Gli1 Research Articles

CSIG-05. IS THERE ANY BRAIN TUMOR PREDISPOSITION IN RELATED PALLISTER-HALL SYNDROMES AT THE NEW GENOMICS ERA

Abstract Pallister-Hall Syndrome (PHS) and its related syndromes are a spectrum of congenital malformations. They are associated to GLI3 mutations and specific brain tumors. Considered as lethal, it is now well established that this disease may be miss-diagnosed with the report of adult cases. Here, we discuss the management of the genetic counselling and the risk of neurologic tumor predisposition in GLI3-related PHS. We comprehensively reviewed the scientific literature using various databases to explore the risk of neurologic tumors and evolving practice of neuro-cancer genetic counseling in GLI3-related PHS. The review of literature showed that GLI3 is involved in tissue development as well as in cancer. GLI3 was identified as either a pro or anti-cancerous protein. In fact, GLI3 is described as upregulated in multiple cancers and is found to positively regulate malignant behavior. In neuro-oncology GLI gene was first identified as a highly expressed gene in human glioma and then as having an anti-cancerous role in medulloblastoma where GLI3 seems act as a tumor suppressor. In mutational studies, GLI3-germinal mutations are characterized by a high recurrence of hypothalamic hamartoma in syndromic patients. GLI3-somatic alterations have been also demonstrated in cell samples of patients with non-syndromic hypothalamic hamartoma. In addition, while inactivation of GLI3 is suggested to be associated with medulloblastoma, mutations are not common in sporadic medulloblastoma. The association of germinal GLI3 variants in syndromic patients with medulloblastoma has been identified in only one patient harboring GLI3 H302X variant. Our review permitted us to conclude that germline GLI3 mutations may predispose to a specific non-severe form of delayed hypothalamic hamartoma, which can be revealed in pauci or asymptomatic and unrecognized mutated individuals. The likelihood of having a neuro-cancer predisposition in GLI3 mutated families seems to be insignificant.

Effects of Isoxazolyl Steroids on Key Genes of Sonic Hedgehog Cascade Expression in Tumor Cells.

Activation of the Hedgehog (Hh) signaling pathway is often associated with the progression of various types of cancer. The purpose of study was to search for inhibitors of the Hh signaling pathway among eight compounds belonging to the group of isoxazolyl steroids. The evaluation of the effectiveness of the compounds was based on the analysis of their cytotoxicity, effect on the cell cycle, on the expression of key Hh-signaling-pathway genes (Ptch1, Smo, and Gli1) and putative target genes MMP-2 and MMP-9. Four compounds with the most pronounced cytotoxic effect were identified: compounds 1, 2 (HeLa cells) and 3, 4 (A549 cells). Compounds 1 and 2 significantly reduced the expression of the Ptch1, Smo, Gli1 genes, but had the opposite effect on MMP-2 gene expression: Compound 1 increased it, and compound 2 decreased it. Compounds 3 and 4 did not have a noticeable inhibitory effect on the expression of the Shh pathway receptors, but significantly inhibited MMP-2 and MMP-9 expression. Thus, it was shown that inhibition of the Shh signaling pathway by isoxazolyl steroids can have the opposite effect on MMPs gene expression, which is what should be taken into account in further studies of these compounds as therapeutic agents.

Targeting GLI1 and BAX by nanonoscapine could impede prostate adenocarcinoma progression

Prostate cancer as a critical global health issue, requires the exploration of a novel therapeutic approach. Noscapine, an opium-derived phthalide isoquinoline alkaloid, has shown promise in cancer treatment thanks to its anti-tumorigenic properties. However, limitations such as low bioavailability and potential side effects have hindered its clinical application. This study introduces nanonoscapine as a novel medication to overcome these challenges, leveraging the advantages of improved drug delivery and efficacy achieved in nanotechnology. We monitored the effects of nanonoscapine on the androgen-sensitive human prostate adenocarcinoma cell line, LNCaP, investigating its impact on GLI1 and BAX genes’ expressions, crucial regulators of cell cycle and apoptosis. Our findings, from MTT assays, flow cytometry, and gene expression analyses, have demonstrated that nanonoscapine effectively inhibits prostate cancer cell proliferation by inducing G2/M phase arrest and apoptosis. Furthermore, through bioinformatics and computational analyses, we have revealed the underlying molecular mechanisms, underscoring the therapeutic potential of nanonoscapine in enhancing patient outcomes. This study highlights the significance of nanonoscapine as an alternative or adjunct treatment to conventional chemotherapy, warranting further investigation in clinical settings.

Abstract We032: FOXK1-Gli2 network regulates cardiomyocyte proliferation and heart regeneration

Unlike adult mammals, the neonatal mouse has an exceptional regenerative capacity following myocardial injury. Our goal was to decipher and define novel factors and mechanisms that have a role in regeneration of the injured adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation, and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. In these studies, we identified a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration through the regulation of Gli2 . Analysis of single nucleus RNAseq datasets of neonatal hearts revealed that FOXK1 was expressed in neonatal CMs. qPCR analysis from P1 hearts of control and Foxk1 null mice confirmed this finding and demonstrated dysregulation of the cell cycle regulators, Ccne1 and p21 , in the absence of Foxk1 . Control and Foxk1 null mice were pulsed with EdU from P3 to P7 which demonstrated that the absence of Foxk1 led to a decrease in CM proliferation in-vivo. Then, using a transgenic mouse model that we engineered to overexpress FOXK1 in muscle ( MCK-Foxk1 ), we observed CM proliferation in adult MCK-Foxk1 mice, which showed increased incorporation of EdU and increased pH3 expression compared to control. Bulk RNA-seq analysis of P1, P7, and P14 hearts indicated increased CM proliferation in the MCK-Foxk1 mouse and revealed Gli2 as a potential downstream target of FOXK1. A gel shift assay was performed, which demonstrated that FOXK1 was able to bind to a FOXK1 motif upstream of the Gli2 gene. ChIP-PCR further verified that Gli2 was a direct downstream target of FOXK1 in the embryonic stem cell-embryoid body (ES-EB) system. We then performed ligation of the left anterior descending coronary artery (LAD) in adult control and MCK-Foxk1 mice. Echocardiography following the LAD-ligation injuries showed improved cardiac function in MCK-Foxk1 transgenic mice compared with controls. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration and suggest that this function is mediated through the regulation of Gli2 gene expression.

Etiology of combined pituitary hormone deficiency: GNAO1 as a novel candidate gene.

Because the causes of combined pituitary hormone deficiency (CPHD) are complex, the etiology of congenital CPHD remains unknown in most cases. The aim of the study was to identify the genetic etiology of CPHD in a well-defined single-center cohort. In total, 34 children (12 girls) with congenital CPHD (growth hormone (GH) deficiency and impaired secretion of at least one other pituitary hormone) treated with GH in our center were enrolled in the study. Their median age was 11.2 years, pre-treatment height was -3.2 s.d., and maximal stimulated GH was 1.4 ug/L. Of them, 30 had central adrenal insufficiency, 27 had central hypothyroidism, ten had hypogonadotropic hypogonadism, and three had central diabetes insipidus. Twenty-six children had a midline defect on MRI. Children with clinical suspicion of a specific genetic disorder underwent genetic examination of the gene(s) of interest via Sanger sequencing or array comparative genomic hybridization. Children without a detected causal variant after the first-tier testing or with no suspicion of a specific genetic disorder were subsequently examined using next-generation sequencing growth panel. Variants were evaluated by the American College of Medical Genetics standards. Genetic etiology was confirmed in 7/34 (21%) children. Chromosomal aberrations were found in one child (14q microdeletion involving the OTX2 gene). The remaining 6 children had causative genetic variants in the GLI2, PROP1, POU1F1, TBX3, PMM2, and GNAO1 genes, respectively. We elucidated the cause of CPHD in a fifth of the patients. Moreover, our study supports the PMM2 gene as a candidate gene for CPHD and suggests pathogenic variants in the GNAO1 gene as a potential novel genetic cause of CPHD.

"GLI1 Subcellular Localization and Overexpression as Prognostic Factors for Disease-Free Survival in Colorectal Carcinoma".

Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21months compared to those with normal expression who had 31months (p < 0.05). Moreover, individuals with early onset disease (15months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease( 23months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes.

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling

Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.

Shh Gene Regulates the Proliferation and Apoptosis of Dermal Papilla Cells to Affect Its Differential Expression in Secondary Hair Follicle Growth Cycle of Cashmere Goats.

Sonic hedgehog (Shh) is a component of the Hedgehog signaling pathway, playing an important role in regulating cell proliferation, differentiation, apoptosis, and the repair of damaged organisms. To further clarify the expression pattern of Shh gene in the secondary hair follicle growth cycle of cashmere goats and its mechanism of action on secondary hair follicle papilla cells, and improve cashmere quality, in this study, we took Inner Mongolia Albas white cashmere goats as the research objects and collected skin samples at different growth stages to obtain secondary hair follicles, detected Shh and its gene expression by RT-qPCR, Western blot, immunohistochemistry, and other techniques, while we also cultured DPCs in vitro. Shh gene overexpression and interference vectors were constructed, and the effects of Shh gene on the proliferation and apoptosis of DPCs were studied through cell transfection technology. The results showed that there are significant differences in Shh and its gene expression in the secondary hair follicle growth cycle skins of cashmere goats, with the highest expression level in anagen, followed by catagen, and the lowest expression level in telogen. Shh was mainly expressed in the inner root sheath, outer root sheath, and secondary hair follicle papilla. After the overexpression of Shh gene, the proliferation and vitality of the hair papilla cells were enhanced compared to the interference group. After Shh gene interference, the apoptosis rate of the cells increased, indicating that Shh gene can regulate downstream Ptch, Smo, and Gli2 gene expression to promote the proliferation of DPCs, and thus form its expression pattern in the secondary hair follicle growth cycle of cashmere goats.

The Chromosome-Scale Genome of Chitala ornata Illuminates the Evolution of Early Teleosts.

Teleosts are the most prolific vertebrates, occupying the vast majority of aquatic environments, and their pectoral fins have undergone remarkable physiological transformations throughout their evolution. Studying early teleost fishes, such as those belonging to the Osteoglossiformes order, could offer crucial insights into the adaptive evolution of pectoral fins within this group. In this study, we have assembled a chromosomal-level genome for the Clown featherback (Chitala ornata), achieving the highest quality genome assembly for Osteoglossiformes to date, with a contig N50 of 32.78 Mb and a scaffold N50 of 40.73 Mb. By combining phylogenetic analysis, we determined that the Clown featherback diverged approximately 202 to 203 million years ago (Ma), aligning with continental separation events. Our analysis revealed the intriguing discovery that a unique deletion of regulatory elements is adjacent to the Gli3 gene, specifically in teleosts. This deletion might be tied to the specialized adaptation of their pectoral fins. Furthermore, our findings indicate that specific contractions and expansions of transposable elements (TEs) in teleosts, including the Clown featherback, could be connected to their adaptive evolution. In essence, this study not only provides a high-quality genomic resource for Osteoglossiformes but also sheds light on the evolutionary trajectory of early teleosts.

A Comprehensive Clinicopathologic and Molecular Reappraisal of GLI1 -altered Mesenchymal Tumors with Pooled Outcome Analysis Showing Poor Survival in GLI1 - amplified Versus GLI1- rearranged Tumors.

GLI1 -altered mesenchymal tumor is a recently described distinct pathologic entity with an established risk of malignancy, being defined molecularly by either GLI1 gene fusions or amplifications. The clinicopathologic overlap of tumors driven by the 2 seemingly distinct mechanisms of GLI1 activation is still emerging. Herein, we report the largest series of molecularly confirmed GLI1 -altered mesenchymal neoplasms to date, including 23 GLI1- amplified and 15 GLI1 -rearranged new cases, and perform a comparative clinicopathologic, genomic, and survival investigation. GLI1- rearranged tumors occurred in younger patients (42 vs. 52y) and were larger compared with GLI1 -amplified tumors (5.6cm vs. 1.5cm, respectively). Histologic features were overall similar between the 2 groups, showing a multinodular pattern and a nested architecture of epithelioid, and less commonly spindle cells, surrounded by a rich capillary network. A distinct whorling pattern was noted among 3 GLI1 -amplified tumors. Scattered pleomorphic giant cells were rarely seen in both groups. The immunoprofile showed consistent expression of CD56, with variable S100, CD10 and SMA expression. Genomically, both groups had overall low mutation burdens, with rare TP53 mutations seen only in GLI1- amplified tumors. GLI1 -amplified mesenchymal tumors exhibit mostly a single amplicon at the 12q13-15 locus, compared with dedifferentiated liposarcoma, which showed a 2-peak amplification centered around CDK4 (12q14.1) and MDM2 (12q15). GLI1 -amplified tumors had a significantly higher GLI1 mRNA expression compared with GLI1 -rearranged tumors. Survival pooled analysis of current and published cases (n=83) showed a worse overall survival in GLI1 -amplified patients, with 16% succumbing to disease compared with 1.7% in the GLI1- rearranged group. Despite comparable progression rates, GLI1 -amplified tumors had a shorter median progression-free survival compared with GLI1 -rearranged tumors (25mo vs. 77mo). Univariate analysis showed that traditional histologic predictors of malignancy (mitotic count ≥4/10 high-power fields, presence of necrosis, and tumor size ≥5cm) are associated with worse prognosis among GLI1 -altered mesenchymal tumors.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

Lasting differential gene expression of circulating CD8 T cells in chronic HCV infection with cirrhosis identifies a role for Hedgehog signaling in cellular hyperfunction.

The impact of chronic hepatic infection on antigen non-specific immune cells in circulation remains poorly understood. We reported lasting global hyperfunction of peripheral CD8 T cells in HCV-infected individuals with cirrhosis. Whether gene expression patterns in bulk CD8 T cells are associated with the severity of liver fibrosis in HCV infection is not known. RNA sequencing of blood CD8 T cells from treatment naïve, HCV-infected individuals with minimal (Metavir F0-1 ≤ 7.0 kPa) or advanced fibrosis or cirrhosis (F4 ≥ 12.5 kPa), before and after direct-acting antiviral therapy, was performed. CD8 T cell function was assessed by flow cytometry. In CD8 T cells from pre-DAA patients with advanced compared to minimal fibrosis, Gene Ontology analysis and Gene Set Enrichment Analysis identified differential gene expression related to cellular function and metabolism, including upregulated Hedgehog (Hh) signaling, IFN-α, -γ, TGF-β response genes, apoptosis, apical surface pathways, phospholipase signaling, phosphatidyl-choline/inositol activity, and second-messenger-mediated signaling. In contrast, genes in pathways associated with nuclear processes, RNA transport, cytoskeletal dynamics, cMyc/E2F regulation, oxidative phosphorylation, and mTOR signaling, were reduced. Hh signaling pathway was the top featured gene set upregulated in cirrhotics, wherein hallmark genes GLI1 and PTCH1 ranked highly. Inhibition of Smo-dependent Hh signaling ablated the expression of IFN-γ and perforin in stimulated CD8 T cells from chronic HCV-infected patients with advanced compared to minimal fibrosis. CD8 T cell gene expression profiles post-DAA remained clustered with pre-DAA profiles and disparately between advanced and minimal fibrosis, suggesting a persistent perturbation of gene expression long after viral clearance. This analysis of bulk CD8 T cell gene expression in chronic HCV infection suggests considerable reprogramming of the CD8 T cell pool in the cirrhotic state. Increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.

A novel missense variant located within the zinc finger domain of the GLI3 gene was identified in a Vietnamese pedigree with index finger polydactyly.

Polydactyly, particularly of the index finger, remains an intriguing anomaly for which no specific gene or locus has been definitively linked to this phenotype. In this study, we conducted an investigation of a three-generation family displaying index finger polydactyly. Exome sequencing was conducted on the patient, with a filtration to identify potential causal variation. Validation of the obtained variant was conducted by Sanger sequencing, encompassing all family members. Exome analysis uncovered a novel heterozygous missense variant (c.1482A>T; p.Gln494His) at the zinc finger DNA-binding domain of the GLI3 protein within the proband and all affected family members. Remarkably, the variant was absent in unaffected individuals within the pedigree, underscoring its association with the polydactyly phenotype. Computational analyses revealed that GLI3 p.Gln494His impacts a residue that is highly conserved across species. The GLI3 zinc finger DNA-binding region is an essential part of the Sonic hedgehog signaling pathway, orchestrating crucial aspects of embryonic development through the regulation of target gene expression. This novel finding not only contributes valuable insights into the molecular pathways governing polydactyly during embryonic development but also has the potential to enhance diagnostic and screening capabilities for this condition in clinical settings.

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Genetic variation in two candidate genes against gastrointestinal parasites in Colombian Hair Sheep

Objective. To characterize two SNP-type genetic polymorphisms in the GLI1 (rs411868094) and IL20RA (rs419463995) genes, candidates for resistance against gastrointestinal parasites in two biotypes of Colombian hair sheep. Materials and methods. From the DNA bank of the Animal Genetics Laboratory of the University of Sucre, 167 samples, belonging to the Ethiopian (n=94) and Sudan (n=73) biotypes, were analyzed by PCR and subsequent bidirectional sequencing of two SNPs in the GLI1 (T&gt;G) and IL20RA (G&gt;A) genes. Allelic and genotypic frequencies, observed (Ho) and expected (He) heterozygosity, F index, and Hardy-Weinberg equilibrium deviations (EHW) were calculated using the GENALEX version 6.5 program. Results. For the GLI1 locus, the mean genotypic frequencies were 0.155±0.07, 0.370±0.07, and 0.475±0.07 for GG, GT, and TT, respectively. In the Ethiopian biotype, the highest frequencies of the genotype of interest (GG) were found. For the IL20RA locus, the AA and AG genotypes had similar and the highest frequency (0.465±0.03) compared to the GG genotype (0.110±0.01). The genotype of interest at this locus (AA) was the most frequent in both OPC biotypes. Conclusions. The alleles of interest associated with low FEC had a low frequency for the GLI1 gene, but a high frequency for IL20RA. The Ethiopian OPC biotype showed the highest frequencies of the genotypes of interest.

Intra-articular injection of miRNA-1 agomir, a novel chemically modified miRNA agonists alleviates osteoarthritis (OA) progression by downregulating Indian hedgehog in rats

Our objective in this study is to determine whether intra-articular injection of miRNA-1 can attenuate the progression of OA in rats by down regulating Ihh. Knee chondrocytes were isolated from male Sprague–Dawley rats aged 2–3 days. Second-generation chondrocytes were transfected with miR-1 mimic and empty vector with lipo3000 for 6 h and then stimulated with 10 ng/mL IL-1β for 24 h. OA-related and cartilage matrix genes were quantified using real-time quantitative polymerase chain reaction (RT-qPCR). Two-month-old male Sprague–Dawley rats were divided into three groups (n = 30?): sham operation group + 50 µL saline, anterior cruciate ligament transection (ACLT) group + 50 µL miR-1 agomir (concentration), and control group ACLT + 50 µL miR-1 agomir. Treatment was started one week after the operation. All animals were euthanized eight weeks after the operation. X-rays and micro-CT were used to detect imaging changes in the knee joints. FMT was used to monitor joint inflammation in vivo. Safranin O staining was used to detect morphological changes in articular cartilage. Immunohistochemistry was used to detect Col2, Col10, metalloproteinase-13 (MMP-13). RT-qPCR was used to detect gene changes includingmiR-1, Col2, Col10, MMP-13, Ihh, Smo, Gli1, Gli2, and Gli3. Overexpression of miR-1 in IL-1β-stimulated chondrocytes reduced the levels of Ihh, MMP-13, and Col10 but increased the levels of Col2 and aggrecan. Intra-articular injection of miR-1 agomir reduced osteophyte formation, inflammation, and prevented cartilage damage. RT-qPCR results indicated that the miR-1 agomir increased articular cartilage anabolism and inhibited cartilage catabonism. miR-1 can attenuate the progression of OA by downregulating Ihh.

The effect of ginger extract on expression of Gli1 and Patched-1 genes involved in breast cancer signaling pathway under in vitro condition

Introduction and Aim: Breast cancer ranks among the leading causes of death in women worldwide. Ginger has shown potential efficacy against certain cancer types, surpassing conventional therapies such as chemotherapy and radiation. However, its molecular mechanisms remain less understood. Materials and Methods: In this study, MCF-7 cancer cells were cultured and treated with various concentrations of aqueous ginger extract (20, 30, 45, 65 microgram/mL) for 12, 48, and 72 hours. The effects were assessed through gene expression analysis and cell vitality assays (MTT). Results: The cell vitality test revealed a direct correlation between cytotoxicity and extract concentration. Concentrations exceeding 30 microgram/mL exhibited significant cell death (IC50 of 104.03 microgram/mL). Gene expression analysis demonstrated an increase in Patched-1 and a decrease in Gli1 expression with rising extract concentrations. The maximum Patched-1 expression occurred at 65 microgram/mL after 72 hours. Patched-1 (oncogene) and Gli1 (tumor suppressor) are pivotal genes in the hedgehog (Hh) signalling pathway associated with breast cancer. Conclusion: It appears that ginger compounds play a substantial role in regulating cancer progression by influencing key components of the hedgehog signalling pathway.

Morphological changes and hub genes screening in rats with anorectal malformations during hindgut development

BackgroundAnorectal malformations (ARMs) are among the most common congenital digestive tract malformations worldwide. Although there have been extensive efforts for elucidating their pathogenesis, the specific molecular mechanisms underlying ARMs remain unclear. MethodsWe generated ARM Wistar rat models using ethylenethiourea. Nine pregnant rats were allocated to the ARM and control groups, respectively. Hindgut tissue was isolated from embryos collected on gestational days 14, 15, and 16, representing the key timepoints of anorectal development. High-throughput sequencing was used to identify differentially expressed genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the obtained genes. Module analysis was performed via Search Tool for the Retrieval of Interacting Genes (STRING), and a protein-protein interaction (PPI) network was constructed using Cytoscape software. Gene Set Enrichment Analysis (GSEA) was conducted to verify the enrichment of development/apoptosis/epithelium/morphogenesis/Wnt pathway-related core gene sets and identify ARM hub genes. Reverse transcription-quantitative polymerase chain reaction assay was employed to verify mRNA levels of hub genes. Results313 GSEA core genes form ten core gene sets were selected. Based on the 281 differentially expressed circRNAs (|Fc| > 2, FDR < 0.05), 42 PPI core genes were obtained. According to the integration of GSEA analysis and PPI network, three hub genes (Cul1, Gli3, and Osr2) highly associated with ETU-induced ARMs were identified. The qRT-PCR validation data were consistent with the sequencing results. ConclusionsThese results provide a theoretical basis for the further identification of potential diagnostic and therapeutic targets in ARMs.

Polyhydramnios associated with rare genetic syndromes: two case reports

BackgroundWe present two genetic causes of polyhydramnios that were challenging to diagnose due to their rarity and complexity. In view of the severe implications, we wish to highlight these rare genetic conditions when obstetricians consider differential diagnoses of polyhydramnios in the third trimester.Case presentationPatient 1 is a 34-year-old Asian woman who was diagnosed with polyhydramnios at 28 weeks’ gestation. First trimester testing, fetal anomaly scan, and intrauterine infection screen were normal. Subsequent antenatal ultrasound scans revealed macroglossia, raising the suspicion for Beckwith–Wiedemann syndrome. Chromosomal microarray analysis revealed a female profile with no pathological copy number variants. The patient underwent amnioreduction twice in the pregnancy. The patient presented in preterm labor at 34 weeks’ gestation but elected for an emergency caesarean section. Postnatally, the baby was noted to have a bell-shaped thorax, coat hanger ribs, hypotonia, abdominal distension, and facial dysmorphisms suggestive of Kagami–Ogata syndrome. Patient 2 is a 30-year-old Asian woman who was diagnosed with polyhydramnios at 30 weeks’ gestation. She had a high-risk first trimester screen but declined invasive testing; non-invasive prenatal testing was low risk. Ultrasound examination revealed a macrosomic fetus with grade 1 echogenic bowels but no other abnormalities. Intrauterine infection screen was negative, and there was no sonographic evidence of fetal anemia. She had spontaneous rupture of membranes at 37 + 3 weeks but subsequently delivered by caesarean section in view of pathological cardiotocography. The baby was noted to have inspiratory stridor, hypotonia, low-set ears, and bilateral toe polysyndactyly. Further genetic testing revealed a female profile with a pathogenic variant of the GLI3 gene, confirming a diagnosis of Greig cephalopolysyndactyly syndrome.ConclusionThese cases illustrate the importance of considering rare genetic causes of polyhydramnios in the differential diagnosis, particularly when fetal anomalies are not apparent at the 20-week structural scan. We would like to raise awareness for these rare conditions, as a high index of suspicion enables appropriate counseling, prenatal testing, and timely referral to pediatricians and geneticists. Early identification and diagnosis allow planning of perinatal care and birth in a tertiary center managed by a multidisciplinary team.

The importance of routine genetic testing in pediatric epilepsy surgery.

Genetic variants in relevant genes coexisting with MRI lesions in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. Still, presurgical evaluation does not include genetic diagnostics routinely. Here, we report our presurgical evaluation algorithm that includes routine genetic testing. We analyzed retrospectively the data of 68 children with DRE operated at a mean age of 7.8 years (IQR: 8.1 years) at our center. In 49 children, genetic test results were available. We identified 21 gene variants (ACMG III: n = 7, ACMG IV: n = 2, ACMG V: n = 12) in 19 patients (45.2%) in the genes TSC1, TSC2, MECP2, DEPDC5, HUWE1, GRIN1, ASH1I, TRIO, KIF5C, CDON, ANKD11, TGFBR2, ATN1, COL4A1, JAK2, KCNQ2, ATP1A2, and GLI3 by whole-exome sequencing as well as deletions and duplications by array CGH in six patients. While the results did not change the surgery indication, they supported counseling with respect to postoperative chance of seizure freedom and weaning of antiseizure medication (ASM). The presence of genetic findings leads to the postoperative retention of at least one ASM. In our cohort, the International League against Epilepsy (ILAE) seizure outcome did not differ between patients with and without abnormal genetic findings. However, in the 7/68 patients with an unsatisfactory ILAE seizure outcome IV or V 12 months postsurgery, 2 had an abnormal or suspicious genetic finding as a putative explanation for persisting seizures postsurgery, and 3 had received palliative surgery including one TSC patient. This study highlights the importance of genetic testing in children with DRE to address putative underlying germline variants as genetic epilepsy causes or predisposing factors that guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning. PLAIN LANGUAGE SUMMARY: Genetic variants in children with drug-resistant epilepsy (DRE) can negatively influence epilepsy surgery outcomes. However, presurgical evaluation does not include genetic diagnostics routinely. This retrospective study analyzed the genetic testing results of the 68 pediatric patients who received epilepsy surgery in our center. We identified 21 gene variants by whole-exome sequencing as well as deletions and duplications by array CGH in 6 patients. These results highlight the importance of genetic testing in children with DRE to guide patient and/or parent counseling on a case-by-case with respect to their individual chance of postoperative seizure freedom and ASM weaning.