| Purpose:Systematicaly analyse the copy number variations (CNVs) in sporadic pheochromocytoma cells genome DNA, discover and accurately locate the tumor-related deletion of chromosome1. And then screen the sporadic pheochromocytoma-associated genes within the deletion area to provide a new reference, for the diagnosis and treatment for sporadic pheochromocytoma.Materials and methods:1.39patients who were clinicly dignosised as sporadic pheochromocytoma from2010-9to2012-1in PUMCH were enrolled, including38benign cases and1malignant cases Take another39cases of their own peripheral blood leucocyte as normal control group. Extract cell DNA.2. Apply Sequenom MassArray to detect wheather or not there is gene mutation of inherited pheochromocytoma. Exclude inherited cases. Assays by Sequenom is for somatic mutation analysis designed to the preferred gene, pathway, or disease of investigation. All assays are designed using Sequenom's Assay Design tools with2-3rounds of optimization to provide you with100%working assays. The assays are then run on the MassARRAY Analyzer system to verify PCR and primer extension.3. Apply Affymetrix Inc. GeneChip SNP6.0chip to detect whole genome DNA copy number variations (including amplification and deletion) in14cases of sporadic pheochromocytoma (including tumor tissue and their own peripheral blood leucocyte). Analysis of SNP6.0chip test results and screen CNV region in14cases of sporadic pheochromocytoma. The Genome-Wide Human SNP Array6.0contains more than906,600single nucleotide polymorphisms (SNPs) and more than946,000probes for the detection of copy number variation(CNV). This array contains a total of946,000non-polymorphic copy number probes. These probes—744,000originally selected for their spacing and202,000selected based on known copy number changes reported in the Toronto Database of Genomic Variants (DGV)—enable us to detect de novo copy number changes and perform association studies by genotyping both SNP and known copy number vatations (CNVs) loci.The median inter-marker distance over all1.8 million SNP and copy number markers combined is less than700bases. All fragments resulting from restriction enzyme digestion, regardless of size, are substrates for adaptor ligation. A generic primer that recognizes the adaptor sequence is used to amplify adaptor-ligated DNA fragments. PCR conditions have been optimized to preferentially amplify fragments in the200to1,100bp size range. PCR amplification products for each restriction enzyme digest are combined and purified using polystyrene beads. The amplified DNA is then fragmented, labeled, and hybridized to a SNP Array6.0.The Genome-Wide Human SNP Nsp/Sty Assay Kit contains validated and qualified reagents for the most critical steps in the assay. This includes the PCR primer and adaptors, reagents to fragment and label the PCR products, and control reagents. Manual processing kits are available for either50or100reactions. An automated assay kit (for processing96reactions) is also available (see ordering information). Whole-genomeamplified material prepared by the Qiagen REPLI-g(?) kits may also be used as the starting material for the Genome-Wide Human SNP Assay Kit. Deletion regions that occurs in no less than50%cases are preliminaryly identified as the hot regions of chromosome1.4. Apply Q-PCR to confirm CNV region in left cases of sporadic pheochromocytoma. Amplification of a cDNA product by quantitative PCR (Q-PCR) is monitored by a fluorescent signal proportional to the amount of produced amplicon. The Q-PCR amplification curve usually displays an exponential phase followed by a non-exponential phase, ending with a plateau. Contrary to prevalent interpretation, we demonstrate that under standard qPCR conditions, the plateau can be explained by depletion of the probe through Taq polymerase catalysed hydrolysis. Knowing the probe concentration and the fluorescence measured at the plateau, a specific fluorescence can thus be calculated. As far as probe hydrolysis quantitatively reflects amplicon synthesis, this, in turn, makes it possible to convert measured fluorescence levels in the exponential phase into concentrations of produced amplicon. It follows that the absolute target cDNA concentration initially engaged in the Q-PCR can be directly estimated from the fluorescence data, with no need to refer to any calibration with known concentrations of target DNA. Missing regions that occurs in no less than50%cases are finally identified as the hot regions of chromosome1.5. Identifie the sporadic pheochromocytoma-associated gene candidates in hot regions of chromosome1by UCSC.Results:1.38cases were identified as sporadic pheochromocytoma, and1as inherited pheochromocytoma.2.Chromosome deletion of pheochromocytoma tissue compared with matched normal cells occurred in1p,3q,17p,22q,11q,Common occurrence (11of14) of deletion of chromosome is1p.The hot regions of1p (in no less than50%cases) include chrl:85592199-85598821, chrl:70877065-70905276, chrl:101491482-101552821.3.The result of Q-PCR confirmed that chrl:85592199-85598821and chrl:70877065-70905276are final hot regions (missing regions occurring in75%cases).4. The hot regions of1p include133genes which may be associated with sporadic pheochromocytomaConclusion:1.SNP6.0chip can effectively detect and precisely locate sporadic pheochromocytoma whole-genome small area DNA copy number changes, can discover new deleted regions of chromosomes.2. Part deletion of1p may occur with tumor development, which may be the basis of sporadic pheochromocytoma pathogenesis.3. Test results for further screening and localization of sporadic pheochromocytoma related genes provide important clues and theoretical information. SNP chips is an effective method of screening sporadic pheochromocytoma tumor-associated genes. |
