Novel GPC3 Gene Mutation in Simpson-Golabi-Behmel Syndrome with Endocrine Anomalies: A Case Report.

Simpson-Golabi-Behmel syndrome (SGBS) represents a rare X-linked recessive syndrome with prenatal and postnatal overgrowth, coarse facial features, congenital malformations, organomegaly and an increased risk of tumors. Mutations on the GPC3 gene, encoding the glypican-3 protein, have previously been shown to cause the disease. In this report, a 12-year-old Chinese boy was hospitalized in our institution for some clinical features of SGBS. His serum endocrine evaluation showed hormone level abnormalities, including high prolactin, high testosterone, high thyroid-stimulating hormone (TSH) levels, and low estradiol levels. Whole exome sequencing (WES) was performed in the patient for mutation analysis and a novel hemizygous mutation, c.185delT, p.(Leu62Cysfs*22), on the GPC3 gene, was identified. The mother was a heterozygous carrier. The SGBS patients might present with endocrine anomalies, which adds to the clinical heterogeneity of the disease. The novel GPC3 mutation c.185delT expands the mutational spectrum of the GPC3 gene.

Prenatal diagnosis of Simpson-Golabi-Behmel syndrome type 1 with an 814 kb Xq26.2 deletion with the initial presentation of a thick nuchal fold.

OBJECTIVE: Simpson-Golabi-Behmel syndrome type 1 (SGBS1) is a rare X-linked recessive disorder characterized by overgrowth and multiple anomalies. Most clinical diagnoses of SGBS1 are made postnatally. We present the case of a pregnant woman in whom the fetus presented with a thick nuchal fold 5.6 mm at 15 weeks of gestation, leading to the prenatal diagnosis of SGBS1 with Xq26.2 (133408101-134221889) deletion. CASE REPORT: We report the case of a 34-year-old pregnant woman with the initial presentation of fetal thick nuchal fold 5.6 mm at 15 weeks of gestation. Amniocentesis of the fetal karyotype revealed a normal 46, XY, and single nucleotide polymorphism array showed Xq26.2 (133408101-134221889) deletion. Prenatal ultrasound at 21 weeks of gestation revealed a thick nuchal fold, hepatomegaly, nephromegaly, congenital diaphragmatic hernia, hypospadias, and polyhydramnios. Fetal magnetic resonance imaging revealed hepatomegaly, nephromegaly, congenital diaphragmatic hernia, and right lung hypoplasia. The woman had her pregnancy terminated at 24 weeks of gestation. The proband had a general appearance of low-set ears, hypertelorism, a large tongue, and hypospadias and some unique findings on autopsy, including hepatomegaly, right hiatal hernia, liver extensive extramedullary hematopoiesis, kidney marked congestion, and focal hemorrhage. DISCUSSION: The main prenatal ultrasound findings that alert clinical doctors about the possible diagnosis of SGBS1 included macrosomia, polyhydramnios, organomegaly, renal malformations, congenital diaphragmatic hernia, and cardiac anomalies. Our case underscores the importance of fetal karyotyping combined with single nucleotide polymorphism array when a thick nuchal fold is found. Genetic counseling is essential in SGBS1, and prenatal testing or preimplantation testing for subsequent pregnancies is necessary to identify possible pathogenic variants.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7.

OBJECTIVES: To build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer. METHODS: Hepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamine(TM2000)) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed. RESULTS: In the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level. CONCLUSIONS: The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

The glypican 3-hosted murine mir717 gene: sequence conservation, seed region polymorphisms and putative targets.

Mir717 (mmu-mir-717) was first reported in mouse and resides in the intron 3 of glypican 3 (Gpc3) gene. Our present study revealed that this microRNA (miRNA) gene is conserved among 26 mammalian species and harbors polymorphic sites within the mature seed region in mice. Our finding represents a rare four layer genomic overlap consisting of growth associated quantitative trait locus (QTL), body mass associated Gpc3 gene, highly conserved miRNA gene and mature miRNA seed single nucleotide polymorphism (SNP) identified in the lean mouse strain 129/Sv. Additionally, genes potentially targeted by Mir717 include 91 genes associated with obesity and related phenotypes in mammals. Our analysis provides a basis for further experiments to causally connect the identified SNP and Mir717 gene itself to obesity regulation. Furthermore, our bioinformatics analysis now enables functional annotation of Mir717 orthologs in other species, thus determining the effect of its target genes on fat-related traits.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

OBJECTIVES@#To build GPC3 gene short hairpin interference RNA (shRNA) slow virus vector, observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth, and provide theoretical basis for gene therapy of liver cancer.@*METHODS@#Hepatocellular carcinoma cell line Huh-7 was transfected by a RNA interference technique. GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR. Targeted GPC3 gene sequences of small interfering RNA (siRNA) PGC-shRNA-GPC3 were restructured. Stable expression cell lines of siRNA were screened and established with the help of liposomes (lipofectamine(TM2000)) as carrier transfection of human liver cell lines. In order to validate siRNA interference efficiency, GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot. The absorbance value of the cells of blank group, untransfection group and transfection group, the cell cycle and cell apoptosis were calculated, and effects of GPC3 gene on Huh-7 cell proliferation and apoptosis were observed.@*RESULTS@#In the liver cancer cell lines Huh-7, GPC3 gene showed high expression. PGC-shRNA-GPC3 recombinant plasmid was constructed successfully via sequencing validation. Stable recombinant plasmid transfected into liver cancer cell lines Huh-7 can obviously inhibit GPC3 mRNA expression level.@*CONCLUSIONS@#The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

Construction of GPC3 green fluorescent protein eukaryotic expression vector and its influence on growth promoting effect of growth factors in SK-Hep-1 cells / 第二军医大学学报

Objective: To construct a GPC3 green fluorescent protein eukaryotic expression vector pEGFP-N2-G-PC3, and analyze its effects on the growth promoting effect of growth factors (fibroblast growth factor-2, FGF2; insulin-like growth factor-2, IGF2; transforming growth factor-β1, TGF-β1; and bone morphogenetic protein-4,BMP4) in human hepatoma cell line GPC3-SK-Hep-1. Methods: A eukaryotic expression vector for GPC3 genes (pEGFP-N2-GPC3) was constructed by recombinant DNA technique and was transfected into SK-Hep-1 cells by Lipofectamine™ 2000; the cells stably expressing GPC3 were screened out by G418 (600 μg/ml). The mRNA expression of GPC3 was detected by RT-PCR method and the protein expression of GPC3 by Western blotting and fluorescence microscope. Effects of GPC3 gene on the growth promoting effects of the above growth factors were examined by MTT. Results: The recombinant plasmid was verified to be correctly constructed by restriction endonuclease analysis and DNA sequencing. The green fluorescence was detected in the transfected SK-Hep-1 cells under fluorescence microscope. RT-PCR and Western blotting both confirmed that GPC3 was successfully expressed in SK-Hep-1 cells. FGF2-induced cell proliferation was significantly decreased by GPC3 gene, whereas the growth promoting effects of IGF2, TGF-β1 and BMP4 were not altered by GPC3 gene. Conclusion: We have successfully obtained the synthetic GPC3 protein, which has the same amino acid sequence as that of human GPC3 protein. The eukaryotic expression vector pEGFP-N2-GPC3 has been correctly constructed and GPC3 protein has been successfully expressed in SK-Hep-1 cells. GPC3 may negatively modulate FGF2 signaling pathway.