ZIC2 Research Articles

Identification and Analysis of ZIC-Related Genes in Cerebellum of Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with significant genetic heterogeneity. The ZIC gene family can regulate neurodevelopment, especially in the cerebellum, and has been implicated in ASD-like behaviors in mice. We performed bioinformatic analysis to identify the ZIC gene family in the ASD cerebellum. We explored the roles of ZIC family genes in ASD by investigating (i) the association of ZIC genes with ASD risk genes from the Simons Foundation Autism Research Initiative (SFARI) database and ZIC genes in the brain regions of the Human Protein Atlas (HPA) database; (ii) co-expressed gene networks of genes positively and negatively correlated with ZIC1, ZIC2, and ZIC3, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and receiver operating characteristic (ROC) curve analysis of genes in these networks; and (iii) the relationship between ZIC1, ZIC2, ZIC3, and their related genes with cerebellar immune cells and stromal cells in ASD patients. (i) ZIC1, ZIC2, and ZIC3 were associated with neurodevelopmental disorders and risk genes related to ASD in the human cerebellum and (ii) ZIC1, ZIC2, and ZIC3 were highly expressed in the cerebellum, which may play a pathogenic role by affecting neuronal development and the cerebellar internal environment in patients with ASD, including immune cells, astrocytes, and endothelial cells. (iii) OLFM3, SLC27A4, GRB2, TMED1, NR2F1, and STRBP are closely related to ZIC1, ZIC2, and ZIC3 in ASD cerebellum and have good diagnostic accuracy. (iv) ASD mice in the maternal immune activation model demonstrated that Zic3 and Nr2f1 levels were decreased in the immune-activated cerebellum. Our study supports the role of ZIC1, ZIC2, and ZIC3 in ASD pathogenesis and provides potential targets for early and accurate prediction of ASD.

FRI285 Evidence For ZIC1 As A Novel Gene For Isolated Hypogonadotropic Hypogonadism With Phenotypic Pleiotropic Intersection With Cerebellar Malformation

Abstract Disclosure: J. Cassin: None. D.L. Keefe: None. K.E. Sung: None. C.C. Bojo: None. G. Santiago: None. L. Plummer: None. K.B. Salnikov: None. S.B. Seminara: None. R. Balasubramania: None. P.L. Mellon: None. Isolated Hypogonadotropic Hypogonadism (IHH) is a condition marked by absent pubertal development and low sex steroids in the setting of low/normal gonadotropins. Mutations in known IHH genes only explain <50% of the genetic etiology suggesting significant missing heritability in IHH. To explore this missing IHH heritability, we examined whole exome sequencing data from approximately 1400 IHH probands for rare genetic variants in novel genes that are enriched in IHH patients. We identified four IHH probands with rare, novel heterozygous variants in ZIC1 (p.H134Rfs*21, p.Y287X, p.E299K, p.S413Y), a gene encoding C2H2-type zinc finger protein (rare variant enrichment p <0.0001 for both truncating and missense alleles). Prior literature review show that heterozygous deletions affecting the ZIC1 gene have been linked to Dandy Walker malformation [DWM], a cerebellar malformation, while gain-of-function ZIC1 single nucleotide variants have been linked to coronal craniosynostosis (premature cranial suture fusion). Notably, two IHH subjects with novel ZIC1 variants in our cohort exhibited posterior fossa abnormalities (DWM [p.H134Rfs*21] and ventriculomegaly [E299K]) but none had any cranial suture defects. Given the above genetic enrichment, phenotypic overlap between DWM and IHH, and ZIC1’s known high expression in the hypothalamus, we explored whether ZIC1 regulates the expression of hypothalamic releasing factors. We determined that ZIC1 overexpression increases GNRH1 luciferase expression in the immortalized GnRH cell line, GT1-7. We next explored whether the rare ZIC1 variants identified in the four IHH probands would affect this phenotype. We discovered that three of the four ZIC1 variants significantly reduced GNRH1-luciferase expression. Of those, one truncating mutation, p.H134Rfs*21, resulted in lower ZIC1 protein content in the cell and failure to localize to the nucleus, suggesting a hypomorphic mechanism of action. These findings implicate ZIC1 variants as putatively causal for IHH. Further studies will define the precise ZIC1 transcriptional targets that relate to ZIC1’s pleotropic effects on GnRH transcription, neuronal function, cerebellar development, and cranial suture fusion. Presentation: Friday, June 16, 2023

DNA Methylation Testing for Endometrial Cancer Detection in Urine, Cervicovaginal Self-Samples and Cervical Scrapes

ABSTRACT Endometrial cancer is the sixth most common cancer in women globally and has a rising incidence, accounting for 417,000 new diagnoses and more than 97,000 deaths in 2020. Early detection is critical because of a poor advanced-stage prognosis and high relapse risk. Postmenopausal bleeding precedes endometrial cancer in 90% of cases, yet only 5% to 10% of patients with this symptom have an underlying malignancy. Recent cytology research has demonstrated endometrial cancer cells in vaginal samples, suggesting urine and cervicovaginal self-samples may allow convenient and minimally invasive screening. In addition, screening samples for DNA methylation in tumor suppressor genes can increase testing efficacy of triaging patients with postmenopausal bleeding as it does not require intact tumor cells. This study aimed to evaluate the diagnostic performance of endometrial cancer detection using DNA methylation analysis in paired urine, cervicovaginal self-samples, and clinician-taken cervical scrapes. Paired samples from participants of the SOLUTION1 study with endometrial cancer were collected between October 2016 and August 2020. A complete urine void and cervicovaginal self-sample was collected at home, whereas the cervical scrape was taken by a clinician in the operating room before surgery. Each patient had a paired urine, cervicovaginal self-sample, and clinician-taken cervical scrape available for methylation analysis. Controls were collected from the Urine Controls (URIC) biobank and Dutch national cervical cancer screening program. Promoter hypermethylation of the ADCYAP1, BHLHE22, CDH13, CDO1, GALR1, GHSR, HAND2, SST, and ZIC1 genes was tested using quantitative methylation-specific polymerase chain reaction. Optimal 3-marker combinations were determined for each sample type using multivariable logistic regression analysis, and diagnostic performances of each marker and 3-marker panels were assessed by leave-one-out cross validation. A total of 103 endometrial cancer patients were included in this study, with 317 unpaired samples of control women. Methylation levels of all markers were significantly higher in all 3 sample types of endometrial cancer patients compared with healthy control women. In urine, the non–cross-validated area under the curve of the DNA methylation markers ranged between 0.61 and 0.93, in cervicovaginal self-samples between 0.62 and 0.91, and in clinician-taken cervical scrapes between 0.61 and 0.95. Most markers (7/9) showed the highest performance in urine, with the remaining 2 showing best performance in clinician-taken cervical scrapes. The optimal 3-marker combination panels showed area under the curve values of 0.95 (05% confidence interval [CI], 0.92–0.98) for urine, 0.94 (95% CI, 0.90–0.97) for cervicovaginal self-samples, and 0.97 (95% CI, 0.96–0.99) for clinician-taken cervical scrapes. The sensitivity and specificity of the optimal urine testing panel were both 90%, were 89% sensitivity and 92% specificity for cervicovaginal self-samples, and were 93% sensitivity and 90% specificity for clinician-taken cervical scrapes. The leave-one-out cross-validation analysis revealed virtually equal performances in marker panels for each of the 3 testing modalities. The results of this study demonstrate high diagnostic potential of DNA methylation testing in minimally and noninvasive samples for endometrial cancer detection, emphasizing the potential for patient-friendly home-based sample collection.

A novel super-enhancer-related gene signature predicts prognosis and immune microenvironment for breast cancer

BackgroundThis study targeted at developing a robust, prognostic signature based on super-enhancer-related genes (SERGs) to reveal survival prognosis and immune microenvironment of breast cancer.MethodsRNA-sequencing data of breast cancer were retrieved from The Cancer Genome Atlas (TCGA), 1069 patients of which were randomly assigned into training or testing set in 1:1 ratio. SERGs were downloaded from Super-Enhancer Database (SEdb). After which, a SERGs signature was established based on the training set, with its prognostic value further validated in the testing set. Subsequently, we identified the potential function enrichment and tumor immune infiltration of the model. Moreover, in vitro experiments were completed to further explore the biological functions of ZIC2 gene (one of the risk genes in the prognostic model) in breast cancer.ResultsA risk score system of prognostic value was constructed with 6 SERGs (ZIC2, NFE2, FOXJ1, KLF15, POU3F2 and SPIB) to find patients in high-risk group with significantly worse prognosis in both training and testing sets. In addition, a multivariate regression was established via integrating the 6 genes with age and N stage, indicating well performance by calibration, time-dependent receiver operating characteristic (ROC) analysis and decision curve analysis (DCA). Further analysis demonstrated that tumor-associated pathological processes and pathways were significantly enriched in the high-risk group. In general, the novel SERGs signature could be applied to screen breast cancer with immunosuppressive microenvironment for the risk score was negatively correlated with ESTIMATE score, tumor-infiltration lymphocytes (such as CD4 + and CD8 + T cell), immune checkpoints and chemotactic factors. Furthermore, down-regulation of ZIC2 gene expression inhibited the cell viability, cellular migration and cell cycle of breast cancer cells.ConclusionsThe novel SERGs signature could predict the prognosis of breast cancer; and SERGs might serve as potential therapeutic targets for breast cancer.

Developmentally regulated expression of integrin alpha-6 distinguishes neural crest derivatives in the skin.

Neural crest-derived cells play essential roles in skin function and homeostasis. However, how they interact with environmental cues and differentiate into functional skin cells remains unclear. Using a combination of single-cell data analysis, neural crest lineage tracing, and flow cytometry, we found that the expression of integrin α6 (ITGA6) in neural crest and its derivatives was developmentally regulated and that ITGA6 could serve as a functional surface marker for distinguishing neural crest derivatives in the skin. Based on the expression of ITGA6, Wnt1-Cre lineage neural crest derivatives in the skin could be categorized into three subpopulations, namely, ITGA6bright, ITGA6dim, and ITGA6neg, which were found to be Schwann cells, melanocytes, and fibroblasts, respectively. We further analyzed the signature genes and transcription factors that specifically enriched in each cell subpopulation, as well as the ligand or receptor molecules, mediating the potential interaction with other cells of the skin. Additionally, we found that Hmx1 and Lhx8 are specifically expressed in neural crest-derived fibroblasts, while Zic1 and homeobox family genes are expressed in mesoderm-derived fibroblasts, indicating the distinct development pathways of fibroblasts of different origins. Our study provides insights into the regulatory landscape of neural crest cell development and identifies potential markers that facilitate the isolation of different neural crest derivatives in the skin.

Annotation of uORFs in the OMIM genes allows to reveal pathogenic variants in 5'UTRs.

An increasing number of studies emphasize the role of non-coding variants in the development of hereditary diseases. However, the interpretation of such variants in clinical genetic testing still remains a critical challenge due to poor knowledge of their pathogenicity mechanisms. It was previously shown that variants in 5'-untranslated regions (5'UTRs) can lead to hereditary diseases due to disruption of upstream open reading frames (uORFs). Here, we performed a manual annotation of upstream translation initiation sites (TISs) in human disease-associated genes from the OMIM database and revealed ∼4.7 thousand of TISs related to uORFs. We compared our TISs with the previous studies and provided a list of 'high confidence' uORFs. Using a luciferase assay, we experimentally validatedthe translation of uORFsin the ETFDH, PAX9, MAST1, HTT, TTN,GLI2andCOL2A1 genes,as well as existence of N-terminal CDS extension in theZIC2 gene. Besides, we created a tool to annotate the effects of genetic variants located in uORFs. We revealed the variants from the HGMD and ClinVar databases that disrupt uORFs and thereby could lead to Mendelian disorders. We also showed that the distribution of uORFs-affecting variants differs between pathogenic and population variants. Finally, drawing on manually curated data, we developed a machine-learning algorithm that allows us to predict the TISs in other humangenes.

Human-gained heart enhancers are associated with species-specific cardiac attributes.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Age-Related DNA Methylation in Normal Kidney Tissue Identifies Epigenetic Cancer Risk Susceptibility Loci in the ANKRD34B and ZIC1 Genes.

Both age-dependent and age-independent alteration of DNA methylation in human tissues are functionally associated with the development of many malignant and non-malignant human diseases. TCGA-KIRC data were biometrically analyzed to identify new loci with age-dependent DNA methylation that may contribute to tumor risk in normal kidney tissue. ANKRD34B and ZIC1 were evaluated as candidate genes by pyrosequencing of 539 tissues, including 239 normal autopsy, 157 histopathologically tumor-adjacent normal, and 143 paired tumor kidney samples. All candidate CpG loci demonstrated a strong correlation between relative methylation levels and age (R = 0.70–0.88, p < 2 × 10−16) and seven out of 10 loci were capable of predicting chronological age in normal kidney tissues, explaining 84% of the variance (R = 0.92). Moreover, significantly increased age-independent methylation was found for 9 out of 10 CpG loci in tumor-adjacent tissues, compared to normal autopsy tissues (p = 0.001–0.028). Comparing tumor and paired tumor-adjacent tissues revealed two patient clusters showing hypermethylation, one cluster without significant changes in methylation, and a smaller cluster demonstrating hypomethylation in the tumors (p < 1 × 10−10). Taken together, our results show the presence of additional methylation risk factors besides age for renal cancer in normal kidney tissue. Concurrent tumor-specific hypermethylation suggests a subset of these loci are candidates for epigenetic renal cancer susceptibility.

Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

EP263: A pathogenic deletion at 13q32.1q33.1 presenting with bilateral sensorineural hearing loss and pigmentation anomalies, mimicking Waardenburg syndrome

13q deletion syndrome is caused by structural and functional monosomy of the long arm of chromosome 13. Individuals with partial deletions of 13q have a wide spectrum of clinical phenotypes, including mild to severe intellectual disability, growth retardation, craniofacial dysmorphism, hand and foot anomalies, brain, cardiac and renal defects. Previous reports suggest that deletions including the 13q32 band are associated with severe congenital malformations. The ZIC2 gene maps to this critical distal region at 13q32.2 and has been considered a possible candidate for brain malformations, such as holoprosencephaly and Dandy-Walker malformation. We present an individual with a 7.9 Mb deletion on chromosome 13, encompassing almost all of band q32, with a relatively mild phenotype. An 11-month-old term male was seen in the medical genetic clinic regarding concerns for possible Waardenburg Syndrome. He presented with bilateral sensorineural hearing loss at 9 months of age and was noted to have multiple hypopigmented lesions on both his feet and chest. Prenatal and post- natal course was unremarkable with normal growth and development. Physical examination revealed mild dysmorphic features such as borderline, low set and posteriorly rotated ears with folded upper helixes and a broad nasal bridge. Hypopigmented lesions consistent with leukoderma was noted over the anterior chest wall, abdominal wall and medial aspect of his right thigh. Family history was positive for a maternal cousin with similar hypopigmented lesions and maternal great, great grandmother with early premature graying of hair. There was no family history of congenital hearing loss or birth defects. A chromosomal microarray (CMA) was performed and revealed a 7.9 megabase pathogenic deletion at chromosome 13q32.1q33.1, involving 65 genes. This deletion encompasses the ZIC2 gene which is associated with holoprosencephaly. This was an unexpected finding and further imaging studies to evaluate for cardiac, renal and brain malformations have been planned. Deletions of 13q are rare and additional cases are needed to better understand the genotype-phenotype correlation. We present an atypical case of a 13q deletion syndrome associated with mild dysmorphism, bilateral sensorineural hearing loss and leukoderma. There have been suggestions that distal deletions are associated with a more severe clinical phenotype and proximal deletions causes less severe major anomalies. This case broadens the clinical spectrum of 13q deletion syndrome as hearing loss is an uncommon finding and dermatological involvement has not been previously reported. There have been one prior report indicating that the loss of Zic2 function results in impaired inner ear development in mice and may be a candidate gene that contributes to the genetics of hearing loss in humans. Additionally, although the region deleted on the long arm of chromosome 13 involves the ZIC2 gene, this patient appears to be growing and developing normally. These findings suggest regions outside of band 13q32 likely contribute to the severe congenital malformations previously associated with distal 13q deletions.

Combined training increases thermogenic fat activity in patients with overweight and type 2 diabetes.

Exercise is an important strategy in the management of diabetes. Experimental studies have shown that exercise acts, at least in part, by inducing the production of myokines that improve metabolic control and activate brown/beige adipose tissue depots. Combined training (CT) is recommended by the major diabetes guidelines due to its metabolic and cardiovascular benefits, however, its impact on brown/beige adipose tissue activities has never been tested in humans with overweight and type 2 diabetes (T2D). Here, we evaluated the effects of 16-week combined training (CT) program on brown adipose tissue activity; browning and autophagy markers, and serum pro-thermogenic/inflammatory inducers in patients with overweight and T2D. Thirty-four patients with overweight and T2D were assigned to either a control group (CG) or a combined training group (CTG) in a randomized and controlled study. Functional/fitness parameters, anthropometry/body composition parameters, blood hormone/biochemical parameters, thermogenic/autophagic gene expression in subcutaneous adipose tissue were evaluated before and at the end of the intervention. In addition, cold-induced 18-Fluoroxyglucose Positron Emission Computed Tomography (18F-FDG PET/CT) was performed in the training group before and after the end of the intervention. CT increased cervical/supraclavicular brown adipose tissue (BAT) thermogenic activity (p = 0.03) as well as in perirenal adipose tissue (p = 0.02). In addition, CT increased the expression of genes related to thermogenic profile (TMEM26: + 95%, p = 0.04; and EPSTI1: + 26%, p = 0.03) and decreased autophagic genes (ULK1: -15%, p = 0.04; LC3: -5%, p = 0.02; and ATG4: -22%, p < 0.001) in subcutaneous adipose tissue. There were positive correlations between Δ% BAT activity with Δ% of post training energy expenditure cold exposure, HDL-c, IL4, adiponectin, irisin, meteorin-like, and TMEM26 and ZIC1 genes, besides negative correlations with LDL-c, total cholesterol and C-reactive protein. This is the first evidence of the beneficial actions of CT on adipose tissue thermogenic activity in humans, and it adds important support for the recommendation of CT as a strategy in the management of diabetes.

Gene-Specific DNA Methylation Profiles in Pediatric Medulloblastomas.

Medulloblastoma is the most common pediatric central nervous tumor of high malignancy that has been classified into both histological subtypes and molecular subgroups by the 2016 World Health Organization classification. However, there is a still need to understand the genomic characteristics and predict the clinical course. The aim of the study is to investigate the significance of the methylation profiles in molecular subclassification and precision medicine of the disease. The study enrolled 47 pediatric medulloblastoma patients. DNA methylation levels of KLF4, SPINT2, RASSF1A, EZH2, ZIC2, and PTCH1 genes were analyzed using methylation-specific pyrosequencing. The significance of the statistical relationship between methylation profiles and clinicopathological parameters including molecular subgroups and histological subtypes, the status of metastasis, and event-free survival were analyzed. DNA methylation analysis demonstrated that KLF4, PTCH1, and ZIC2 hypermethylation were associated with the SHH-activated subgroup, whereas both SPINT2 and RASSF1A hypermethylation were associated with metastatic disease. EZH2 gene was not methylated in any of the samples. We think that customized DNA methylation profiling may be a useful tool in the molecular subclassification of pediatric medulloblastoma and a potential technical approach in precision medicine.

Anti Zic4 Paraneoplastic Neurological Syndrome Presenting as Oscillopsia.

Anti Zic4 Paraneoplastic Neurological Syndrome Presenting as Oscillopsia.

New SHH and Known SIX3 Variants in a Series of Latin American Patients with Holoprosencephaly

Holoprosencephaly (HPE) is the failure of the embryonic forebrain to develop into 2 hemispheres promoting midline cerebral and facial defects. The wide phenotypic variability and causal heterogeneity make genetic counseling difficult. Heterozygous variants with incomplete penetrance and variable expressivity in the SHH, SIX3, ZIC2, and TGIF1 genes explain ∼25% of the known causes of nonchromosomal HPE. We studied these 4 genes and clinically described 27 Latin American families presenting with nonchromosomal HPE. Three new SHH variants and a third known SIX3 likely pathogenic variant found by Sanger sequencing explained 15% of our cases. Genotype-phenotype correlation in these 4 families and published families with identical or similar driver gene, mutated domain, conservation of residue in other species, and the type of variant explain the pathogenicity but not the phenotypic variability. Nine patients, including 2 with SHH pathogenic variants, presented benign variants of the SHH, SIX3, ZIC2, and TGIF1 genes with potential alteration of splicing, a causal proposition in need of further studies. Finding more families with the same SIX3 variant may allow further identification of genetic or environmental modifiers explaining its variable phenotypic expression.

Correlation of zinc finger protein 2, a prognostic biomarker, with immune infiltrates in liver cancer.

Purpose: The expression and clinical value of zinc finger protein 2 gene (ZIC2) in hepatocellular carcinoma (HCC) were analyzed by mining gene information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases.Methods: Gene chip data sets were retrieved from GEO and TCGA and screened for differentially expressed genes in HCC. Gene expression profile interaction analysis (GEPIA) and Kaplan–Meier curves were used to analyze the relationship between differentially expressed genes (DEGs) and survival and prognosis in patients with HCC. Moreover, the Genecards database was used to extract ZIC2-related proteins and to analyze the physiological process of protein enrichment. Furthermore, the relationships between ZIC2 gene and tumor cell immune invasion and that between immune cell infiltration and the 5-year survival rate were studied using the tumor immune evaluation resource (TIMER) database.Results: Datasets from GEO and TCGA revealed that ZIC2 was differentially expressed in HCC tissues and normal tissues (P<0.05). High ZIC2 expression was associated with overall survival (OS) and progress-free survival in HCC patients. Overall, 25 ZIC2 related proteins, including Gli3, PRKDC, and rnf180 were identified and protein enrichment analysis indicated these were associated with four types of cell components, six types of cell functions, and eight types of biological processes. ZIC2 was positively correlated with immune infiltration cells in patients with HCC, and higher expression of ZIC2 mRNA CD4+T cells is associated with a better 5-year survival.Conclusion: ZIC2 gene may be used as an immune response marker in liver cancer to predict the prognosis of HCC.

Methylation Profiling of Specific Genes in Ependymomas.

Ependymomas are neuroepithelial tumors of the central nervous system with heterogeneous biology and clinical course. The aim of the present study is to investigate the relationship between the methylation status and clinicopathological parameters in ependymomas. DNA methylation status of CDKN2A, RASSF1A, KLF4 and ZIC2 genes were quantitatively analyzed with pyrosequencing in 44 ependymoma tumor tissues. The relationship of methylation profiles with tumor subtype, histological grade and patient age was statistically analyzed. DNA methylation analyses for CDKN2A revealed no difference in methylation levels. Of the 31 included samples for optimal ZIC2 methylation analysis, 10 were hypermethylated (32.3%) and this change was significantly found in the adult spinal ependymomas (p=0.01). KLF4 hypermethylation was observed in 6 of the overall included 35 samples (17.1%); however, there was no statistically significant relation of the methylation status with tumor subtype, histological grade or age group. RASSF1A hypermethylation was observed in overall 40 included samples with varying methylation levels. Higher levels of hypermethylation were significantly related to the grade 3 histology (p=0.01) and spinal ependymomas (p=0.006). The pediatric cases with grade 3 ependymomas and ependymomas of adulthood showed significantly increased RASSF1A hypermethylation levels (p < 0.001 and p=0.001, respectively). DNA methylation changes are likely to have biological importance in ependymomas. Both ZIC2 and RASSF1A methylation status may be useful parameters in the subclassification of these tumors.

Deregulation of the Epithelial-to-Mesenchymal Transition Process Underlies Zic2-Linked Holoprosencephaly

Mutations in the Zic2 gene cause holoprosencephaly type 5, but the mechanisms that translate Zic2 mutations into this devastating pathology remain unclear. Here, we report that Zic2 is expressed in a few epiblast cells during gastrulation to become transiently upregulated in the primitive streak. Later, during neurulation, Zic2 is expressed in neural crest cells and downregulated as they delaminate from the neural tube. In combination with transcriptomic data from mutant embryos, chromatin occupancy profiles in gastrula and neural crest cells reveal that Zic2 regulates a large number of genes associated with the Wnt, cadherin and TGF-β pathways. Functional experiments in chick and mice demonstrate that Zic2 blocks the activation of the Wnt canonical pathway at the time that induces the TGF-β pathway to trigger cell delamination. All these experiments together demonstrate that Zic2 initiates epithelial-to-mesenchymal transition at early developmental stages.

Diagnostic Outcomes for Genetic Testing of 54 Genes in Pregnancy Loss Using Array CGH Method: A Two-Year Retrospective Study

Objective: The current study aimed to retrospectively evaluate different diag- nostic approaches for the array genetic analysis of the cases from all trimester fetal loss in the medical genetics clinic between 2016 and 2017. The Quantitati- ve Fluorescent Polymerase Chain Reaction(QFPCR) test was performed on 50 samples, and aneuploidy was detected in 11 samples as a result of the test, and the array-CGH was performed when 39 QF-PCR resulted in normal test results. Under this purpose, we aimed to analyze and determine the possible copy num- ber variation(CNV), gene deletions, and/or duplications involved in embryonic cell division, tissue differentiation, intended.Materials and methods: DNA isolation from cases of this retrospective study was completed using the PureLink Genomic DNA isolation kit. DNA samples were then genoty - ped for molecular etiological reasons by oligonücleotid microarray -CGH method (aCGH , 60 K ISCA design , Agilent , Germany ). Hybridized probe correlations of the case and reference DNAs were evaluated with databases (Database of Genomic Variants Analysis ) used in genomic variation analysis in terms of 54 functional genes CNVs associated with intrauterine losses.Results: CNV was detected in 30 (77%) of 39 fetal samples analyzed within the scope of the research. Fifty-five percent of CNVs were found to be duplication (55%) and forty-five percent were deletions (45%). As a result of the evaluation, deletion was detected in 19 (35%) of 54 genes, duplication was detected in 26 (48%), while in 3 (6%) both deletion and duplication were detected. Although CNV detected in autosomal chromosomes (chromosome 1, 2, 3, 4, 5, 7, 8, 10, 12, 13, 14, 15 and 20), CNV was established the most common in X chromosome. In our study, CNVs associated with COX7B, ZIC1, MECP2, FMR1, HOXD13, JAG1, MSX2, NEXN, and SIX3 genes were found to be more frequent in terms of fetal loss etiology.Conclusions: Based on our experience, the array-CGH method can be used to investigate the etiology of the normal results of QF-PCR in cases of fetal loss. The array CGH method will be preferred more and more due to the ease of application and the data obtained. When we look at the literature, it is seen that there is not enough research on array CGH about fetal loss and more studies are needed to increase the experience in this field.

Analysis of Cross-Association between mRNA Expression and RNAi Efficacy for Predictive Target Discovery in Colon Cancers.

Simple SummaryThis study focused on finding genes for which mRNA expression was able to predict the anticancer efficacy of its RNAi treatment. Predictive target discovery is of critical importance in developing biomarker-based strategies of precision medicine. We demonstrated this carrying out cross-association analysis on collateral mRNA expression and RNAi treatment data of ~12,000 genes on a colon cell line panel. The analysis revealed several genes with significant association between mRNA expression level and the inhibitory efficacy of its RNAi treatment. The experimental validation confirm that this simple approach has general applications for studying gene association between omics data from diverse cancer lineages.The availability of large-scale, collateral mRNA expression and RNAi data from diverse cancer cell types provides useful resources for the discovery of anticancer targets for which inhibitory efficacy can be predicted from gene expression. Here, we calculated bidirectional cross-association scores (predictivity and descriptivity) for each of approximately 18,000 genes identified from mRNA and RNAi (i.e., shRNA and sgRNA) data from colon cancer cell lines. The predictivity score measures the difference in RNAi efficacy between cell lines with high vs. low expression of the target gene, while the descriptivity score measures the differential mRNA expression between groups of cell lines exhibiting high vs. low RNAi efficacy. The mRNA expression of 90 and 74 genes showed significant (p < 0.01) cross-association scores with the shRNA and sgRNA data, respectively. The genes were found to be from diverse molecular classes and have different functions. Cross-association scores for the mRNA expression of six genes (CHAF1B, HNF1B, HTATSF1, IRS2, POLR2B and SATB2) with both shRNA and sgRNA efficacy were significant. These genes were interconnected in cancer-related transcriptional networks. Additional experimental validation confirmed that siHNF1B efficacy is correlated with HNF1B mRNA expression levels in diverse colon cancer cell lines. Furthermore, KIF26A and ZIC2 gene expression, with which shRNA efficacy displayed significant scores, were found to correlate with the survival rate from colon cancer patient data. This study demonstrates that bidirectional predictivity and descriptivity calculations between mRNA and RNAi data serve as useful resources for the discovery of predictive anticancer targets.

Cognitive Stimulation Induces Differential Gene Expression in Octopus vulgaris: The Key Role of Protocadherins.

Octopuses are unique invertebrates, with sophisticated and flexible behaviors controlled by a high degree of brain plasticity, learning, and memory. Moreover, in Octopus vulgaris, it has been demonstrated that animals housed in an enriched environment show adult neurogenesis in specific brain areas. Firstly, we evaluated the optimal acclimatization period needed for an O. vulgaris before starting a cognitive stimulation experiment. Subsequently, we analyzed differential gene expression in specific brain areas in adult animals kept in tested (enriched environment), wild (naturally enriched environment), and control conditions (unenriched environment). We selected and sequenced three protocadherin genes (PCDHs) involved in the development and maintenance of the nervous system; three Pax genes that control cell specification and tissue differentiation; the Elav gene, an earliest marker for neural cells; and the Zic1 gene, involved in early neural formation in the brain. In this paper, we evaluated gene expression levels in O. vulgaris under different cognitive stimulations. Our data shows that Oct-PCDHs genes are upregulated in the learning and lower motor centers in the brain of both tested and wild animals (higher in the latter). Combining these results with our previous studies on O. vulgaris neurogenesis, we proposed that PCDH genes may be involved in adult neurogenesis processes, and related with their cognitive abilities.