Rapid Identification of Promoter Hypermethylation in Hepatocellular Carcinoma by Pyrosequencing of Etiologically Homogeneous Sample Pools
2007; Elsevier BV; Volume: 9; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2007.060209
ISSN1943-7811
AutoresEmelyne Dejeux, Vincent Audard, Catherine Cavard, Marta Gut, Benoı̂t Terris, Jörg Tost,
Tópico(s)Cancer-related gene regulation
ResumoAberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of ∼1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material. Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of ∼1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material. DNA methylation occurring at the 5-position of cytosines in the context of the dinucleotide CpG is of particular importance for proper development and gene regulation and is strongly implicated in the pathogenesis of various diseases.1Robertson KD DNA methylation and human disease.Nat Rev Genet. 2005; 6: 597-610Crossref PubMed Scopus (2041) Google Scholar Epigenetic deregulation, ie, aberrant DNA methylation levels, altered patterns of histone tail modifications, and chromatin structure, has now been recognized as a hallmark of cancer.2Coleman WB Rivenbark AG Quantitative DNA methylation analysis: the promise of high-throughput epigenomic diagnostic testing in human neoplastic disease.J Mol Diagn. 2006; 8: 152-156Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar3Jones PA Baylin SB The fundamental role of epigenetic events in cancer.Nat Rev Genet. 2002; 3: 415-428Crossref PubMed Google Scholar4Laird PW Cancer epigenetics.Hum Mol Genet. 2005; : R65-R76Crossref PubMed Scopus (437) Google Scholar An overall decrease in DNA methylation (global hypomethylation) is accompanied by a region- and gene-specific hypermethylation of CpG islands. Genes involved in DNA repair, detoxification, cell-cycle regulation, and apoptosis are thereby often inappropriately inactivated. Epigenetic changes occur early in the progression process and often precede malignancy, and epigenetic lesions in normal tissue might set the stage for neoplasia.5Cui H Horon IL Ohlsson R Hamilton SR Feinberg AP Loss of imprinting in normal tissue of colorectal cancer patients with microsatellite instability.Nat Med. 1998; 4: 1276-1280Crossref PubMed Scopus (266) Google Scholar Methylation patterns can be shared by different types of tumors as well as being tumor type-specific.6Costello JF Fruhwald MC Smiraglia DJ Rush LJ Robertson GP Gao X Wright FA Feramisco JD Peltomaki P Lang JC Schuller DE Yu L Bloomfield CD Caligiuri MA Yates A Nishikawa R Su Huang H Petrelli NJ Zhang X O'Dorisio MS Held WA Cavenee WK Plass C Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.Nat Genet. 2000; 24: 132-138Crossref PubMed Scopus (1156) Google Scholar Analysis of DNA methylation patterns has proven useful as biomarker for the early diagnosis, classification, prognosis, and therapy of human cancers.7Brena RM Huang TH Plass C Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings.J Mol Med. 2006; 84: 365-377Crossref PubMed Scopus (87) Google Scholar8Laird PW Early detection: the power and the promise of DNA methylation markers.Nat Rev Cancer. 2003; 3: 253-266Crossref PubMed Scopus (1251) Google Scholar9Yoo CB Jones PA Epigenetic therapy of cancer: past, present and future.Nat Rev Drug Discov. 2006; 5: 37-50Crossref PubMed Scopus (1113) Google Scholar Realizing the important participation of DNA methylation in the pathogenesis of cancer and other diseases, a variety of techniques for the study of DNA methylation has been developed in the last few years.2Coleman WB Rivenbark AG Quantitative DNA methylation analysis: the promise of high-throughput epigenomic diagnostic testing in human neoplastic disease.J Mol Diagn. 2006; 8: 152-156Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,7Brena RM Huang TH Plass C Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings.J Mol Med. 2006; 84: 365-377Crossref PubMed Scopus (87) Google Scholar,8Laird PW Early detection: the power and the promise of DNA methylation markers.Nat Rev Cancer. 2003; 3: 253-266Crossref PubMed Scopus (1251) Google ScholarOne of the methods that has recently received much attention for the simultaneous analysis and quantification of the degree of methylation at several CpG positions in close proximity is pyrosequencing.10Colella S Shen L Baggerly KA Issa JP Krahe R Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites.Biotechniques. 2003; 35: 146-150PubMed Google Scholar11Dupont JM Tost J Jammes H Gut IG De novo quantitative bisulfite sequencing using the pyrosequencing technology.Anal Biochem. 2004; 333: 119-127Crossref PubMed Scopus (215) Google Scholar12Tost J Dunker J Gut IG Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing.Biotechniques. 2003; 35: 152-156PubMed Google Scholar13Uhlmann K Brinckmann A Toliat MR Ritter H Nürnberg P Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis.Electrophoresis. 2002; 23: 4072-4079Crossref PubMed Scopus (145) Google Scholar The pyrosequencing technology is based on the luminometric detection of pyrophosphate that is released on nucleotide incorporation and converted into a light signal by a cascade consisting of four enzymes.14Ronaghi M Uhlen M Nyren P A sequencing method based on real-time pyrophosphate.Science. 1998; 281: 363-365Crossref PubMed Scopus (1185) Google Scholar Its ease-of-use, high reliability, and flexibility have made pyrosequencing an analysis platform that has been widely used for various diagnostic applications such as routine (multiplex) genotyping,15Langaee T Ronaghi M Genetic variation analyses by pyrosequencing.Mutat Res. 2005; 573: 96-102Crossref PubMed Scopus (104) Google Scholar,16van der Straaten T Kweekel D Tiller M Bogaartz J Guchelaar HJ Multiplex pyrosequencing of two polymorphisms in DNA repair gene XRCC1.J Mol Diagn. 2006; 8: 444-448Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar bacterial typing,17Clarke SC Pyrosequencing: nucleotide sequencing technology with bacterial genotyping applications.Expert Rev Mol Diagn. 2005; 5: 947-953Crossref PubMed Scopus (27) Google Scholar18Gharizadeh B Oggionni M Zheng B Akom E Pourmand N Ahmadian A Wallin KL Nyren P Type-specific multiple sequencing primers: a novel strategy for reliable and rapid genotyping of human papillomaviruses by pyrosequencing technology.J Mol Diagn. 2005; 7: 198-205Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar19Jordan JA Butchko AR Durso MB Use of pyrosequencing of 16S rRNA fragments to differentiate between bacteria responsible for neonatal sepsis.J Mol Diagn. 2005; 7: 105-110Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar and sensitive detection of mutations.20Ogino S Kawasaki T Brahmandam M Yan L Cantor M Namgyal C Mino-Kenudson M Lauwers GY Loda M Fuchs CS Sensitive sequencing method for KRAS mutation detection by pyrosequencing.J Mol Diagn. 2005; 7: 413-421Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar One of its major strengths is the quantitative nature of the results. The bioluminometric response is linear (R2 > 0.99) for the sequential addition of up to five identical nucleotides (C, G, and T) or three α-S-dATPs. Pyrosequencing has therefore been used as a method to determine allele frequencies of SNPs in pools of samples. Deviations from results obtained by genotyping of individual samples were as low as 1.6% (determined from peak height) in a pool of 1126 samples, and the technology was found to be able to detect differences in allele frequencies of less than 2% between pools of DNA.21Gruber JD Colligan PB Wolford JK Estimation of single nucleotide polymorphism allele frequency in DNA pools by using pyrosequencing.Hum Genet. 2002; 110: 395-401Crossref PubMed Scopus (100) Google Scholar22Neve B Froguel P Corset L Vaillant E Vatin V Boutin P Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.Biotechniques. 2002; 32: 1138-1142PubMed Google Scholar23Wasson J Skolnick G Love-Gregory L Permutt MA Assessing allele frequencies of single nucleotide polymorphisms in DNA pools by pyrosequencing technology.Biotechniques. 2002; 32: 1144-1150PubMed Google ScholarPyrosequencing is ideally suited for DNA methylation analysis after bisulfite treatment of DNA because it combines the ability of direct quantitative sequencing, reproducibility, speed, and ease-of-use and is becoming more and more used. Besides the identification of genes aberrantly silenced by promoter hypermethylation in cancer,24Mirmohammadsadegh A Marini A Nambiar S Hassan M Tannapfel A Ruzicka T Hengge UR Epigenetic silencing of the PTEN gene in melanoma.Cancer Res. 2006; 66: 6546-6552Crossref PubMed Scopus (208) Google Scholar,25Xinarianos G McRonald FE Risk JM Bowers NL Nikolaidis G Field JK Liloglou T Frequent genetic and epigenetic abnormalities contribute to the deregulation of cytoglobin in non-small cell lung cancer.Hum Mol Genet. 2006; 15: 2038-2044Crossref PubMed Scopus (56) Google Scholar pyrosequencing has been used as a reference method for validation of newly developed methods for DNA methylation analysis,26Schatz P Distler J Berlin K Schuster M Novel method for high throughput DNA methylation marker evaluation using PNA-probe library hybridization and MALDI-TOF detection.Nucleic Acids Res. 2006; 34: e59Crossref PubMed Scopus (43) Google Scholar to monitor chemically induced demethylation in leukemia patients,27Yang AS Doshi KD Choi SW Mason JB Mannari RK Gharybian V Luna R Rashid A Shen L Estecio MR Kantarjian HM Garcia-Manero G Issa JP DNA methylation changes after 5-aza-2′-deoxycytidine therapy in patients with leukemia.Cancer Res. 2006; 66: 5495-5503Crossref PubMed Scopus (241) Google Scholar as well as for a diagnostic test for aberrant methylation in the imprinting disorders Prader-Willi and Angelman syndromes.28White HE Durston VJ Harvey JF Cross NC Quantitative analysis of SNRPN gene methylation by pyrosequencing as a diagnostic test for Prader-Willi syndrome and Angelman syndrome.Clin Chem. 2006; 52: 1005-1013Crossref PubMed Scopus (56) Google Scholar One of the major limitations for the identification of novel genes involved in tumorigenesis is the amount of available sample that is in most cases derived from primary tumors. The successive use of several sequencing primers on the same DNA template (serial pyrosequencing) significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of a specific region amplified in a single polymerase chain reaction (PCR).29Tost J El Abdalaoui H Gut IG Serial pyrosequencing for quantitative DNA methylation analysis.Biotechniques. 2006; 40: 721-726Crossref PubMed Scopus (60) Google Scholar However, it still requires prior knowledge of the presence of epimutations in the target. Using a candidate gene approach, we found in various projects that only 5 to 10% of genes hypothesized to display aberrant methylation actually show differential methylation in a certain tumor type (J.T., unpublished data). Here, we present a novel method that enables rapid screening for differential methylation using etiologically homogeneous pools of samples. We analyzed quantitatively the DNA methylation levels at 112 CpGs in the promoter or differentially methylated region (DMR) of five genes that have previously been found to be implicated in the pathogenesis of hepatocellular carcinoma (HCC): the cell-cycle regulator CDKN2A (p16),30Liew CT Li HM Lo KW Leow CK Chan JY Hin LY Lau WY Lai PB Lim BK Huang J Leung WT Wu S Lee JC High frequency of p16INK4A gene alterations in hepatocellular carcinoma.Oncogene. 1999; 18: 789-795Crossref PubMed Scopus (165) Google Scholar glutathione S-transferase π (GSTP1) involved in detoxification and drug resistance,31Zhong S Tang MW Yeo W Liu C Lo YM Johnson PJ Silencing of GSTP1 gene by CpG island DNA hypermethylation in HBV-associated hepatocellular carcinomas.Clin Cancer Res. 2002; 8: 1087-1092PubMed Google Scholar the insulin-like growth factor 2 (IGF2),32Poirier K Chalas C Tissier F Couvert P Mallet V Carrie A Marchio A Sarli D Gicquel C Chaussade S Beljord C Chelly J Kerjean A Terris B Loss of parental-specific methylation at the IGF2 locus in human hepatocellular carcinoma.J Pathol. 2003; 201: 473-479Crossref PubMed Scopus (26) Google Scholar the DNA mismatch repair gene MLH1,33Matsukura S Soejima H Nakagawachi T Yakushiji H Ogawa A Fukuhara M Miyazaki K Nakabeppu Y Sekiguchi M Mukai T CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection.Br J Cancer. 2003; 88: 521-529Crossref PubMed Scopus (59) Google Scholar and β-catenin (CTNNB1), which is mutated in 26 to 40% of human HCCs and not regulated by methylation of its promoter.34Buendia MA Genetics of hepatocellular carcinoma.Semin Cancer Biol. 2000; 10: 185-200Crossref PubMed Scopus (283) Google Scholar,35de La Coste A Romagnolo B Billuart P Renard CA Buendia MA Soubrane O Fabre M Chelly J Beldjord C Kahn A Perret C Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas.Proc Natl Acad Sci USA. 1998; 95: 8847-8851Crossref PubMed Scopus (972) Google Scholar Samples were grouped into pools after bisulfite treatment depending on the etiology (hepatitis B or C virus infection and alcohol consumption) and on the tumoral and nontumoral tissue status: control liver, noncirrhotic or cirrhotic adjacent nontumoral liver, hepatic adenoma, and HCC with and without β-catenin mutation. All genes displaying variable methylation patterns were successfully detected in the pools, and no false-negative results occurred. The high quantitative concordance of the data accumulated by single sample analysis compared with the pooled sample amplification clearly demonstrates the ability of our method to preserve precious sample resources from clinical specimens without loss of accuracy. Our method therefore provides the possibility to screen a large number of potential target genes even with a limited amount of sample.Materials and MethodsSamplesTumor samples were collected in accordance with French law and ethical guidelines, and their use was approved by the institutional ethics committee. Operative notes and pathology reports were subjected to exhaustive review. Samples used in this study were 27 HCCs and three hepatic adenomas and their nontumoral counterparts and 10 normal control livers without cirrhosis. DNA from peritumoral liver tissue, a hepatic adenoma, and the carcinoma was available from one patient. The degree of hepatic fibrosis in noncancerous liver was graded according to the METAVIR classification.36Bedossa P Poynard T An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group.Hepatology. 1996; 24: 289-293Crossref PubMed Google Scholar Seventeen HCCs developed on extensive fibrosis or cirrhosis (F3 and F4). Etiologies of the tumors were hepatitis C virus (HCV) infection for 11 cases (41%), hepatitis B virus (HBV) infection for four cases (15%), and alcohol consumption for seven cases (26%). Five cases (18%) developed in the absence of known etiology. Fourteen HCC and the three hepatic adenomas displayed β-catenin mutations after RNA isolation and further sequencing with the primers F1/R1, F1/R2, and F2/R2, as described previously.35de La Coste A Romagnolo B Billuart P Renard CA Buendia MA Soubrane O Fabre M Chelly J Beldjord C Kahn A Perret C Somatic mutations of the β-catenin gene are frequent in mouse and human hepatocellular carcinomas.Proc Natl Acad Sci USA. 1998; 95: 8847-8851Crossref PubMed Scopus (972) Google ScholarPreparation of DNAsDNAs were extracted from frozen tissue samples using standard methods, and DNA concentrations of extracted DNA were determined using the Quant-iT dsDNA broad range assay kit (Invitrogen, Cergy Pontoise, France) on a SpectraMAX Gemini XPS microplate spectrofluorometer (Molecular Devices, St. Grégoire, France) and normalized to a concentration of 50 ng/μl. Unmethylated DNA was obtained by whole genome amplification of a DNA extracted from a lymphoblastoid cell line from the CEPH/Utah collection using the REPLI-g kit (Qiagen, Courtaboeuf, France) using 50 ng as input DNA.Highly methylated DNA was obtained by treating genomic DNA of the same lymphoblastoid cell line with the methylase SssI (Ozyme, St. Quentin-en-Yvelines, France). In brief, 7.5 μl of NE-buffer 2, 10 nmol of S-adenosylmethionine (SAM), and 6 U of SssI were added to 4.5 μg of human genomic DNA in a final volume of 67.5 μl. The solution was incubated at 37°C in a water bath. After 3 hours and again after an additional 2 hours, 10 nmol of SAM and 6 U of SssI were added, and the reaction was incubated overnight at 37°C. The enzyme was inactivated at 95°C for 5 minutes and DNA stored at −20°C until further use.Bisulfite Treatment and Adjustment of ConcentrationOne μg of DNA was bisulfite converted using the MethylEasy HT kit for Centrifuge (Human Genetic Signatures, North Ryde, NSW, Australia) according to the manufacturer's instructions. Concentration of the bisulfite converted DNAs was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and normalized to a concentration of 20 ng/μl. Pools were constructed from bisulfite-converted DNAs.DNA Methylation Analysis by PyrosequencingQuantitative DNA methylation analysis of the bisulfite-treated DNA was performed by pyrosequencing or, in case of several sequencing primers, by serial pyrosequencing.29Tost J El Abdalaoui H Gut IG Serial pyrosequencing for quantitative DNA methylation analysis.Biotechniques. 2006; 40: 721-726Crossref PubMed Scopus (60) Google Scholar Primers for PCR amplification and pyrosequencing were purchased from Biotez (Buch, Germany). Regions of interest were amplified using 20 ng of bisulfite-treated human genomic DNA and 5 to 7.5 pmol of forward and reverse primer, one of them being biotinylated. Sequences of the oligonucleotides for PCR amplification and pyrosequencing are given in Table 1. Reaction conditions were 1× HotStar Taq buffer supplemented with 1.6 mmol/L MgCl2, 200 mmol/L dNTPs, and 2.0 U of HotStar Taq polymerase (Qiagen) in a 25-μl volume. The PCR program consisted of a denaturing step of 15 minutes at 95°C followed by 50 cycles of 30 seconds at 95°C, 30 seconds at the respective annealing temperature (Table 1), and 20 seconds at 72°C, with a final extension of 5 minutes at 72°C. Amplification products were purified and rendered single-stranded on a Pyrosequencing workstation (Pyrosequencing AB, Uppsala, Sweden). PCR products were incubated for 10 minutes at room temperature with 51 μl of binding buffer (10 mmol/L Tris, 2 mol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid, and 0.1% Tween 20, pH 7.6, adjusted with 1 mol/L HCl) and 4 μl of streptavidin-coated Sepharose beads (GE Health Care, Uppsala, Sweden). The binding mix was aspirated, and the template was successively washed with 70% ethanol, rendered single-stranded with 0.2 mol/L NaOH, and neutralized with washing buffer (10 mmol/L Tris, pH 7.6, adjusted with 4 mol/L acetic acid). Beads were released into 40 μl of annealing buffer (20 mmol/L Tris and 2 mmol/L magnesium acetate, pH 7.6, adjusted with 4 mol/L acetic acid) containing 15 pmol of the respective sequencing primer (Table 1). Primers were annealed to the target by incubation at 80°C for 2 minutes. Quantitative DNA methylation analysis was performed on a PSQ 96MA system with the PyroGold SQA reagent kit (Pyrosequencing), and results were analyzed using the Q-CpG software (V1.0.9; Pyrosequencing). Stripping of the template strand for subsequent annealing of a new sequencing primer (serial pyrosequencing) was performed by adding 20 μl of binding buffer to the completed sequencing reaction and resuspending the Sepharose beads. The binding mix was then purified without further incubation and the biotinylated template strand rendered again single-stranded using the above-described purification protocol. This process completely removes all DNA strands that have been de novo synthesized during the last sequencing run as well as remaining sequencing primers.29Tost J El Abdalaoui H Gut IG Serial pyrosequencing for quantitative DNA methylation analysis.Biotechniques. 2006; 40: 721-726Crossref PubMed Scopus (60) Google ScholarTable 1Sequences of Primers Used for Amplification and Pyrosequencing Reactions, Including Genbank Accession Numbers and Nucleotides (Nt) Corresponding to the Amplified Fragments as Well as the Annealing Temperatures for the Respective PCR AmplificationsGenePCR primer forwardPCR primer reverseTemperature PCR (°C)CDKN2A (262 bp) AF527803 Nt 19893 to 201545′-GAGGGGTTGGTTGGTTATTAGA-3′5′-Biotin-TACAAACCCTCTACCCACCTAAAT-3′64CTNNB1 (294 bp) AY463360 Nt 1786 to 20795′-TGGGGTGTTTAGGTATTTTATTT-3′5′-Biotin-TAAAACTACTCCTCAAACCTTCCTC-3′64.2GSTP1 (301 bp) AY324387 Nt 1670 to 19705′-GAAAGAGGGAAAGGTTTTTT-3′5′-Biotin-CCATACTAAAAACTCTAAACCCCATC-3′58GSTP1 (301 bp) AY324387 Nt 1670 to 19705′-Biotin-GAAAGAGGGAAAGGTTTTTT-3′5′-CCATACTAAAAACTCTAAACCCCATC-3′58GSTP1 (212 bp) AY324387 Nt 1845 to 20565′-GGGATTATTTTTATAAGGTT-3′5′-Biotin- TCCTAAATCCCCTAAACCCC-3′54MLH1 (297 bp) AY217549 Nt 1504 to 18005′-GGGAGGTTATAAGAGTAGGGTTAA-3′5′-Biotin-TCTCAACTCTATAAATTACTAAATCTCTTC-3′61.4IGF2 DMR2 (255 bp) AF125183 Nt 7881 to 81005′-GGGAAAGGGGTTTAGGATTTTTAT-3′5′-Biotin-ATAATTTACTCCCCCTTCAACCTC-3′60Pyrosequencing primerCpG numberCpGs5′-TGGTTATTAGAGGGTG-3′1 to 775′-GAGGGGGAGAGTAGGTAG-3′8 to 1365′-GGGGAGTAGTATGGAGTTTT-3′14 to 2185′-GGGTYGGGTAGAGGA-3′20 to 2895′-CGGTAGTAGATTGTTGGG-3′4 to 20175′-GTTGYGTYGGTGG-3′21 to 2775′-TTTYGGTTTGTGGTAGTA-3′28 to 3475′-GTAGTTTTYGTTATTAGTGA-3′33 to 3865′-GGATTATTTTTATAAGGT-3′25 to 3285′-CGAACCTTATAAAAATAATC-3′17 to 1175′-GTAGTTTTYGTTATTAGTGA-3′9 to 1465′-GAGAGGAGGAGTTTGAGA-3′3 to 755′-TTTTTATTGGTTGGATATTT-3′8 to 1885′-AAAACGAATTAATAGGAAGA-3′11 to 18115′-GATTTTTATYGGAAGTA-3′2 to 655′-GGGTYGATAYGTTTTTTT-3′6 to 1385′-TTTTATAGTATAGAGAGAGT-3′16 to 183CpGs are numbered in the order of appearance from the 5′ end of an amplification product. Y, pyrimidine. (table continues) Open table in a new tab StatisticsQuantitative DNA methylation values are represented by boxplots in which the distribution of data points in a sample set is displayed without any assumption of statistical distribution. Lower and upper quartiles of the data delimit the box, the median is represented by the bold black dot in the box, and minimal and maximum values are indicated by the lines. A Mann-Whitney U-test was used to compare quantitative methylation values between two groups. For IGF2, the average of all samples was calculated, and for each sample, the absolute distance of the methylation value from this average was used to define values for ranking because both hypo- and hypermethylation occur in the tumors.ResultsThe rationale behind this study was to evaluate pyrosequencing-based DNA methylation analysis in pools of samples that are homogenized for distinct etiologies to enable rapid and cost-effective screening of multiple genes potentially displaying differential methylation in a candidate region. As a proof-of-principle, we chose to analyze the methylation patterns in the promoter regions of four genes (CDKN2A, GSTP1, MLH1, and CTNNB1) and the differentially methylated region 2 (DMR2) of IGF2 in a panel of 71 samples (10 control livers, 27 paired HCC samples, three paired adenomas, and two additional peritumoral livers). Aberrant methylation in hepatocellular carcinogenesis had previously been reported for CDKN2A, GSTP1, MLH1, and IGF2, but not for CTNNB1, which is mutated in a subset of HCCs.Limit of DetectionWe and others12Tost J Dunker J Gut IG Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing.Biotechniques. 2003; 35: 152-156PubMed Google Scholar,21Gruber JD Colligan PB Wolford JK Estimation of single nucleotide polymorphism allele frequency in DNA pools by using pyrosequencing.Hum Genet. 2002; 110: 395-401Crossref PubMed Scopus (100) Google Scholar have previously shown that pyrosequencing-based DNA analysis has a limit of detection of ∼3% for the minor component of a quantitative signal and a quantitative resolution of 5% or better. However, because this parameter is of utmost importance for the approach, we chose to reverify the limit of detection using the CDKN2A amplicon as a model system. Completely in vitro methylated and unmethylated DNAs were bisulfite-treated and normalized to a concentration of 20 ng/μl using a NanoDrop spectrophotometer. The use of the more accurate fluorescent dyes as used in the Quant-iT kit is no longer possible because these are highly selective for double-stranded DNA, and strands are no longer complementary after bisulfite treatment. The maximal error in the determination of the concentration after normalization because of pipetting and other random fluctuations was estimated to be 20 ± 1.3 ng/μl (6.5%). Completely methylated DNA was diluted into the unmethylated DNA to create mixtures with a methylation degree of 0, 2, 5, and 10%. Figure 1 confirms the limit of detection being at 2% and clearly demonstrates the ability of our approach to detect methylation differences as low as 2 to 5%. We therefore decided to divide the samples into pools consisting of a maximum of eight samples. This approach permits the identification of aberrant methylation if a single sample displays a methylation degree of 20 to 25% against a background of seven unmethylated samples. If more than one sample is methylated, methylation levels of ∼10% are sufficient to be detected.To assess the variation induced by bisulfite treatment, two independent bisulfite treatments were performed, and quantitative DNA methylation levels were analyzed at the CDKN2A promoter. Methylation degrees at individual CpG positions between showed a high degree of reproducibility with a mean variation of 1.9% (range, −5.9 to +6.3 for individual CpG positions). This variability is in the same range as the variation observed in independent PCR amplifications. These results are in good concordance with recently published results demonstrating a SD of 3.5% between seven independent bisulfite treatments.37Ogino S Kawasaki T Brahmandam M Cantor M Kirkner GJ Spiegelman D Makrigiorgos GM Weisenberger DJ Laird PW Loda M Fuchs CS Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis.J Mol Diagn. 2006; 8: 209-217Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar Bisulfite treatment does therefore not have a major influence on quantitative methylation values if well standardized.Construction of PoolsFor proof-of-principle, samples were divided into 12 pools. Seven of the 10 normal liver tissues were grouped together into one pool and the six available peritumoral liver samples without known etiology into the second pool. A third pool was constructed from peritumoral noncirrhotic livers from alcoholic individuals (n = 3), and the fourth pool was built from DNA extracted from peritumoral noncirrhotic liver from HBV- or HCV-infected individuals (n = 4). Peritumoral cirrhotic liver tissues were divided into three pools, one for patients with increased alcohol consumption (n = 5) and two pools for cirrhotic peritumoral liver tissues from patients with HBV or HCV infection (n = 6 each). The three hepatic adenomas were pooled together for further analysis. HCCs were divided into four
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