Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

Genetic analysis of all-fish growth hormone gene transferred carp (Cyprinus carpio L.) and its F-1 generation

Recombinant "all-fish" growth hormone gene (GH) was microinjected Into the fertilized eggs of carp. A comparison between the growth traits of transgenics and non-transgenics was carried out, and the transgenic individuals with significant "fast-growing" effect were successfully gained. A comparison on the reproductivities was also given out between the transgenics and their non-transgenic siblings, and showed that the reproductive capacity of transgenics was substantially equivalent to those of the non-transgenics. On the other hand, the genetic separation and the characteristic distribution of the F-1 generation were genetically analyzed, which gave solid evidence for the hypothesis that 2-3 chromosomes are integrated with transgene. In addition, the distinct biological effects for multisite-integrated transgenes were further discussed. The present study opens a door for the breeding of "fast-growing" transgenic fish.

Growth hormone 1 gene (GH1) polymorphisms as possible markers of the production potential of beef cattle using the Brazilian Canchim breed as a model

The growth hormone 1 gene (GH1) is a candidate gene for body weight and weight gain in cattle since it plays a fundamental role in growth regulation. We investigated the GH1 gene AluI and DdeI restriction enzyme polymorphisms, located 149 bp apart in the cattle genome, as possible markers of the production potential of Canchim crossbreed cattle, a 5/8 Charolais (Bos taurus) and 3/8 Nelore (Bos indicus) breed developed in Brazil, by evaluating the birth weight, weaning weight, yearling weight and plasma insulin-like growth factor-1 (IGF-1) concentration of 7 month to 10 months old Canchim calves (n = 204) of known genealogy and which had been genotyped for the AluI and DdeI markers. Our results showed significant effect (p < 0.05) between the homozygous DdeI+/DdeI+ polymorphism and the estimated breeding value for weaning weight (ESB-WW), while the AluI leucine homozygous (L/L) and leucine/valine (L/V) heterozygous polymorphisms showed no significant effect on the traits studied. The restriction sites of the two enzymes led to the formation of haplotypes which also exerted a significant effect (p < 0.05) on the ESB-WW, with the largest difference being 8.5 kg in favor of the homozygous L plus DdeI+/L plus DdeI+ genotype over the heterozygous L plus DdeI-/V plus DdeI+ genotype.

Growth hormone pharmacogenetics: the interactive effect of a microsatellite in the IGF1 promoter region with the GHR-exon 3 and-202 A/C IGFBP3 variants on treatment outcomes of children with severe GH deficiency

Insulin-like growth factor type 1 (IGF1) is a mediator of growth hormone (GH) action, and therefore, IGF1 is a candidate gene for recombinant human GH (rhGH) pharmacogenetics. Lower serum IGF1 levels were found in adults homozygous for 19 cytosine-adenosine (CA) repeats in the IGF1 promoter. The aim of this study was to evaluate the influence of (CA)n IGF1 polymorphism, alone or in combination with GH receptor (GHR)-exon 3 and -202 A/C insulin-like growth factor binding protein-3 (IGFBP3) polymorphisms, on the growth response to rhGH therapy in GH-deficient (GHD) patients. Eighty-four severe GHD patients were genotyped for (CA) n IGF1, -202 A/C IGFBP3 and GHR-exon 3 polymorphisms. Multiple linear regressions were performed to estimate the effect of each genotype, after adjustment for other influential factors. We assessed the influence of genotypes on the first year growth velocity (1st y GV) (n = 84) and adult height standard deviation score (SDS) adjusted for target-height SDS (AH-TH SDS) after rhGH therapy (n = 37). Homozygosity for the IGF1 19CA repeat allele was negatively correlated with 1st y GV (P = 0.03) and AH-TH SDS (P = 0.002) in multiple linear regression analysis. In conjunction with clinical factors, IGF1 and IGFBP3 genotypes explain 29% of the 1st y GV variability, whereas IGF1 and GHR polymorphisms explain 59% of final height-target-height SDS variability. We conclude that homozygosity for IGF1 (CA) 19 allele is associated with less favorable short-and long-term growth outcomes after rhGH treatment in patients with severe GHD. Furthermore, this polymorphism exhibits a non-additive interaction with -202 A/C IGFBP3 genotype on the 1st y GV and with GHR-exon 3 genotype on adult height. The Pharmacogenomics Journal (2012) 12, 439-445; doi:10.1038/tpj.2011.13; published online 5 April 2011

Growth hormone receptor polymorphism and growth hormone therapy response in children: a bayesian meta-analysis

Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphism (GHR(d3)) may account for some of this variability. The authors performed a systematic review (to April 2011), including investigator-only data, to quantify the effects of the GHR(fl-d3) and GHR(d3-d3) genotypes on rhGH therapy response and used a recently established Bayesian inheritance model-free approach to meta-analyze the data. The primary outcome was the 1-year change-in-height standard-deviation score for the 2 genotypes. Eighteen data sets from 12 studies (1,527 children) were included. After several prior assumptions were tested, the most appropriate inheritance model was codominant (posterior probability = 0.93). Compared with noncarriers, carriers had median differences in 1-year change-in-height standard-deviation score of 0.09 (95% credible interval (CrI): 0.01, 0.17) for GHR(fl-d3) and of 0.14 (95% CrI: 0.02, 0.26) for GHR(d3-d3). However, the between-study standard deviation of 0.18 (95% CrI: 0.10, 0.33) was considerable. The authors tested by meta-regression for potential modifiers and found no substantial influence. They conclude that 1) the GHR(d3) polymorphism inheritance is codominant, contrasting with previous reports; 2) GHR(d3) genotypes account for modest increases in rhGH effects in children; and 3) considerable unexplained variability in responsiveness remains.

Human epidermal growth factor receptor-2 gene amplification in gastric cancer using tissue microarray technology

To assess human epidermal growth factor receptor-2 (HER2)-status in gastric cancer and matched lymph node metastases by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH).

Isolated growth hormone deficiency type 2: from gene to therapy

Isolated growth hormone deficiency type-2 (IGHD-2), the autosomal-dominant form of GH deficiency, is mainly caused by specific splicing mutations in the human growth hormone (hGH) gene (GH-1). These mutations, occurring in and around exon 3, cause complete exon 3 skipping and produce a dominant-negative 17.5 kD GH isoform that reduces the accumulation and secretion of wild type-GH (wt-GH). At present, patients suffering from IGHD-2 are treated with daily injections of recombinant human GH (rhGH) in order to reach normal height. However, this type of replacement therapy, although effective in terms of growth, does not prevent toxic effects of the 17.5-kD mutant on the pituitary gland, which can eventually lead to other hormonal deficiencies. Considering a well-known correlation between the clinical severity observed in IGHD-2 patients and the increased expression of the 17.5-kD isoform, therapies that specifically target this isoform may be useful in patients with GH-1 splicing defects. This chapter focuses on molecular strategies that could represent future directions for IGHD-2 treatment.

Fgfrl1, a fibroblast growth factor receptor-like gene, is found in the cephalochordate Branchiostoma floridae but not in the urochordate Ciona intestinalis

FGFRL1 is a novel member of the fibroblast growth factor receptor family that controls the formation of musculoskeletal tissues. Some vertebrates, including man, cow, dog, mouse, rat and chicken, possess a single copy the FGFRL1 gene. Teleostean fish have two copies, fgfrl1a and fgfrl1b, because they have undergone a whole genome duplication. Vertebrates belong to the chordates, a phylum that also includes the subphyla of the cephalochordates (e.g. Branchiostoma floridae) and urochordates (tunicates, e.g. Ciona intestinalis). We therefore investigated whether other chordates might also possess an FGFRL1 related gene. In fact, a homologous gene was found in B. floridae (amphioxus). The corresponding protein showed 60% sequence identity with the human protein and all sequence motifs identified in the vertebrate proteins were also conserved in amphioxus Fgfrl1. In contrast, the genome of the urochordate C. intestinalis and those from more distantly related invertebrates including the insect Drosophila melanogaster and the nematode Caenorhabditis elegans did not appear to contain any related sequences. Thus, the FGFRL1 gene might have evolved just before branching of the vertebrate lineage from the other chordates.

Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Autosomal-dominant isolated growth hormone deficiency (IGHD type II) with normal GH-1 gene

BACKGROUND: Autosomal-dominant isolated GH deficiency (IGHD) is a rare disorder that is commonly believed to be due to heterozygous mutations in the GH-1 gene (GH-1). These mutations cause the production of a protein that affects the release of the product of the normal allele. Rarely, heterozygous mutations in the gene encoding for HESX-1 gene (HESX-1) may cause autosomal-dominant IGHD, with penetrance that has been shown to be variable in both humans and mice. SUBJECTS AND METHODS: We have sequenced the whole GH-1 in the index cases of 30 families with autosomal-dominant IGHD. In all the families other possible causes of GH deficiency and other pituitary hormones deficits were excluded. We here describe the clinical, biochemical and radiological picture of the families without GH-1 mutations. In these families, we also sequenced the HESX-1. RESULTS: The index cases of the five families with autosomal-dominant IGHD had normal GH-1, including the intronic sequences. They had no HESX-1 mutations. CONCLUSION: This study shows that GH-1 mutations are absent in 5/30 (16.6%) of the families with autosomal-dominant IGHD and raises the possibility that mutations in other gene(s) may be involved in IGHD with this mode of transmission.