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Molecular Metabolism May 2018Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a...
OBJECTIVE
Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized.
METHODS
CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes. Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue.
RESULTS
GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation of JAK2 was significantly increased, possibly due to the increased serum leptin levels and increased hepatic leptin receptor expression and activation in GHR-KO pigs. In addition, increased mTOR phosphorylation was observed in GHR-KO liver samples, and phosphorylation studies of downstream substrates suggested the activation of mainly mTOR complex 2.
CONCLUSION
GHR-KO pigs resemble the pathophysiology of LS and are an interesting model for mechanistic studies and treatment trials.
Topics: Adiposity; Animals; Body Weight; Growth Hormone; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Janus Kinase 2; Laron Syndrome; Liver; Mechanistic Target of Rapamycin Complex 2; Receptors, Somatotropin; STAT5 Transcription Factor; Signal Transduction; Swine
PubMed: 29678421
DOI: 10.1016/j.molmet.2018.03.006 -
Journal of Translational Medicine Feb 2018Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is...
BACKGROUND
Laron syndrome is an autosomal disease resulting from mutations in the growth hormone receptor (GHR) gene. The only therapeutic treatment for Laron syndrome is recombinant insulin-like growth factor I (IGF-I), which has been shown to have various side effects. The improved Laron syndrome models are important for better understanding the pathogenesis of the disease and developing corresponding therapeutics. Pigs have become attractive biomedical models for human condition due to similarities in anatomy, physiology, and metabolism relative to humans, which could serve as an appropriate model for Laron syndrome.
METHODS
To further improve the GHR knockout (GHRKO) efficiency and explore the feasibility of precise DNA deletion at targeted sites, the dual-sgRNAs/Cas9 system was designed to target GHR exon 3 in pig fetal fibroblasts (PFFs). The vectors encoding sgRNAs and Cas9 were co-transfected into PFFs by electroporation and GHRKO cell lines were established by single cell cloning culture. Two biallelic knockout cell lines were selected as the donor cell line for somatic cell nuclear transfer for the generation of GHRKO pigs. The genotype of colonies, cloned fetuses and piglets were identified by T7 endonuclease I (T7ENI) assay and sequencing. The GHR expression in the fibroblasts and piglets was analyzed by confocal microscopy, quantitative polymerase chain reaction (q-PCR), western blotting (WB) and immunohistochemical (IHC) staining. The phenotype of GHRKO pigs was recapitulated through level detection of IGF-I and glucose, and measurement of body weight and body size. GHRKO F1 generation were generated by crossing with wild-type pigs, and their genotype was detected by T7ENI assay and sequencing. GHRKO F2 generation was obtained via self-cross of GHRKO F1 pigs. Their genotypes of GHRKO F2 generation was also detected by Sanger sequencing.
RESULTS
In total, 19 of 20 single-cell colonies exhibited biallelic modified GHR (95%), and the efficiency of DNA deletion mediated by dual-sgRNAs/Cas9 was as high as 90% in 40 GHR alleles of 20 single-cell colonies. Two types of GHR allelic single-cell colonies (GHR, GHR) were selected as donor cells for the generation of GHRKO pigs. The reconstructed embryos were transferred into 15 recipient gilts, resulting in 15 GHRKO newborn piglets and 2 fetuses. The GHRKO pigs exhibited slow growth rates and small body sizes. From birth to 13 months old, the average body weight of wild-type pigs varied from 0.6 to 89.5 kg, but that of GHRKO pigs varied from only 0.9 to 37.0 kg. Biochemically, the knockout pigs exhibited decreased serum levels of IGF-I and glucose. Furthermore, the GHRKO pigs had normal reproduction ability, as eighteen GHRKO F1 piglets were obtained via mating a GHRKO pig with wild-type pigs and five GHRKO F2 piglets were obtained by self-cross of F1 generation, indicating that modified GHR alleles can pass to the next generation via germline transmission.
CONCLUSION
The dual-sgRNAs/Cas9 is a reliable system for DNA deletion and that GHRKO pigs conform to typical phenotypes of those observed in Laron patients, suggesting that these pigs could serve as an appropriate model for Laron syndrome.
Topics: Animals; Base Sequence; CRISPR-Associated Protein 9; DNA; Disease Models, Animal; Embryo, Mammalian; Fetus; Fibroblasts; Gene Knockout Techniques; Germ Cells; Growth and Development; Laron Syndrome; Nuclear Transfer Techniques; Receptors, Somatotropin; Swine; RNA, Guide, CRISPR-Cas Systems
PubMed: 29482569
DOI: 10.1186/s12967-018-1409-7 -
Clinical Case Reports Feb 2018We report a case of short stature irresponsive to growth hormone (GH) replacement therapy. Low GH response to provocative tests and undetectable IGF-1 levels had...
We report a case of short stature irresponsive to growth hormone (GH) replacement therapy. Low GH response to provocative tests and undetectable IGF-1 levels had suggested GH deficiency, while response to therapy indicated GH insensitivity. Molecular evaluation of the GH/IGF-1 axis should be performed in these cases to improve diagnosis and therapy.
PubMed: 29445490
DOI: 10.1002/ccr3.1349 -
Proceedings of the National Academy of... Jan 2018Laron syndrome (LS), or primary growth hormone (GH) insensitivity, is the best-characterized entity among the congenital insulin-like growth factor 1 (IGF1)...
Laron syndrome (LS), or primary growth hormone (GH) insensitivity, is the best-characterized entity among the congenital insulin-like growth factor 1 (IGF1) deficiencies. Life-long exposure to minute endogenous IGF1 levels is linked to low stature as well as a number of endocrine and metabolic abnormalities. While elevated IGF1 is correlated with increased cancer incidence, epidemiological studies revealed that patients with LS do not develop tumors. The mechanisms associated with cancer protection in LS are yet to be discovered. Recent genomic analyses identified a series of metabolic genes that are overrepresented in patients with LS. Given the augmented expression of these genes in a low IGF1 milieu, we hypothesized that they may constitute targets for IGF1 action. Thioredoxin-interacting protein (TXNIP) plays a critical role in cellular redox control by thioredoxin. TXNIP serves as a glucose and oxidative stress sensor, being commonly silenced by genetic or epigenetic events in cancer cells. Consistent with its enhanced expression in LS, we provide evidence that gene expression is negatively regulated by IGF1. These results were corroborated in animal studies. In addition, we show that oxidative and glucose stresses led to marked increases in expression. Supplementation of IGF1 attenuated TXNIP levels, suggesting that IGF1 exerts its antiapoptotic effect via inhibition of Augmented expression in LS may account for cancer protection in this condition. Finally, TXNIP levels could be potentially useful in the clinic as a predictive or diagnostic biomarker for IGF1R-targeted therapies.
Topics: Animals; Carrier Proteins; Cell Line; Gene Expression; Glucose; Humans; Insulin; Insulin-Like Growth Factor I; Laron Syndrome; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neoplasms; Oxidative Stress; Promoter Regions, Genetic; RNA, Messenger
PubMed: 29339473
DOI: 10.1073/pnas.1715930115 -
Endokrynologia Polska 2017Short stature is the main problem that paediatric endocrinologists have to grapple with. Endocrine disorders account for only 5% of patients with short stature, but this... (Review)
Review
Short stature is the main problem that paediatric endocrinologists have to grapple with. Endocrine disorders account for only 5% of patients with short stature, but this is still one of the most common causes of reports to the endocrine clinic and hospitalisation in the endocrine department. A properly functioning growth hormone/insulin-like growth factor (GH/IGF) axis is one of the most important factors in proper growth. A lot of genetic defects in this axis lead to syndromes marked by impaired growth, like Laron syndrome, muta-tions in the STAT5B, insulin-like growth factor 1 (IGF1), and insulin-like growth factor 1 receptor (IGF1R) and mutations in the acid labile subunit (ALS). Two proteases important for the proper functioning of the GH/IGF axis: pregnancy-associated plasma protein-A (PAPP-A) and pregnancy-associated plasma protein-A2 (PAPP-A2), have been described. The first description of the new syndrome of growth failure associated with mutation in the PAPP-A2 gene was given by Andrew Dauber et al. This review evaluates the current data concerning PAPP-A2 function, and particularly the effect of PAPP-A2 mutation on growth.
Topics: Animals; Female; Growth; Growth Hormone; Humans; Insulin-Like Growth Factor I; Male; Mice; Mutation; Pregnancy-Associated Plasma Protein-A
PubMed: 29238946
DOI: 10.5603/EP.a2017.0060 -
Clinical Case Reports Nov 2017Glucose and lipid profile together with blood pressure should always be considered for low sera-IGF-1 patients. Even when adulthood is reached, IGF-1 therapy in these...
Glucose and lipid profile together with blood pressure should always be considered for low sera-IGF-1 patients. Even when adulthood is reached, IGF-1 therapy in these patients should be pursued as metabolic and protective cellular effects could be triggered. Real incidence of growth hormone insensitivity is still to be uncovered.
PubMed: 29152285
DOI: 10.1002/ccr3.1193 -
Endocrinology, Diabetes & Metabolism... 2017Herein, we present a 14-year-old patient with short stature (134 cm) referred from Paediatrics to our department for complementary evaluation since growth hormone (GH)...
UNLABELLED
Herein, we present a 14-year-old patient with short stature (134 cm) referred from Paediatrics to our department for complementary evaluation since growth hormone (GH) treatment failed to show any improvement. He was born premature and small for gestational age. Genital examination classified the patient as Tanner I-II with small penis and testicular size for his age. Biochemical analyses revealed normal GH levels with low serum insulin-like growth factor-1 (IGF-1). Molecular diagnosis confirmed several mutations in and , and so he was diagnosed with Laron Syndrome or GH insensibility and treated with IGF-1 substitutive therapy.
LEARNING POINTS
Evaluation of the GH/IGF-1 axis when short stature does not respond to conservative treatment must be included in the ordinary practice.Laron Syndrome real incidence should be calculated once undiagnosed cases arise, as treatment, due to lack of market, is unaffordable.Even when adulthood is reached, and no longitudinal growth can be achieved, still IGF-1 treatment in Laron Syndrome patients should be pursued as metabolic and protective derangements could arise.
PubMed: 29147569
DOI: 10.1530/EDM-17-0126 -
Italian Journal of Pediatrics Oct 2017Mutations localized in the Growth Hormone Receptor (GHR) gene are often associated with the pathogenesis of Laron Syndrome, an autosomal recessive hereditary disorder... (Review)
Review
BACKGROUND
Mutations localized in the Growth Hormone Receptor (GHR) gene are often associated with the pathogenesis of Laron Syndrome, an autosomal recessive hereditary disorder characterized by severe growth retardation. Biochemically, patients present normal to high circulating GH levels, in presence of very low or undetectable IGF-I levels, which do not rise after rhGH treatment.
CASE PRESENTATION
We describe the case of a 3.8 years old girl with symmetrical short stature (-3.76 SDS), low IGF-1 and IGFBP-3, in presence of normal GH levels. Parents were not relatives and there was no family history of short stature. During the second day of birth, she developed severe hypoglycaemia that required glucose infusion. She presented frontal bossing and depressed nasal bridge. IGF-1 generation test showed no response, suggesting a GH resistance evidence. In the hypothesis of Laron Syndrome, we decided to perform a molecular analysis of Growth Hormone Receptor (GHR) gene. This analysis demonstrated that the patient was compound heterozygote for two missense mutations.
CONCLUSIONS
GHR gene mutations are a well demonstrated cause of GH insensitivity. In heterozygous patients, probably the normal stature may be achieved by a compensatory mechanism of GH secretion or signalling. On the contrary, in homozygous or compound heterozygous patients these compensatory mechanisms are inadequate, and short stature may be the consequence.
Topics: Child; Child, Preschool; Female; Genetic Predisposition to Disease; Heterozygote; Humans; Italy; Laron Syndrome; Mutation, Missense; Prognosis; Receptors, Somatotropin; Severity of Illness Index
PubMed: 29025428
DOI: 10.1186/s13052-017-0411-7 -
International Journal of Molecular... Jul 2017Obesity is an excessive accumulation or expansion of adipose tissue (AT) due to an increase in either the size and/or number of its characteristic cell type, the... (Review)
Review
Obesity is an excessive accumulation or expansion of adipose tissue (AT) due to an increase in either the size and/or number of its characteristic cell type, the adipocyte. As one of the most significant public health problems of our time, obesity and its associated metabolic complications have demanded that attention be given to finding effective therapeutic options aimed at reducing adiposity or the metabolic dysfunction associated with its accumulation. Growth hormone (GH) has therapeutic potential due to its potent lipolytic effect and resultant ability to reduce AT mass while preserving lean body mass. However, AT and its resident adipocytes are significantly more dynamic and elaborate than once thought and require one not to use the reduction in absolute mass as a readout of efficacy alone. Paradoxically, therapies that reduce GH action may ultimately prove to be healthier, in part because GH also possesses potent anti-insulin activities along with concerns that GH may promote the growth of certain cancers. This review will briefly summarize some of the newer complexities of AT relevant to GH action and describe the current understanding of how GH influences this tissue using data from both humans and mice. We will conclude by considering the therapeutic use of GH or GH antagonists in obesity, as well as important gaps in knowledge regarding GH and AT.
Topics: Adipocytes; Adipose Tissue; Animals; Human Growth Hormone; Humans; Mice; Obesity; Receptors, Somatotropin
PubMed: 28933734
DOI: 10.3390/ijms18081621 -
The American Journal of the Medical... May 2017Fanconi anemia (FA) is a condition characterized by genetic instability and short stature, which is due to growth hormone (GH) deficiency in most cases. However, no...
BACKGROUND
Fanconi anemia (FA) is a condition characterized by genetic instability and short stature, which is due to growth hormone (GH) deficiency in most cases. However, no apparent relationships have been identified between FA complementation group genes and GH. In this study, we thereby considered an association between FA and Laron syndrome (LS) (insulin-like growth factor 1 [IGF-1] deficiency).
METHODS
A 21-year-old female Mexican patient with a genetic diagnosis of FA was referred to our research department for an evaluation of her short stature. Upon admission to our facility, her phenotype led to a suspicion of LS; accordingly, serum levels of IGF-1 and IGF binding protein 3 were analyzed and a GH stimulation test was performed. In addition, we used a next-generation sequencing approach for a molecular evaluation of FA disease-causing mutations and genes involved in the GH-IGF signaling pathway.
RESULTS
Tests revealed low levels of IGF-1 and IGF binding protein 3 that remained within normal ranges, as well as a lack of response to GH stimulation. Sequencing confirmed a defect in the GH receptor signaling pathway.
CONCLUSIONS
To the best of our knowledge, this study is the first to suggest an association between FA and LS. We propose that IGF-1 administration might improve some FA complications and functions based upon IGF-1 beneficial actions observed in animal, cell and indirect clinical models: erythropoiesis modulation, immune function improvement and metabolic regulation.
Topics: Body Height; Fanconi Anemia; Female; Human Growth Hormone; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Laron Syndrome; Mexico; Receptors, Somatotropin; Signal Transduction; Young Adult
PubMed: 28502327
DOI: 10.1016/j.amjms.2017.02.001