Keith N. Rand

Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives

Methylated cytosines appear as sequence variations following bisulfite treatment and polymerase chain reaction (PCR) amplification. By using methylation-specific PCR (MSP), it is possible to detect methylated sequences in a background of unmethylated DNA with a high level of sensitivity. MSP is frequently used to identify methylated alleles in carcinogenesis, and may be combined with the TaqMan real-time PCR system, which uses fluorescence-based detection of amplification products during the amplification phase of the PCR and increases the sensitivity of detection (MethyLight). Sequences that have been incompletely converted during the bisulfite treatment are frequently coamplified during MSP, resulting in an overestimation of DNA methylation. The presence of amplified sequences originating from partially unconverted material may be determined by sequencing or by restriction digests or Southern blots of MSPs. Alternately, we have developed a method where the PCR and conversion assay are combined within a single TaqMan reaction by using an additional fluorescent probe directed against unconverted DNA (ConLight-MSP). We recommend that MSP detection always should include a step to detect unconverted DNA to avoid overestimation of the frequency or level of methylated DNA in the sample.

A panel of genes methylated with high frequency in colorectal cancer

BackgroundThe development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.MethodsCombined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).ResultsCombined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.ConclusionsThis study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Hypomethylation of repeated DNA sequences in cancer

An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5′ extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5′ extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 105-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences.

Relative Distribution of Folate Species is Associated with Global DNA Methylation in Human Colorectal Mucosa

Folate exists as functionally diverse species within cells. Although folate deficiency may contribute to DNA hypomethylation in colorectal cancer, findings on the association between total folate concentration and global DNA methylation have been inconsistent. This study determined global, LINE-1, and Alu DNA methylation in blood and colon of healthy and colorectal cancer patients and their relationship to folate distribution. Blood and normal mucosa from 112 colorectal cancer patients and 114 healthy people were analyzed for global DNA methylation and folate species distribution using liquid chromatography tandem mass spectrometry. Repeat element methylation was determined using end-specific PCR. Colorectal mucosa had lower global and repeat element DNA methylation compared with peripheral blood (P < 0.0001). After adjusting for age, sex and smoking history, global but not repeat element methylation was marginally higher in normal mucosa from colorectal cancer patients compared with healthy individuals. Colorectal mucosa from colorectal cancer subjects had lower 5-methyltetrahydrofolate and higher tetrahydrofolate and formyltetrahydrofolate levels than blood from the same individual. Blood folate levels should not be used as a surrogate for the levels in colorectal mucosa because there are marked differences in folate species distribution between the two tissues. Similarly, repeat element methylation is not a good surrogate measure of global DNA methylation in both blood and colonic mucosa. There was no evidence that mucosal global DNA methylation or folate distribution was related to the presence of cancer per se, suggesting that if abnormalities exist, they are confined to individual cells rather than the entire colon. Cancer Prev Res; 5(7); 921–9. ©2012 AACR.

Abstract 3125: Relative distribution of folate species is associated with global DNA methylation in human colorectal mucosa

Folate, an important cellular methyl donor, exists as functionally diverse species within cells. Folate deficiency is thought to contribute to DNA hypomethylation in colorectal cancer (CRC) however findings on the relationship between total folate concentration and global DNA methylation have been inconsistent. The aim of this study was to determine if global DNA methylation in blood and colorectal mucosa from healthy and CRC patients is related to folate species distribution. Blood and colorectal mucosa was collected from 112 CRC patients, 114 healthy individuals and 82 low folate individuals. Global methylation and folate was determined using liquid chromatography tandem mass spectrometry and repeat element methylation was determined using end-specific polymerase chain reaction. In colorectal mucosa mean global methylation was 8% lower compared to blood while LINE-1 and Alu elements were 3.3-fold and 1.9-fold hypomethylated respectively (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3125. doi:1538-7445.AM2012-3125

Evolutionary evidence suggests that CpG island-associated Alus are frequently unmethylated in human germline

Most Alu elements are considered to belong to the methylated fraction of the genome that has undergone CpG depletion, whereas CpG islands are characteristically unmethylated. By analysing the CpG content of >12,000 autosomal CpG island-Alu Sx pairs we wanted to study what happens when an Alu is situated close to a CpG island. We have found that many Alus located close to CpG islands have retained a high proportion of CpG sites, which is consistent with these Alus being unmethylated in the human germline.

Bisulphite differential denaturation PCR for analysis of DNA methylation.

Differential denaturation during PCR can be used to selectively amplify unmethylated DNA from a methylated DNA background. The use of differential denaturation in PCR is particularly suited to amplification of undermethylated sequences following treatment with bisulphite, since bisulphite selectively converts cytosines to uracil while methylated cytosines remain unreactive. Thus amplicons derived from unmethylated DNA retain less cytosines and their lower G + C content allows for their amplification at the lower melting temperatures, while limiting amplification of the corresponding methylated amplicons (Bisulphite Differential Denaturation PCR, BDD-PCR). Selective amplification of unmethylated DNA of four human genomic regions from three genes, GSTP1, BRCA1 and MAGE-A1, is demonstrated with selectivity observed at a ratio of down to one unmethylated molecule in 105 methylated molecules. BDD-PCR has the potential to be used to selectively amplify and detect aberrantly demethylated genes, such as oncogenes, in cancers. Additionally BDD-PCR can be effectively utilised in improving the specificity of methylation specific PCR (MSP) by limiting amplification of DNA that is not fully converted, thus preventing misinterpretation of the methylation versus non-conversion.

Chromatography and generation of specific antisera to synthetic peptides from a protective Boophilus microplus antigen

Four oligopeptides corresponding to predicted antigenic regions of the protective Bm86 glycoprotein of the cattle tick Boophilus microplus were synthesized and purified. Three were conjugated to carrier proteins and antisera raised in rabbits and cows. All elicited antipeptide antibodies that recognized Bm86 and recombinant derived products in Western blots; however, only one produced antiserum capable of recognizing native Bm86 in an indirect immunofluorescence assay. Ticks fed in vitro on this antiserum showed no obvious gut damage.

Abstract 91: Analysis of global and repeat sequence DNA methylation in the blood and normal colonic mucosa of colorectal cancer patients, healthy individuals and patients with folate deficiency

Aberrant DNA methylation is an early event in colorectal tumorigenesis and in colorectal cancer (CRC) patients these changes are detected in their normal colonic mucosa. DNA methylation changes may arise from aging or environmental exposures e.g. folate deficiency. We aimed to compare global and repeat-sequence DNA methylation in the colonic mucosa and blood of three groups of individuals – CRC patients, healthy people and patients with folate deficiency. We conducted a multisite cohort study collecting mucosa and blood from 176 patients who had undergone CRC resection and 194 healthy individuals at screening colonoscopy. Blood was also collected from 30 individuals with megaloblastic anaemia secondary to folate deficiency (red cell folate 1 . Alu repeat methylation was determined using a novel, ultra-sensitive real-time PCR assay based upon methylation-sensitive restriction enzyme digestion coupled with a fluorescence-based readout 2 . Genotyping for a common polymorphism in the folate metabolism enzyme MTHFR (C677T) was performed using pyrosequencing. The mean age of CRC patients (68 ± 14 years) was significantly higher than healthy subjects (55 ± 14 years; p To date, our results suggest that Alu repeat methylation reflect global DNA methylation and that this holds true across tissue types irrespective of cancer status. Global and Alu repeat demethylation in individuals with folate deficiency and overrepresentation of the T MTHFR allele suggests a possible link between diet and the epigenome that warrants further study. 1. Quinlivan, E.P. & Gregory, J.F., 3rd. (2008) Nucleic acids research 36, e119. 2. Rand, K. & Molloy, P. (2010) BioTechniques 49, xiii-xvii. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 91. doi:10.1158/1538-7445.AM2011-91