Novel genetic and epigenetic alterations in ... - Ous-research.no

Quantitative MSPPrimers and probes for quantitative MSP (qMSP)were designed to specifically amplify fullymethylated bisulfite-converted complementarysequences of the promoter of interest. The primersand probe sequences used for the MGMT[GenBank: NM_002412] are listed in Additional file3. To normalize for DNA input in each sample, areference gene (ACTB [12]) was used.Fluorescence based real-time PCR assays werecarried out in a reaction volume of 20 L, consistingof 16.6mM ammonium sulphate; 67mM trizmapreset; 6.7mM MgCl 2; 10mM mercaptoethanol; 0.1%DMSO; 200μM each of dATP, dCTP, dGTP, anddTTP; 600nM of each primer; 0.4 L of Rox dye;200nM of probe; 1 unit of platinum Taq polymerase(Invitrogen, Carlsbad, CA, USA), and 2 μl ofbisulfite-modified DNA as a template. PCR wasperformed in separate wells for each primer/probeset and each sample was run in triplicate.Additionally, multiple water blanks were used, andas positive and negative control we usedcommercial methylated and unmethylated DNA(Millipore, Temecula, CA, USA). A series of dilutionsof methylated DNA after bisulfite conversion wereused for constructing a standard curve to quantifythe amount of fully methylated alleles in eachreaction. All amplifications were carried out in 96-well plates on an 7000 Sequence Detection System(Applied Biosystems, Foster City, CA, USA), at95ºC for 2 min followed by 45 cycles of 95ºC for 15s, and 60ºC for 1 min.In order to adjust for the possible various amountsof bisulfite treated DNA input in each PCR, theqMSP levels were normalized against therespective values of the internal reference gene(ACTB). The ratio thus generated constitutes anindex of the percentage of input copies of DNA thatare fully methylated at the primer- and probebindingsites. The ratio was multiplied by 100 foreasier tabulation (methylation level = target gene /reference gene x 100).A given sample was considered positive forpromoter hypermethylation when amplification wasdetected in at least 2 of the triplicates of therespective qMSP analysis. The qMSP thresholdwas determined by adjusting the best fit of theslope and R2, using the calibration curve.Selection criteria for the 11 gene promotersanalyzed in the present studySome of the genes analyzed were known to betargeted through promoter methylation in cancer,including colorectal cancer (SCGB3A1, RUNX3,CDKN2A, MLH1, and MGMT). HOXA9 was apotential new methylation target in colorectalcancer. ADAMTS1, CRABP1, MAL, and NR3C1were identified as novel epigenetically silencedtarget genes in colorectal cancer by our group[9,10,13] They were selected to be tested incombination with known methylated genes in a largeseries of colorectal lesions to check forinterdependencies. The methylation status of allincluded genes was compared in a series of normalmucosa from individuals without cancer with thoseof normal, benign and malignant tissue from thelarge bowel of cancer patients. Only two previousstudies have compared gene methylation amongthe same four types of sample groups asinvestigated here.[14,15] The first only investigatedone gene and the latter 10 genes among which onlythree overlapping the present selected gene list.Gene mutation status of BRAF, KRAS and TP53.The present carcinoma series form a part of a seriespreviously studied for genetic changes, includingBRAF, KRAS and TP53.[16,17] The specificmutation status of the individual tumors includedhere can be found in Additional file 4.StatisticsThe 2x2 contingency tables were analyzed usingFisher’s exact test and 3x2 tables were analyzed bythe Pearson 2 test. Non-parametric analyses wereperformed using the Kruskal-Wallis and Mann-Whitney tests. An independent T-test wasperformed when comparing continuous normallydistributed data with two groups. The bivariatecorrelation analysis was performed with Pearson’scorrelation. In order to determine age-specificmethylation for the genes we used logisticregression analysis. All two-tailed P-values werederived from statistical tests using the SPSS15.0software (SPSS, Chicago, IL, USA), and consideredstatistically significant at P < 0.05. The methylationheat-map was generated by average linkagehierarchical clustering and Pearson correlationdistance measure, using the SpotFireDecisionSite®9.0 software.Seven individuals had multiple polyps in the colon,and to exclude potential bias when analyzing patientdata such as sex and age, one polyp from eachindividual was randomly selected for statisticalanalyses.ResultsMSI status of colorectal tumorsTwo of sixty-three (3%) polyps displayed MSI. Bothwere large (>10mm) and located in the proximalcolon. The carcinomas were pre-selected accordingto MSI-status and 27/52 (52%) were MSI-positive.DNA promoter methylation in normal mucosa,adenomas, and carcinomasThe results of the MSP analyses of all samples andeach gene are summarized in Figure 2, Table 1,and Additional file 5. The mean number of genesmethylated per sample was 0.4 for the N1 group,1.2 for N2, 2.2 for adenomas, and 3.9 forcarcinomas, and was significantly different amongthe groups using Kruskal-Wallis test; P < 0.0001(mean rank N1, 10.2; N2, 17.3; adenomas, 23.1;and carcinomas, 31.4). Overall, 6/21 (29%) of theN1 samples, 9/18 (50%) of the N2 samples, 52/63(83%) of the adenomas, and 48/52 (92%) of thecarcinomas, were methylated in one or more of theeleven analyzed genes.3