 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 31, 2018 |
| Title |
Comparative tissue gene expression profiling and alternative splicing by exon-sensitive microarrays in non-syndromic craniosynostosis |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
|
| Summary |
Craniosynostosis (CS) is the congenital premature fusion of one or more cranial sutures and represents the more prevalent craniofacial malformation in humans, with an overall incidence of 1 out of 2000-3000 live births. Non-syndromic craniosynostoses (NSC) are believed to be multifactorial disorders, with a strong genetic component, due to possible gene–gene or gene–environment interactions that remain to be clearly identified. In this study we delved into the molecular signaling acting in calvarial tissue and cells from patients affected by nonsynodromic midline craniosynostosis, using a comparative analysis between fused and unfused sutures of each affected individuals. Using comparative microarray tissue gene expression profiling we have identified a subset of genes involved in the structure and function of the primary cilium, including the Bardet-Biedl syndrome 9 (BBS9) gene, which was recently associated to sagittal synostosis in a GWAS study. We therefore characterized BBS9 expression and cilium-related signaling in cells isolated from patients’ calvarial bone.
|
| |
|
| Overall design |
5 patients affected by nonsynodromic sagittal synostosis or nonsyndromic metopic synostosis were enrolled in this study. Tissue specimens were collected, from each patients, both from patent and fused calvarial sutures and cryopreservated in liquid nitrogen. Total RNA was extracted from snap-frozen calvarial tissue specimens using pestel and TRIzol Reagent (Invitrogen), and subsequently purified using silica membrane spin columns from RNeasy Mini kit (Qiagen). The yield, quality and integrity of RNA were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies). The resulting total RNA was then used to created the biotin-labeled library to be hybridized on GeneChip® Exon 1.0 ST human microarrays following the procedure described by the manufacturer (Affymetrix). The CEL files resulting from the hybridization were analyzed using oneChannelGUI 1.6.5 (Sanges et al. Bioinformatics 2007, 23, 3406-3408). Exon and gene-level probeset summarization was done by mean of RMA and sketch quantile normalization. Gene-level differential expression: to assess differential expression at gene-level, we used an empirical Bayes method together with a false discovery rate (FDR) correction of the p-value Thus, the list of differentially expressed genes was generated using an FDR ≤ 0.05 together with an absolute log2(fold-change) threshold of 1. Exon-level analysis: an intensity filter was subsequently applied at gene-level to remove not expressed and low expressed genes, i.e. genes were retained for exon-level analysis if in all biological replication gene-level signal was greater than 5. Subsequently, only genes characterized to have at least two RNA isoforms annotated in Ensembl database were retained for further analysis. The Splicing Index value was calculated by taking the log2 ratio of the normalized exon intensity (NI) in Sample 1 and the NI in Sample 2. The normalized exon intensity (NI) is the ratio of the probe set intensity to the gene intensity. Alternative splicing events (ASEs) were detected as described in (Della Beffa et al. BMC Genomics 2008, 9, 571).
|
| |
|
| Contributor(s) |
Barba M, Bernardini C, Di Pietro L, Lattanzi W |
| Citation(s) |
29674126 |
| Submission date |
Mar 18, 2016 |
| Last update date |
Feb 18, 2019 |
| Contact name |
camilla bernardini |
| E-mail(s) |
camilla.bernardini@unicatt.it
|
| Phone |
+390630154711
|
| Organization name |
UCSC
|
| Department |
Human Anatomy
|
| Street address |
L.go F.Vito 1
|
| City |
Rome |
| ZIP/Postal code |
00186 |
| Country |
Italy |
| |
|
| Platforms (1) |
| GPL5175 |
[HuEx-1_0-st] Affymetrix Human Exon 1.0 ST Array [transcript (gene) version] |
|
| Samples (10)
|
|
| Relations |
| BioProject |
PRJNA315672 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE79386_RAW.tar |
228.7 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
|
|
|
|
 |
Less...
More...