The variation of insulin like growth factor 2 maker is associated with growth traits in Thai native (Kradon) pigs.

OBJECTIVE: This study was conducted to investigate polymorphisms of the melanocortin-4 receptor (MC4R) and insulin like growth factor 2 (IGF2) genes and to evaluate the growth traits affected by such polymorphisms in Thai native (Kradon) pigs. METHODS: Blood samples and productive data from 91 Kradon pigs were collected. DNA was extracted and quantified, the IGF2 and MC4R genes were amplified, and the polymerase chain reaction (PCR) produces were digested using the PCR-restriction fragment length polymorphism (PCR-RFLP) technique. Genotyping was performed, and the association between genotypes and growth traits on the birth and weaning weights were evaluated. RESULTS: The IGF2 intron7 g.162G>C variations in Kradon pigs were found in three genotypes: i) GG, ii) GC, and iii) CC. The GG genotype frequency was the highest followed by the GC and CC genotypes. The frequencies of the G and C alleles were 0.703 and 0.297, respectively. The MC4R genotype was found in only one genotype (GG). The IGF2 gene pattern was not associated with birth weight traits, whereas the IGF2 gene pattern was related to the weaning weight trait in Kradon pigs. Pigs with the CC and GC genotypes had higher weaning weights than ones with the GG genotype (p<0.001). CONCLUSION: Thai native Kradon pigs with the CC and GC genotypes of the IGF2 gene have higher weaning weights than pigs with the GG genotype.

Association analysis of IGF2 gene polymorphisms with growth traits of Dezhou donkey.

IGF2 is an insulin-like growth factor that plays an important role in the development of animal embryos. In order to determine whether IGF2 gene is associated with important economic characteristics of donkeys, we investigated the association between single nucleotide polymorphisms (SNPs) of IGF2 gene and body size traits of Chinese Dezhou donkeys and analyzed the expression level of IGF2 gene in different tissues of juvenile and adult Dezhou donkeys. In this study, two SNPs (g.281766 G > A and g.291322 C > T) were detected in IGF2 gene, both of which were in Hardy-Weinberg equilibrium (P > 0.05) and were moderately polymorphic (0.25 < PIC < 0.50). Association analysis showed that the two SNP loci were significantly correlated with body length and rump height (p < 0.05) of female Dezhou donkeys. Quantitative results showed that the expression of IGF2 gene was higher in heart, liver, spleen, lung, kidney, stomach and muscle tissues of juvenile donkeys than that of adult donkeys. Together, IGF2 can be considered as a candidate gene for growth and development of female Dezhou donkey, and its polymorphism can be used as a molecular marker for the Dezhou donkey breeding.

Autocrine IGF-II-Associated Cancers: From a Rare Paraneoplastic Event to a Hallmark in Malignancy.

The paraneoplastic syndrome referred in the literature as non-islet-cell tumor hypoglycemia (NICTH) and extra-pancreatic tumor hypoglycemia (EPTH) was first reported almost a century ago, and the role of cancer-secreted IGF-II in causing this blood glucose-lowering condition has been widely established. The landscape emerging in the last few decades, based on molecular and cellular findings, supports a broader role for IGF-II in cancer biology beyond its involvement in the paraneoplastic syndrome. In particular, a few key findings are constantly observed during tumorigenesis, (a) a relative and absolute increase in fetal insulin receptor isoform (IRA) content, with (b) an increase in IGF-II high-molecular weight cancer-variants (big-IGF-II), and (c) a stage-progressive increase in the IGF-II autocrine signal in the cancer cell, mostly during the transition from benign to malignant growth. An increasing and still under-exploited combinatorial pattern of the IGF-II signal in cancer is shaping up in the literature with respect to its transducing receptorial system and effector intracellular network. Interestingly, while surgical and clinical reports have traditionally restricted IGF-II secretion to a small number of solid malignancies displaying paraneoplastic hypoglycemia, a retrospective literature analysis, along with publicly available expression data from patient-derived cancer cell lines conveyed in the present perspective, clearly suggests that IGF-II expression in cancer is a much more common event, especially in overt malignancy. These findings strengthen the view that (1) IGF-II expression/secretion in solid tumor-derived cancer cell lines and tissues is a broader and more common event compared to the reported IGF-II association to paraneoplastic hypoglycemia, and (2) IGF-II associates to the commonly observed autocrine loops in cancer cells while IGF-I cancer-promoting effects may be linked to its paracrine effects in the tumor microenvironment. Based on these evidence-centered considerations, making the autocrine IGF-II loop a hallmark for malignant cancer growth, we here propose the functional name of IGF-II secreting tumors (IGF-IIsT) to overcome the view that IGF-II secretion and pro-tumorigenic actions affect only a clinical sub-group of rare tumors with associated hypoglycemic symptoms. The proposed scenario provides an updated logical frame towards biologically sound therapeutic strategies and personalized therapeutic interventions for currently unaccounted IGF-II-producing cancers.

sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9.

The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 SNP site, which we called "SNP editing." The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from Streptococcus pyogenes using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based IGF2 SNP editing by silencing the LIG4 gene, which is a key molecule of the HDR's competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the IGF2 SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective IGF2 SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases.

Missense mutations in exon 2 of the MED12 gene are involved in IGF-2 overexpression in uterine leiomyoma.

Uterine leiomyoma (UL), the most common benign tumour found in females, is associated with many recurrent genetic aberrations, such as translocations, interstitial deletions and specific germline mutations. Among these, mutations affecting exon 2 of the mediator complex subunit 12 (MED12) gene are commonly detected in the majority of ULs. Mutational analysis of the MED12 gene, performed on 36 UL samples, revealed that 12 leiomyomas (33.4%) exhibited heterozygous missense mutations in codon 44 of exon 2 of the MED12 gene, four leiomyomas (11.1%) showed internal in-frame deletions, and two leiomyomas (5.5%) exhibited deletions involving intron 1-exon 2 junction, which caused a predicted loss of the splice acceptor. No mutations were detected in uterine myometrium (UM) and pseudocapsule (PC) samples, including those from women with a MED12 mutation in UL. These data showed that the PC is a healthy tissue that surrounds the UL to maintain UM integrity. Analysis of insulin-like growth factor 2 (IGF-2) and collagen type IV alpha 2 (COL4A2) mRNA expression levels in the same set of ULs revealed that only those with MED12 missense mutations expressed significantly higher levels of IGF-2 mRNA. In contrast, MED12 gene status does not appear to affect mRNA expression levels of the COL4A2 gene. On the basis of this finding, we suggest that the MED12 status stratifies the ULs into two mutually exclusive pathways of leiomyoma genesis, one with IGF-2 overexpression and the other with no IGF-2 activation. The occurrence of IGF-2 overexpression could be therapeutically targeted for the non-surgical treatment of leiomyomas.

Investigation of IGF2, Hedgehog and fusion gene expression profiles in pediatric sarcomas.

UNLABELLED: The childhood sarcomas are malignant tumors with high mortality rates. They are divided into two genetic categories: a category without distinct pattern karyotypic changes and the other category showing unique translocations that originate gene rearrangements. This category includes rhabdomyosarcoma (RMS), Ewing's sarcoma (ES) and synovial sarcoma (SS). Diverse studies have related development genes, such as; IGF2, IHH, PTCH1 and GLI1 and sarcomatogenesis. OBJECTIVE: To characterize the RMS, ES and SS rearrangements, we quantify the expression of IGF2 IHH, PTCH1 and GLI1 genes and correlate molecular data with clinical parameters of patients. DESIGN: We analyzed 29 RMS, 10 SS and 60 ES tumor samples by RT-PCR (polymerase chain reaction-reverse transcription) and qPCR (quantitative PCR). RESULTS: Among the samples of ARMS, 50% had rearrangements of PAX3/7-FOXO1, 60% of ES samples were EWS-FLI1 positive and 90% of SS samples were positive for SS18-SSX1/2. In relation to the control reference samples (QPCR Human Reference Total RNA-Stratagene, Human Skeletal Muscle Total RNA-Ambion, Universal RNA Human Normal Tissues-Ambion), RMS samples showed a high IGF2 gene expression (p<0.0001). Moreover, ES samples showed a low IGF2 gene expression (p<0.0001) and high IHH (p<0.0001), PTCH1 (p=0.0173) and GLI1 (p=0.0113) gene expressions. CONCLUSIONS: The molecular characterization of IGF and Hedgehog pathway in these pediatric sarcomas may collaborate to enable a better understanding of the biological behavior of these neoplasms.

Effects of a novel SNP of IGF2R gene on growth traits and expression rate of IGF2R and IGF2 genes in gluteus medius muscle of Egyptian buffalo.

Insulin-like growth factor 2 receptor (IGF2R) is responsible for degradation of the muscle development initiator, IGF2, and thus it can be used as a marker for selection strategies in the farm animals. The aim of this study was to search for polymorphisms in three coding loci of IGF2R, and to analyze their effect on the growth traits and on the expression levels of IGF2R and IGF2 genes in the gluteus medius muscle of Egyptian buffaloes. A novel A266C SNP was detected in the coding sequences of the third IGF2R locus (at nucleotide number 51 of exon 23) among Egyptian water buffaloes. This SNP was non-synonymous mutation and led to replacement of Y (tyrosine) amino acid (aa) by D (aspartic acid) aa. Three different single-strand conformation polymorphism patterns were observed in the third IGF2R locus: AA, AC, and CC with frequencies of 0.555, 0.195, and 0.250, respectively. Statistical analysis showed that the homozygous AA genotype significantly associated with the average daily gain than AC and CC genotypes from birth to 9 mo of age. Expression analysis showed that the A266C SNP was correlated with IGF2, but not with IGF2R, mRNA levels in the gluteus medius muscle of Egyptian buffaloes. The highest IGF2 mRNA level was estimated in the muscle of animals with the AA homozygous genotype as compared to the AC heterozygotes and CC homozygotes. We conclude that A266C SNP at nucleotide number 51 of exon 23 of the IGF2R gene is associated with the ADG during the early stages of life (from birth to 9 mo of age) and this effect is accompanied by, and may be caused by, increased expression levels of the IGF2 gene.

Frequent Biallelic Expression of Insulin-like Growth Factor II (IGF2) in Gynogenetic Ovarian Teratomas: Uncoupling of H19 and IGF2 Imprinting

Human uniparental gestations such as gynogenetic ovarian teratomas provide a model to evaluate the integrity of parent-specific gene expression - i.e. imprinting - in the absence of a complementary parental genetic contribution. The few imprinted genes characterized so far include the insulin-like growth factor-2 gene (IGF2) coding for a fetal growth factor and H19 gene whose normal function is unknown but it is likely to act as an mRNA. IGF2 is expressed by the paternal allele and H19 by the maternal allele. This reciprocal expression is quite interesting because both H19 and IGF2 genes are located close to each other on chromosome 11p15.5. In situ RNA hybridization analysis has shown variable expression of the H19 and IGF2 alleles according to the tissue origin in 11 teratomas. Especially, Skin, derivative of ectoderm, is expressed conspicuously. We examined imprinting of H19 and IGF2 in teratomas using PCR and RT-PCR of exonic polymorphism. H19 and IGF2 transcript could be expressed either biallelically or monoallelically in the teratomas. Biallelic expression (i.e., loss of imprinting) of IGF2 occured in 5 out of 6 mature teratomas and 1 out of 1 immature teratoma. Biallelic expression of H19 occured in 4 out of 10 mature teratomas and 1 out of 1 immature teratoma. Expression levels of H19 and IGF2 transcript using the semi-quantitative RT-PCR had no relation between monoallelic and biallelic expression. Moreover, IGF2 biallelic expression did not affect allele-specificity or levels of H19 expression. These results demonstrate that both genes, H19 and IGF2, can be imprinted, expressed and regulated independently and individually of each other in ovarian teratoma.

Role of IGF2 Gene in Developing Human Ovary / 체질인류학회지

To understand the role of IGF2 gene in development of human ovary, IGF2 expression was detected by monoclonal antibody for IGF2 to its producted protein with immunohistochemical technique on human ovarian tissues. The results was as follows. IGF2 was highly expressed in ovum of mature follicle, IGF2 expression, however, was not high in granulosa and the cells. IGF2 was not highly expressed in ovum of primary follicle. Highly expressed IGF2 was found on corpus luteum and no expression of IGF2 was found in stroma and epithelial cells. These results suggest that IGF2 is important role in ovulation and in production of progesterone. Abnormal IGF2 expression may be concerned to carcinogenesis of ovarian tumor because most of all tumor from ovary is originated from epithelium.