disease. Acta Universitatis Upsaliensis. <i>Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine</i> 1037. 60 pp. Uppsala. ISBN 91-554-5021-0. </p><p>Osteoporosis is a well-known and frequently reported complication of chronic liver disease (CLD) with a high fracture rate contributing to significant morbidity after liver transplantation. The pathogenesis is unknown and controversy exists about many risk factors for osteoporosis in CLD. </p><p>In the present thesis, bone mineral density (BMD) was found to be significantly lower at the lumbar spine (<i>p</i><0.01) in a cohort of patients with CLD compared with age- and gender -matched individuals. Osteoporosis was found in 30% of the patients and 15% of the controls, respectively. Low body mass index (BMI), corticosteroid treatment, prothrombin time, age and female gender were independent risk factors for osteoporosis in the patients. </p><p>In a follow-up study, 43 of 72 patients were available for a second BMD measurement 25 months (median) after the first. Bone loss at the femoral neck was 1.5 ± 2.4% in females and 2.9 ± 2.0% in males with a significant decrease in BMD Z-score over time (<i>p</i>=0.005 and <i>p</i>=0.02 for females and males, respectively), indicating increased bone loss at this site. Hyperbilirubinaemia and low circulating levels of 25-hydroxy vitamin D₃ predicted increased bone loss at the femoral neck. These findings suggest that cortical bone, in addition to trabecular bone, may be affected in CLD and bilirubin and vitamin D₃ may be involved in the pathophysiology of osteoporosis in CLD. </p><p>In order to elucidate the suggested role of insulin-like growth factors (IGFs) and leptin in the pathophysiology of osteoporosis in CLD, we studied the relationship between these factors and BMD. Levels of IGFs were extremely low (<i>p</i><0.0001 compared with the controls) and related to liver function but no correlation was found between the IGFs and BMD. Serum leptin adjusted for BMI correlated negatively with BMD in female patients (<i>p</i>=0.003 and <i>p</i>=0.04 at the lumbar spine and the femoral neck, respectively) and in male patients at the femoral neck (<i>p</i>=0.04). Thus, the IGFs appear not to be involved in the pathophysiology of osteoporosis in CLD but a role of circulating leptin is possible. </p>

Ormarsdóttir, S. 2001. Osteoporosis in Chronic Liver Disease. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1037. 60 pp. Uppsala. ISBN 91-554-5021-0. Osteoporosis is a well-known and frequently reported complication of chronic liver disease (CLD) with a high fracture rate contributing to significant morbidity after liver transplantation. The pathogenesis is unknown and controversy exists about many risk factors for osteoporosis in CLD. In the present thesis, bone mineral density (BMD) was found to be significantly lower at the lumbar spine (p&lt;0.01) in a cohort of patients with CLD compared with age- and gender -matched individuals. Osteoporosis was found in 30% of the patients and 15% of the controls, respectively. Low body mass index (BMI), corticosteroid treatment, prothrombin time, age and female gender were independent risk factors for osteoporosis in the patients. In a follow-up study, 43 of 72 patients were available for a second BMD measurement 25 months (median) after the first. Bone loss at the femoral neck was 1.5 ± 2.4% in females and 2.9 ± 2.0% in males with a significant decrease in BMD Z-score over time (p=0.005 and p=0.02 for females and males, respectively), indicating increased bone loss at this site. Hyperbilirubinaemia and low circulating levels of 25-hydroxy vitamin D3 predicted increased bone loss at the femoral neck. These findings suggest that cortical bone, in addition to trabecular bone, may be affected in CLD and bilirubin and vitamin D3 may be involved in the pathophysiology of osteoporosis in CLD. In order to elucidate the suggested role of insulin-like growth factors (IGFs) and leptin in the pathophysiology of osteoporosis in CLD, we studied the relationship between these factors and BMD. Levels of IGFs were extremely low (p&lt;0.0001 compared with the controls) and related to liver function but no correlation was found between the IGFs and BMD. Serum leptin adjusted for BMI correlated negatively with BMD in female patients (p=0.003 and p=0.04 at the lumbar spine and the femoral neck, respectively) and in male patients at the femoral neck (p=0.04). Thus, the IGFs appear not to be involved in the pathophysiology of osteoporosis in CLD but a role of circulating leptin is possible.

In this thesis, the importance of the growth hormone (GH)/insulin-like growth factor (IGF) system and sex steroids for male bone metabolism has been investigated, and the effects of continuous low dose GH replacement in GH deficient (GHD) adults. In a population-based sample of men, positive correlations were found between bone mineral density (BMD) and IGF-I, IGF-II, IGF binding protein (IGFBP)-3 and the testosterone/sex hormone binding globulin (SHBG) ratio. Serum IGFBP-3 and testosterone levels and weight accounted for 34% to 48% of the variation in BMD at different sites. Compared to healthy age matched controls, men with idiopathic osteoporosis had lower estradiol/SHBG ratio and higher SHBG levels. There were no differences between the groups in serum levels of IGF-I, IGFBP-3, 24 hour cumulated GH secretion or peak GH secretion. In the patients, there was a positive correlation between the estradiol/SHBG ratio and BMD in femoral neck. Treatment of patients and controls with GH 0.8 mg/day for one week resulted in similar increases in serum markers for bone turnover in both groups. Several positive correlations between indices of GH secretion and markers for bone turnover were found in the patients. Men with idiopathic osteoporosis were treated with GH, continuously (0.4 mg/day) or intermittently (0.8 mg/day for two weeks every third month), for two years followed by one year of follow-up. After two years, the BMD and bone mineral content in lumbar spine and total body and serum osteocalcin levels were increased in both groups. This increase was sustained one year post treatment. Treatment of GHD adults with a low fixed dose of GH (0.17 mg/day) for three months, resulted in increases in serum IGF-I and IGFBP-3 levels and lean body mass, and a reduction in fat mass and total and low-density lipoprotein cholesterol levels. These beneficial effects were accomplished without serious side effects. These findings indicate that: i) the sex hormone and GH/IGF systems are important in male bone metabolism, ii) a combination of subtle disturbances in these two systems could contribute to the development of male idiopathic osteoporosis, iii) GH treatment could be considered as a treatment option in this condition.

Η διαδικασία της αύξησης ελέγχεται από έναν πολύπλοκο συνδυασμό πολλών παραγόντων σε διάφορα επίπεδα, που περιλαμβάνουν ενδογενείς παράγοντες, όπως είναι ο γονότυπος, οι ορμόνες, οι παράγοντες αύξησης και εξωγενείς παράγοντες, όπως είναι η διατροφή και η επίδραση του περιβάλλοντος. Οι ορμονικοί παράγοντες, που επηρεάζουν την αύξηση είναι κυρίως η αυξητική ορμόνη (GH) και οι ινσουλινόμορφοι αυξητικοί παράγοντες (IGFs). Στην διαδικασία της αύξησης συμμετέχουν, όμως, και άλλες ορμόνες, όπως η θυροξίνη, τα επινεφριδιακά ανδρογόνα, τα στεροειδή του φύλου, τα γλυκοκορτικοειδή, η βιταμίνη D, η λεπτίνη και η ινσουλίνη, που αλληλεπιδρούν με τον άξονα GH-IGF. Η αυξητική ορμόνη εκκρίνεται στην κυκλοφορία από τα σωματότροπα κύτταρα του πρόσθιου λοβού της υπόφυσης, υπό την επίδραση δύο υποθαλαμικών ορμονών του εκλυτικού παράγοντα της αυξητικής ορμόνης (GHRH), που διεγείρει την έκκριση της GH και της σωματοστατίνης (SS), που αναστέλλει την έκκρισή της. Μέχρι σήμερα στην διεθνή βιβλιογραφία έχουν περιγραφεί πολλές μεταλλάξεις του γονιδίου της GH ως αιτία κοντού αναστήματος στα παιδιά. Η παρούσα μελέτη εξέτασε ομάδα 11 παιδιών με κοντό ανάστημα, ρυθμό αύξησης κάτω από την 2η εκατοστιαία θέση και καθυστερημένη οστική ηλικία. Όλοι οι ασθενείς υπεβλήθησαν σε λεπτομερή κλινική εξέταση και πλήρη εργαστηριακό έλεγχο. Από την κλινική εξέταση και τον εργαστηριακό έλεγχο αποκλείστηκε η παρουσία κάποιας συστηματικής πάθησης. Στην συνέχεια υπεβλήθησαν σε προκλητές δοκιμασίες έκκρισης της GH, με κλονιδίνη και L-Dopa, σε έλεγχο της 24ωρης έκκρισης της GH και τη δοκιμασία γένεσης του IGF-I. Με βάση τα εργαστηριακά αποτελέσματα της έκκρισης της GH η ομάδα των ασθενών διαχωρίστηκε σε αυτούς με ιδιοπαθές κοντό ανάστημα (10 περιπτώσεις) και ένα ασθενή με νευροεκκριτική δυσλειτουργία της GH (GHND), ο οποίος είχε μειωμένη 24ωρη έκκριση GH. Από τους ασθενείς αυτούς ελήφθησαν βιοψίες ούλων, στους καλλιεργημένους ινοβλάστες των οποίων έγιναν οι μελέτες αύξησης των ινοβλαστών και περιφερικό αίμα, από το οποίο έγινε εξαγωγή γονιδιωματικού DNA. Έγινε πολλαπλασιασμός των γονιδίων του υποδοχέα της GH (GHR) και του γονιδίου της GH (GH1) με την αλυσιδωτή αντίδραση πολυμεράσης (PCR) και προσδιορισμός της αλληλουχίας τους. Ανιχνεύτηκαν μεταλλαγές στους 6 από τους 11 ασθενείς, που μελετήθηκαν, οι οποίες εντοπίζονταν στο ιντρόνιο 4 του γονιδίου GH1 και ένας ακόμη ασθενής που έφερε μεταλλάξεις στα ιντρόνια 1 και 2. Οι μεταλλάξεις αυτές δεν επηρέαζαν την διαδικασία του ματίσματος και τον σχηματισμό του mRNA και απομακρύνονταν με το μάτισμα. Στην βιβλιογραφία αναφέρονται περισσότεροι από 10 πολυμορφισμοί του γονιδίου GH1 που εντοπίζονται κυρίως στα ιντρόνια του γονιδίου και κάποιοι από αυτούς έχουν συσχετιστεί με ελαττωμένη έκφραση του γονιδίου GH1. Στον ασθενή με την GHND περιγράφηκε μια μεταλλαγή στη θέση +7 του ιντρονίου 4 του γονιδίου GH1. RT-PCR του GH1 cDNA έδειξε ότι η μετάλλαξη αυτή είναι υπεύθυνη για το εσφαλμένο μάτισμα του mRNA, με αποτέλεσμα την απαλοιφή του εξονίου 5 από το ώριμο μετάγραφο. Ο ασθενής με τη μεταλλαγή είναι ετεροζυγώτης και η ίδια μεταλλαγή σε ετερόζυγη κατάσταση, βρέθηκε και στους δύο γονείς του ασθενούς, οι οποίοι έχουν επίσης κοντό ανάστημα. Η μεταλλαγή αυτή οδηγεί στην παραγωγή μικρότερου μορίου GH. Η βιοδραστικότητα του παραγόμενου ανώμαλου μορίου της GH εκτιμήθηκε με την προσθήκη ορού του ασθενούς σε καλλιέργειες φυσιολογικών ινοβλαστών, με τη μέθοδο ενσωμάτωσης στο DNA της βρώμο-δεοξυουριδίνης (BrDU), η οποία έδειξε μειωμένη σύνθεση DNA συγκρινόμενη με την σύνθεση DNA παρουσία ορού φυσιολογικών ατόμων. Δηλαδή η περίπτωση αυτή οικογενούς κοντού αναστήματος, το οποίο κληρονομείται κατά τον επικρατούντα χαρακτήρα, οφείλεται σε μεταλλαγή στο ιντρόνιο 4 του γονιδίου GH1. / Growth can be defined as an increase in size by accretion of tissue. The control of the growth process is affected by many complex interacting factors including internal cues such as the genotype, external factors such as nutrition and environment, and internal signaling systems such as hormones and growth factors. The principal hormones influencing growth are Growth Hormone (GH) and the Insulin-like Growth Factors (IGFs), but many other hormones contribute, such as thyroxine, adrenal androgens, sex steroids, glucocorticoids, vitamin D, leptin and insulin, often channeled through interaction with the GH-IGF axis. GH is secreted from the anterior pituitary into the circulation. The pattern of GH secretion is determined primarily by the interaction between the hypothalamic peptides Growth Hormone Releasing Hormone (GHRH) and somatostatin (SS). Many mutations of the GH1 gene have been described as the cause of short stature in children. The present study examined 11 children with severe short stature, growth velocity below the 2nd centile and delayed bone age. All patients underwent thorough clinical examination and laboratory investigation in order to exclude an underlying chronic disease. Also GH secretion provocative studies, 24 hr endogenous secretion studies and IGF-I generation test were carried out. According to the results of these tests the patients we studied were divided in two groups: 10 of the patients had idiopathic short stature (ISS) and 1 patient had GH neurosecretory dysfunction (GHND). Fibroblast cultures were established from gingival biopsies obtained from the patients and genomic DNA was extracted from peripheral blood leukocytes. GH1 and GH receptor (GHR) genes were amplified by PCR and sequenced. Hot spot mutations were detected in GH1 intron 4 in 6 patients and mutations in introns 1 and 2 were detected in 1 patient. These mutations did not affect the splicing of the primary RNA transcript. A novel deletion of thymine 7 bp downstream from the 3' splice site of intron 4 was found in the patient who had GHND. RT-PCR of GH1 cDNA showed that this mutation causes aberrant GH mRNA splicing, changes the read frame, creates a new stop codon and results in the deletion of exon 5. This was also confirmed by restriction enzyme analysis of the mutant cDNA. Both short parents and the patient are heterozygotes for this mutation. BrDU incorporation in the DNA of normal fibroblast cultures in the presence of the patient’s blood serum showed reduced DNA synthesis compared to fibroblasts cultured in medium with normal human serum. Addition of high concentrations of GH (4 μg/ml) to the culture medium containing the patient’s serum led to a near normal DNA synthesis. This is a new case of familial short stature inherited as a dominant trait, due to a mutation in intron 4 of the GH1 gene.