Establishment Of High Resolution Meltinganalysis For Detecting IDH Gene Mutations In Gliomas

The IDH1gene is located on chromosome2q33.3, full-length18841bp, including9introns and10exons,and IDH2gene is located on chromosome15q26.1,full-length18498bp,including11introns and12exons, encoding isocitrate dehydrogenase in the TCA catalyticcycle, catalyzing isocitrate generating alpha ketoglutarate, and concerned with hypoxiainducible factor (HIF) stability, while HIF expression and tumor different malignantbehavior[1]. Research shows that IDH mutation in the low level of diffuse gliomas,gemistocytic astrocytoma and secondary glioblastomas have a good prognosis effect.Therefore, the detection of glioma IDH mutation, make a prediction for patients withoperation, radiotherapy and chemotherapy response and prognosis, which help to guideclinical work. In addition, IDH mutation and can also be used as glioma type identificationand classification standards.This study aims to use the current real-time PCR, combined with saturated fluorescentdyes and HRM analysis software, build HRM method on detecting IDH gene mutations ingliomas, and apply to clinical test. Detecting IDH gene mutations by sensitive, specific,simple, rapid and accurate HRM analysis through optimizing test conditions, and to evaluatethe advantages of HRM analysis on mutation scanning and the feasibility of clinicalapplication through comparing with DNA sequencing and immunohistochemistry.Analysisthe statistical correlation of specimen parameters and HRM IDH1gene mutation detectionresults.Firstly, temperature gradient PCR, orthogonal design experiments and response surfaceexperiment, were performed to optimize qPCR-HRM reaction system and reaction conditionswith IDH gene. The minimum detection limit testing was conducted by mixing a series ofdilutions of100%,50%,25%,12.5%,6%,3%,1%,0.5%and0.25%of mutant IDH DNAfrom wild-type IDH DNA to mutation-type. In addition, these DNA were also used as positiveand negative controls. After HRM analysis, the PCR products were directly sequenced, andthen compared the the minimum detection limit of two methods. For the intraassay variation, one wild-type and one mutant of IDH were tested as15replicates for each sample at the sametime. To test interassay variation, a series of samples were tested every30days and threetimes totally. In order to analyze the minimum template quantity, the IDH gene mutant DNAwas diluted to different concentrations of HRM detection, analysis of minimum detectiontemplate.Therefore, IDH mutation detection in gliomas were be detected by HRM. We collected51paraffin-embedded tissue specimens of gliomas in Guangzhou General Hospital ofGuangzhou Military Command. Before DNA extraction, representative sections from tissueswere stained with HE. DNA concentration was adjusted to30ng. Gliomas in mutations weredetected by optimized qPCR-HRM reaction conditions and direct sequencing.51cases ofgliomas specimens were detected by HRM, and compared with IHC method and sequencing.Finally, we wanted to determine whether the mutational status of IDH1gene was associatedwith any of the histological parameters evaluated (tumor cells) and tumor stages and ages andsex factors. The statistical software SPSS13.0was used for data analysis, comparisonsbetween groups were made using the chi-square test, correlation analysiso determine whethermutationional status of IDH1gene was associated with any of the level of statisticalsignificance was established at0.05.As a result, temperature gradient PCR, orthogonal experiment and response analysisresults showed that: the optimal annealing temperature of IDH1gene was53℃and52.5℃for IDH2gene, the optimization of20μl qPCR-HRM reaction system for IDH1includingDNA template60ng, MgCl22.5mmol/L and Primer0.6μmol/L; The optimization of20μlqPCR-HRM reaction system for IDH2including DNA template45ng, MgCl22.75mmol/Land Primer0.6μmol/L. The HRM detection for IDH1mutation limit was0.25%, namely itcould detect mutation when0.5%mutant alleles. Sequencing could not detect mutant allelesbelow a level of40%; The HRM detection for IDH2mutation limit was1%, namely it coulddetect mutation when1%mutant alleles. Sequencing could not detect mutant alleles below alevel of80%, indicating that HRM was more sensitive than direct sequencing. The repetitionof HRM detection of IDH showed interassayand intraassay repeatability was good. Detection of IDH1and IDH2gene HRM DNA template was the lowest which are respectively0.5ngand1ng. HRM was detected in33cases of paraffin embedded tissue of glioma IDH1genemutation,2cases of paraffin embedded tissue of glioma IDH2gene mutation, the rest are wildtype. Through the statistical analysis of the results of HRM IDH gene mutation detectionmethod in accordance with the direct sequencing and immunohistochemistry. Logisticregression analysis showed that the number of specimens of tumor cells, patient age, gender,turmor grade and IDH1gene mutation has a significant relationship between the detectionrate (P>0.05).In summary, glioma IDH gene in the mutant HRM detection method has the advantagesof high sensitivity, good repeatability, simple operation, accurate result, which provides a newmethod for clinical glioma in paraffin embedded tissue samples to detect the mutation of IDHgene.