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Status |
Public on Feb 10, 2017 |
Title |
EFFECTS OF IDURSULFASE® TREATMENT ON EXTRACELLULAR MATRIX COMPONENTS, INTRACELLULAR SIGNALING AND PROLIFERATION/DIFFERENTIATION REGULATION IN CALVARIA-DERIVED BONE TISSUE OF APERT SYNDROME PATIENTS |
Organism |
Homo sapiens |
Experiment type |
Expression profiling by array
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Summary |
Experimental in vtiro approach to molecular pathways implicated in Apert Syndrome’s craniosynostosis through transcriptomics techniques. FGFR2 activation requires a tridimensional configuration between ligand, receptor and heparan sulfate(HS). Mutations in the extracellular region of this receptor affect the balance between proliferation and differentiation of osteoprogenitor cells, leading to craniosynostosis as Apert Syndrome (AS). We postulate that the degradation of HS in periosteal tissue in patients with AS can modify the gene expression profile and modulate cell behavior. Based on previous evidence we propose that the treatment with an enzyme that degrades HS, as Idursulfase, in periosteal tissue of patients with AS, could be affect the formation of the ternary complex (FGF / FGFR / HS), triggering changes in gene profiles expression in several molecular pathways, regarding their basal state with the mutated receptor.
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Overall design |
To delineate this, fibroblasts were obtained from three patients, these were cultivated and stimulated with FGF2 for 24 hours and with Idursulfase®( Elaprase-Shire) (0.025mg / ml) for 48 hours in triplicate in biological replicas for treatment conditions (E) y untreatment conditions (ST). Total RNA (> 50 ng / uL) was obtained to validate gene expression using GeneChip Human Gene 2.0 ST Array (Affymetrix). The Array GeneChip Human Gene 2.0 ST (Whole transcriptome,Affymetrix®) was chosen for this study. The methodological process was carried out according to manufacturer's instructions (GeneChip Whole Transcript PLUS reagent kit). The cDNA sense was fragmented and biotinylated using TdT (terminal deoxynucleotidyl transferase) and using the GeneChip WT Terminal labeling kit. Approximately 5.5μg of target DNA marked was hybridized to the GeneChip at 45° C for 16 hours. The hybridized arrays were washed and stained in the fluidics station for GeneChip450 and scanned with the Scanner GCS3000 (Affymetrix). Signal values were computed using the GeneChip™ Comand Console® software. Raw data were extracted automatically using the data extraction protocol included in the software. After importing the CEL files, the data were summarized and normalized using the RMA method implemented in the Affymetrix® Expression Console™. The results were exported using the RMA analysis and then the gene level protocol was performed for determining differentially expressed genes (DEG). The statistical significance of the expression data was determined using fold change. For each SDR set, hierarchical cluster analysis was performed using complete linkage and Euclidean distance as similarity measures. Enrichment analysis of genes and functional annotation was performed using DAVID (http://david.abcc.ncifcrf.gov/home.jsp). Each term was assigned a score (EASA), which consists of the p-value modified by the Fisher Exact Test. If the value was less than 0.05 it was classified as enrichment. All data analysis and visualization of differentially expressed genes were performed using R 3.1.2 (www.r-project.org). The genes obtained in functional annotations were grouped into clusters and those that had a fold change of2and significant p value were selected. Corrections of p value for the statistical significance of the findings were performed by using Bonferroni, Benjamini, and FDR tests. To delineate this, fibroblasts were obtained from three patients, these were cultivated and stimulated with FGF2 for 24 hours and with Idursulfase®( Elaprase-Shire) (0.025mg / ml) for 48 hours in triplicate in biological replicas for treatment conditions (E) y untreatment conditions (ST). Total RNA (> 50 ng / uL) was obtained to validate gene expression using GeneChip Human Gene 2.0 ST Array (Affymetrix). The Array GeneChip Human Gene 2.0 ST (Whole transcriptome,Affymetrix®) was chosen for this study. The methodological process was carried out according to manufacturer's instructions (GeneChip Whole Transcript PLUS reagent kit).
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Contributor(s) |
Ramirez DA, Muñoz AJ, Torres-Tobar LA, Gutierrez LD, Velasco HM, Prada R, Carracedo A |
Citation missing |
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Submission date |
Feb 09, 2017 |
Last update date |
Mar 15, 2019 |
Contact name |
Diana Ramirez |
Organization name |
Universidad Nacional de Colombia
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Department |
Medicine Faculty
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Lab |
3
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Street address |
Calle 53 Cra 37 Ed. 426
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City |
Bogota |
State/province |
Bogota |
ZIP/Postal code |
110000 |
Country |
Colombia |
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Platforms (1) |
GPL16686 |
[HuGene-2_0-st] Affymetrix Human Gene 2.0 ST Array [transcript (gene) version] |
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Samples (12)
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Relations |
BioProject |
PRJNA373844 |
Supplementary file |
Size |
Download |
File type/resource |
GSE94718_160304AE-01_Diana_Carolina_QC_report.xls.gz |
1.6 Mb |
(ftp)(http) |
XLS |
GSE94718_Analysis_Result.html.gz |
7.8 Kb |
(ftp)(http) |
HTML |
GSE94718_Data_Quality_Check.tar.gz |
137.1 Kb |
(ftp)(http) |
TAR |
GSE94718_RAW.tar |
91.4 Mb |
(http)(custom) |
TAR (of CEL) |
GSE94718_data3.xlsx |
5.2 Mb |
(ftp)(http) |
XLSX |
GSE94718_raw_data.xlsx |
26.0 Mb |
(ftp)(http) |
XLSX |
Processed data included within Sample table |
Processed data provided as supplementary file |
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