Genetic diagnosis of congenital hypopituitarism in Turkish patients by a target gene panel: novel pathogenic variants in GHRHR, GLI2, LHX4 and POU1F1 genes

ABSTRACT Objective: Congenital hypopituitarism (CH) is a rare disease characterized by one or more hormone deficiencies of the pituitary gland. To date, many genes have been associated with CH. In this study, we identified the allelic variant spectrum of 11 causative genes in Turkish patients with CH. Materials and methods: This study included 47 patients [21 girls (44.6%) and 26 boys (55.4%)] from 45 families. To identify the genetic etiology, we screened 11 candidate genes associated with CH using next-generation sequencing. To confirm and detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. Results: We identified 12 possible pathogenic variants in GHRHR, GH1, GLI2, PROP-1, POU1F1, and LHX4 in 11 patients (23.4%), of which six were novel variants: two in GHRHR, two in POU1F1, one in GLI2, and one in LHX4. In all patients, these variants were most frequently found in GLI2, followed by PROP-1 and GHRHR. Conclusion: Genetic causes were determined in only 23.4% of all patients with CH and 63% of molecularly diagnosed patients (7/11) from consanguineous families. Despite advances in genetics, we were unable to identify the genetic etiology of most patients with CH, suggesting the effect of unknown genes or environmental factors. More genetic studies are necessary to understand the etiology of CH.

Anti-pancreatic fibrosis of oxymatrine by promoting expression of transcription factors Gli2 in LTC-14 and PANC-1 pancreatic stellate cells / 中草药

Objective: To investigate the effect of oxymatrine (OM) on the expression of Gli2 in LTC-14 cells and PANC-1 cells induced by TGF-β1. Methods: LTC-14 cells and PANC-1 cells were divided into four groups:control group, TGF-β1 group, TGF-β1 + OM group, and OM group. The protein was extracted after 12 h, and the expressions of related proteins were detected by Western blot. Results: Compared with control group, the expression of Gli2 in LTC-14 cells was significantly decreased induced by TGF-β1, while the expressions of α-SMA, FN, and CoL-I were significantly increased. The expressions of Gli2, FN, CoL-I, and α-SMA were increased significantly induced by TGF-β1when compared with those of control group in PANC-1 cells, and the expression of Gli2 was inhibited by OM pretreatment in TGF-β1 + OM group compared with TGF-β1 group. OM pretreatment can increase the expression of Gli2 before stimulating with TGF-β1 in TGF-β1 + OM group in comparison with TGF-β1 group. Conclusion: OM plays an protected role in pancreatic fibrosis through promoting the expression of Gli2 in LTC-14 cells.

Effect of Gli2 on apoptosis and ROS level in renal tubular epithelial cells under high glucose / 中国免疫学杂志

Objective:To investigate the effect of Gli2 on apoptosis and ROS level in renal tubular epithelial cells under high glucose. Methods:The renal tubular epithelial cell NRK-52E was used as the object of study,Gli2 overexpression vector and control vector were transfected,the non transfected cells were also used as controls,RT-PCR and Western blot detected Gli2 levels,culture of non transfected cells by high glucose and low glucose cell culture medium, cells transfected with Gli2 overexpression vector were cultured in high glucose medium,apoptosis was detected by flow cytometry,the ROS content was detected by DCFH-DA probe method, the content of SOD was detected by xanthine oxidase method; Bax, Cleaved Caspase-3 and Smo levels were detected by Western blot. Results:The Gli2 mRNA and protein levels of NRK-52E cells transfected with Gli2 overexpression vector were significantly higher than that of control cells(P＜0. 01),the Gli2 mRNA and protein levels in the NRK-52E cells transfected with the control vector were not different from those of the control cells(P＞0. 05). The apoptosis rate of NRK-52E cells without transfection was elevated after high glucose culture,elevated ROS content,SOD content decreased,the levels of Bax,Cleaved Caspase-3 were elevated in the cells,Smo levels declined,compared with the control cells,the difference was statistically significant(P＜0. 01). After overexpression of Gli2 over-expression vector,NRK-52E cells were cultured with high glucose,the rate of apoptosis decreased,ROS content decreased,elevated SOD content,the levels of Bax,Cleaved Caspase-3 decreased,elevated levels of Smo,compared with NRK-52E cells cultured with simple high glucose,the difference was statistically significant(P＜0. 01). Conclusion:Apoptosis and oxidative damage in renal tubular epithelial cells induced by high glucose, Gli2 can decrease the apoptosis of renal tubular epithelial cells induced by high glucose, mitigate oxidative damage.

Two novel heterozygous missense variations within the GLI2 gene in two unrelated argentine patients / Two novel heterozygous missense variations within the GLI2 gene in two unrelated Argentine patients

Several heterozygous GLI2 gene mutations have been reported in patients with isolated GH deficiency (IGHD) or multiple pituitary hormone deficiency (MPHD) with or without other malformations. The primary aim of this study was to analyze the presence of GLI2 gene alterations in a cohort of patients with IGHD or MPHD and ectopic/absent posterior pituitary. The coding sequence and flanking intronic regions of GLI2 gene were amplified and directly sequenced from gDNA of 18 affected subjects and relatives. In silico tools were applied to identify the functional impact of newly found variants (Polyphen2, SIFT, Mutation Taster). We identified two novel heterozygous missense variations in two unrelated patients, p.Arg231Gln and p.Arg226Leu, located in the repressor domain of the protein. Both variations affect highly conserved amino acids of the Gli2 protein and were not found in the available databases. In silico tools suggest that these variations might be disease causing. Our study suggests that the GLI2 gene may be one of the candidate genes to analyze when an association of pituitary hormone deficiency and developmental defects in posterior pituitary gland. The highly variable phenotype found suggests the presence of additional unknown factors that could contribute to the phenotype observed in these patients.

Mutaciones heterocigotas en el gen GLI2 fueron previamente comunicadas como causa de déficit aislado de hormona de crecimiento (IGHD) o déficit múltiple de hormonas hipofisarias (MPHD), con o sin otras malformaciones. El objetivo del estudio fue analizar la presencia de alteraciones en el gen GLI2 en un grupo de pacientes con IGHD o MPHD acompañado de neurohipófisis ectópica o ausente. La secuencia codificante y las regiones intrónicas flanqueantes del gen GLI2 fueron amplificadas y secuenciadas de manera directa a partir de ADN genómico extraído de sangre periférica proveniente de 18 sujetos afectados y sus familiares. Se utilizaron herramientas informáticas para predecir el impacto funcional de las nuevas variantes encontradas (Polyphen2, SIFT, Mutation Taster). Identificamos dos nuevas variantes heterocigotas con pérdida de sentido en dos pacientes no relacionados, p.Arg231Gln y p.Arg226Leu, localizadas en el dominio represor de la proteína. Estas variantes afectan aminoácidos altamente conservados en la secuencia proteica de GLI2 y no se encuentran informadas en las bases de datos disponibles. Las herramientas informáticas utilizadas sugieren que estas variantes pueden ser la causa del desarrollo de la enfermedad. Nuestro resultados indican que el gen GLI2 es uno de los genes candidatos a estudiar cuando existe una asociación entre déficit de hormonas hipofisarias y alteraciones en el desarrollo de la neurohipófisis. Se sugiere la existencia de otros factores adicionales que podrían contribuir a la variabilidad del fenotipo observado.

Effects of TNF-α and TGF-β1 on Gli2 expression in primary leukemia cells / 实用医学杂志

Objective To investigate whether TNF-α can promote, but TGF-β can reduce Gli2 expression in primary leukemia cells. Methods (1)Western blot was used to detect Gli2 protein expression in leukemic cells. (2)Primary leukemia cells were treated with TGF-β1 or TNF-α,and expressions of Gli2 and TGF-βR were detected. (3)Primary leukemia cells were treated with TGF-β1,TNF-α or/and SIS3, and Gli2 protein expression was detected. Results (1)The primary leukemia cells could express Gli2 protein. (2)Significant reduction of Gli2 mRNA was observed in 1 ~ 10 ng/mL TGF-β1-treated primary leukemia cells for 12 ~ 24 h, No significant differences of Gli2 mRNA was found between the control group and TNF-α group. (3)Protein expresson of TGF-βRⅠand TGF-βRⅡ in 5 ng/mL TNF-α-treated primary leukemia cells for 24 h was higher than that of the control group. (4)Gli2 portein expression was lower in TGF-β1 with or without TNF-α-treated primary leukemia cells for 24 h than that in the control group, and Gli2 portein expression in the TGF-β1 plus TNF-α group was lower than that in TGF-β1 group. Conclusion Combinations of TGF-β and TNF-α can efficiently inhibit Gli2 expression than TGF-β alone in primary leukemia cells.

The reverse effect of Gli2 gene silencing on epithelial-mesenchymal transition in hepatocellular carcinoma cell line SMMC-7721 / 基础医学与临床

Objective To explore the effect and mechanism of Gli2 on EMT and invasion in the hepatocellular car-cinoma cell line SMMC-7721.Methods shRNA of Gli2 and Negative control (NC) shRNA were constructed and transfected into SMMC-7721 cells.shRNA-Gli2 group,shRNA-NC group and blank group were set up .Transwell chambers assay , adhesion experiments were used to detect the ability of invasion ,homogeneous and heterogeneous cells intercellular adhesion of each group .Meanwhile, the qRT-PCR, Western blot were used to examine Gli2, E-cadherin ,N-cadherin and vimentin mRNA and protein expression .Results In different hepatocellular carcinoma cell lines and be along with the increasing ability of the invasion in hepatocellular carcinoma cell lines , Gli2 expres-sion was higher .Compared with the shRNA-NC group and blank control group ,the interfered group extensive cells invasion ability was inhibited ( P nism may be related to the up-regulation of E-cadherin and the dow N-regulation of N-cadherin, vimentin.

Gli-2 expression in thymic tumor tissues and its relationship with Ki-67 / 中国肿瘤临床

Objective:To investigate the expression of Gli-2 protein and nuclear proliferation marker Ki-67 in human thymic tu-mors, as well as its relationship with clinicopathological features and prognosis. Methods: Immunohistochemical staining was per-formed to determine the expressions of Gli-2 and Ki-67 in 64 thymic tumor cases, in which 9 were type A, 6 were type AB, 11 were type B1, 22 were type B2, and 16 were type C. Results:1) Positive expression rates of Gli-2 in types A, AB, B1, B2, and C thymomas were 1/9 (11.11%), 2/6 (33.33%), 2/11 (18.18%), 5/22 (22.73%), and 13/16 (81.25%), respectively. Statistically significant differences existed in the two sets of data (P0.05). 3) The positive labeling indexes of Ki-67 in invasive and non-inva-sive thymomas were 7.14 ± 6.99 and 15.24 ± 9.13, respectively. The differences between both thymomas were statistically significant (P<0.05). 4) A positive correlation existed in the expression of Ki-67 and Gli-2 in thymomas. Five-year progression-free survival (PFS) rate was lower in the Gli-2 positive group (56.5%, 13/23) than in the negative group (92.7%, 38/41). The Ki-67-positive group (61.5%, 16/26) also showed a lower five-year PFS than that in the negative group (92.1%, 35/38), with statistically significant differences be-tween the two groups (P<0.05). Multivariate Cox's proportional hazard regression analysis indicated that the Gli-2-positive group ex-pression, Ki-67-positive group expression, and invasion of the pleura were independent prognostic factors of thymic tumors. Conclu-sion:The expression of Gli-2 and Ki-67 showed a positive correlation with thymic tumors. Detecting the combined expression of Gli-2 and Ki-67 may elucidate the clinicopathological features and prognoses of patients with thymic tumors.

Hedgehog Related Protein Expression in Breast Cancer: Gli-2 Is Associated with Poor Overall Survival

BACKGROUND: The hedgehog (Hh) signaling pathway is known to play a critical role in various malignancies, but its clinicopathologic role in breast cancer is yet to be established. METHODS: Tissue microarray blocks from 334 cases of breast cancer were prepared. The expression of six Hh signaling proteins including sonic hedgehog (Shh), patched (Ptch), smoothened (Smo), and the glioma-associated oncogene (Gli)-1, Gli-2, and Gli-3 were analyzed immunohistochemically. RESULTS: The expression of Hh signaling proteins was significantly correlated with some prognostic factors including the correlation of lymph node metastasis with the expression of Shh (p=0.001) and Ptch (p=0.064), the correlation of the stages with Shh and Gli-3 expression (p=0.007 and p=0.024, respectively), the correlation of the nuclear grade with the Smo (p=0.004) and Gli-3 (p=0.000), and the correlation of the histologic grade with the Ptch (p=0.016), Smo (p=0.007), and Gli-3 (p=0.000). The Shh, Ptch, Smo, Gli-1, and Gli-2 expression was significantly different between the phenotypes (p=0.000, p=0.001, p=0.004, p=0.039, and p=0.031, respectively). Gli-2 expression was correlated with a worse overall survival outcome (p=0.012). CONCLUSIONS: Hh pathway activation is correlated with a more aggressive clinical behavior in breast carcinomas. The comparison of phenotypes suggested that the Hh pathway may be a useful therapeutic target for breast carcinoma. Patients with Gli-2 expression had a significantly lower overall survival rate and, therefore, it showed promise as a prognostic marker.