Array CGH analysis in gastric gastrointestinal stromal tumors (GISTs)
ABSTRACT: To explore mechanisms underlying wild-type GISTs, array CGH was performed on 32 gastric GISTs (4 risk groups stratified by tumor size and mitotic counts comprised 8 cases each). 27 KIT-mutant, 1 PDGFRA-mutant and 4 wild-type GISTs
Project description:For the purpose of characterization of the 9p24 amplicon, we carried out high-resolution array CGH (Agilent 244K chip) analysis of four cancer cell lines, including three breast cancer cell lines, Colo824, HCC1954 and HCC70, and one esophageal cancer cell line, KYSE150. For each array, female DNA (Promega, Madison, WI) was used as a reference sample and labelled with Cy-3. The samples of interest were each labelled with Cy-5.
Project description:To investigate the cytogenetic and large-scale chromosomal changes in involuted or non-involuted microGISTs using post-whole genome amplification (WGA) FFPE DNA materials Sixteen patients, total 19 FFPE tumor samples (block storage time 4 months to 9 years), including 16 microGISTs and 3 GISTs larger than 1 cm from the same patients harboring microGISTs. All FFPE tumor samples underwent DNA extraction and WGA (modified degenerate oligonucleotide PCR (DOP) method, provided by Sigma). For each tumor sample, a post-WGA DNA extract from the normal tissue in the same block (or block from the same patient with a block storage time differences less than 2 years) was obtained for tumor sample DNA co-hybridization. Tumor and normal areas of interest were marked and collected from 5- to 10-micron unstained or hematoxylin-stained sections by manual or laser (PixCell IITM, Arcturus Bioscience, CA, USA) microdissection. DNAs were then extracted. WGA was performed using GenomePlex® Tissue Whole Genome Amplification WGA5 kit (Sigma, Saint Louis, MO, USA; http://www.sigmaaldrich.com/) in parallel in accordance with the manufacturer's protocols. At least four independent experiments were concurrently performed per template amplification. Four separate WGA reaction products were pooled for each sample.
Project description:To allow acute characterization of ABCB4 locus constitutional deletion in two patients with cholelithiasis/cholestasis, an Agilent 2X400 array CGH was used. Goal was to obtain genomic rearrangements fine characterization in order to describe ABCB4 constitutional deletions. ABCB4 locus microdeletions characterization vs reference sample (pool of six normal control DNAs)
Project description:To allow accute charaterization of NF1 locus constitutional microdeletion in 70 NF1 patients, a custom array CGH was developped. Goal was to obtain genomic rearrangements fine characterization in order to perform genotype-phenotype correlation in NF1 microdeleted patients. To serve as a reference group in our genotype-phenotype correlation study in NF1 microdeletion patients, non-deleted NF1 patients (i.e. patients with an intragenic NF1 mutation) were also selected from our database. A total of 389 NF1 patients were included in the reference group of non-deleted patients. Multiple logistic regression was performed to test the association of each clinical feature individually with the type of constitutional NF1 mutation (intragenic mutation vs. microdeletion). The phenotypic traits of the 389 reference patients are available in the "GSE19730_control_patient_characteristics.txt" supplementary file on the Series record. NF1 locus microdeletions characterization vs reference sample (pool of six normal control DNAs)
Project description:ArrayCGH profiling and examination of IgH disruption by FISH on two myeloma B-cell lineage subpopulations: CD138+ and CD19+ to confirm existence of abnormal bone marrow B-cell subset contaning genetic background for primary oncogenic events.
Project description:To clarify the microRNA (miRNA) expression signatures in gastrointestinal stromal tumors (GISTs), a series of 32 GIST specimens were analyzed using Agilent miRNA microarray V3. Unsupervised hierarchical clustering revealed that GISTs can be categorized into 2 groups according to the expression of a miRNA cluster encoded on chromosome 14q32.31. Total RNA was extracted from 32 fresh frozen GIST specimens obtained from surgical resections. Expression of 851 miRNAs was analyzed using Human miRNA Microarray V3 (Rel 12.0 G4470C; Agilent technologies, Santa Clara, CA, USA). Risk grade was assessed according to the risk definition system proposed by Fletcher et al. (Hum Pathol. 2002;33:459-65.)
Project description:The Goto-Kakizak (GK) rat, a nonobese animal model of Type 2 diabetes (T2D), were developed by repeated inbreeding of glucose-intolerent individuals selected from Wistar rats. During their development, GK rats suffer from reduced beta-cell mass and insulin resistance spontaneously (T2D phenotype), which are supposed to be caused by loci holding different genotypes between GK and Wistar rats. This array CGH experiment can detect loci which show different copy numbers (genotype) between GK and Wistar rats. These loci serve as a valuable repository for mining candidates contributing to the pathogenesis of T2D. The genomic DNA taken from 3 male GK rats as 3 test samples while pooled genomic DNA from 8 male Wistar rats as the common referrence. For each of the hybridization, a dye-swap was designed as well.
Project description:KIT, PDGFRA, NF1, and SDH mutations are alternate initiating events, fostering hyperplasia in gastrointestinal stromal tumors (GISTs), and additional genetic alterations are required for progression to malignancy. The most frequent secondary alteration, demonstrated in ~70% of GISTs, is chromosome 14q deletion. Here we report hemizygous or homozygous inactivating mutations of the chromosome 14q MAX gene in 16 of 76 GISTs (21%). We find MAX mutations in 17% and 50% of sporadic and NF1-syndromic GISTs, respectively, and we find loss of MAX protein expression in 48% and 90% of sporadic and NF1-syndromic GISTs, and in 3 of 8 micro GISTs, which are early GISTs. MAX genomic inactivation is associated with p16 silencing in the absence of p16 coding sequence deletion, and MAX induction restores p16 expression and inhibits GIST proliferation. Hence, MAX inactivation is a common event in GIST progression, fostering cell cycle activity in early GISTs. Overall design: High molecular weight genomic DNA was isolated from 9 GISTs using QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) and analyzed by Affymetrix Cytoscan HD 2,600K-SNP array (Affymetrix, Santa Clara, CA, USA) with Affymetrix Chromosome Analysis Suite 2.0.
Project description:8-oxoguanine (8-oxoG) is one of the most common DNA lesions generated by reactive oxygen species. Here we show the genome-wide distribution of 8-oxoG by coupling immunoprecipitation by antibodies specific for the DNA fragments containing 8-oxoG with microarray that covers rat genome. Genome-wide mapping of 8-oxoG in rat kidney reveals that 8-oxoG preferentially located at gene deserts and depleted in genic regions. We did not observe the difference in 8-oxoG level between highly- and lowly-expressed groups of genes, may be because of the low level of 8-oxoG in genic regions in general comparing to gene deserts. Rather, the distribution of 8-oxoG highly correlates with the distribution of lamina associated domains (LADs), suggesting that the spatial location of the genomic regions in the nucleus is the major determinant for the susceptibility to oxidative modifications. One possible explanation for the enrichment of 8-oxoG in LADs is that the nuclear periphery is more susceptible to the oxidative damage coming from outside the nucleus. Another explanation is that LADs take rather compact conformation, which might prevent the recruitment of the repairing complex to the modified bases. Normal condition. Biological replicate 2