A simplified method to determine methylated cytosines in a target gene

Vaishnavi V., Aarthi R., Smitha S., and Jaffar Ali B.M. Life Sciences Division, AU-KBC Research Centre,

Anna University, Chrompet, Chennai 600 044. India. jaffar@au-kbc.org

Abstract—In this paper we discuss methylation sequencing of DNA target in E-Cadherin gene in head and neck cancer and colorectal cancer by bisulfite-PCR. Bisulfite converted genomic DNA were purified prior to amplification by PCR. Primers are designed to amplify both methylated and unmethylated strands of target DNA sequence. Status of methylation was ascertained by native PAGE. Intriguingly, we have observed methylated, unmethylated and partially methylated amplicons in the tissue samples. Occurrence of positive results in unmethylated primers of cancer tissues as well as positive results for both methylated and unmethylated primers in some tissue samples reveal the limitation of existing methodology in capturing real methylation status. In partially overcoming this limitation, we have designed and implemented specific fluorescence detection based sequencing protocol namely, methylation-sequencing which capture complete methylation status of target gene. In that we have combined dideoxy termination at specific methylation site with fluorescently tagged primer in the PCR reaction which allows the fluorescence detection of specifically terminated oligos. Using a target template DNA and custom-built fluorescence detection apparatus, we demonstrate utility of this approach in obtaining accurate methylation status of given gene target. By design, the methodology eliminates need to sequence entire target DNA.

Keywords-E-cadherin; Bisulphite conversion; Methylation; Sequencing; Fluorescence detection

I. INTRODUCTION DNA methylation occurs in almost all higher eukaryotic

organisms and plays a central role in the control of many genetic functions [1]. Changes in the methylation pattern play an important role in tumorigenesis. In addition to contributing to tumorigenesis, there is growing evidence that epigenetic changes are specifically associated with activation or silencing of genes. In the genome of higher eukaryotes, cytosines may be methylated in CpG islands at locations throughout the genome [2]. The majority of CpG islands are associated with ‘house-keeping genes’, some of them are located in ‘tissue-specific genes’. Availability of wide range of techniques helps to study the occurrence and localization of methyl cytosine in the genome [3-4]. Increasing evidence of methylation mediated gene regulation implicated in disease make the study of methylation status an important tool in both clinical diagnostics and therapeutics.

Among many methods available for studying methylation in genomic DNA, bisulfite-PCR-sequencing offers the highest degree of resolution of the methylation status of a given sample

[5]. It allows the determination of positional CpG genotype for individual samples. However, the existing process of methylation mapping by PCR-sequencing is highly cumbersome and has inherent limitations such as necessity to carry out complete sequencing [6]. On the other hand, determining the promoter methylation status by Methylation Specific PCR (MSP) gives limited information. For instance, it cannot report methylation status on sequence other than the primer chosen. It should also be noted that although the most significant proportion of CpG islands is located in the 5’-untranslated region and the first exon of the genes, certain CpG islands can occasionally be found within the body of the gene, or even in 3’-region. CpG islands in these atypical locations are more prone to methylation [7]. It is likely that methylation at non-promoter region can also retain compaction of genome leading to nuclease-resistant chromatin and the subsequent repression of gene activity. So it is necessary to study the methylation status in the non-promotor region also. It is noted that increasing attention is given to determine the methylation status of single genes or single cytosine residues [8].

One of the reliable approaches to study methylation of cytosine is sodium bisulfite conversion. Bisulphite treatment efficiently deaminates unmethylated cytosine to uracil without affecting 5-methyl cytosine and thereby transforming epigenetic information into sequence information. In fact, PCR amplification and sequencing of bisulfite-converted genomic DNA has emerged as the gold standard for analyzing and comparing methylation patterns at specific loci [9].

Methylation-specific PCR (MSP) allow for the detection of single copies of methylated DNA in CpG islands in the promoter region [10]. In MSP, primers bind specifically to bisulfite converted methylated or unmethylated DNA, leading to specific amplification. Advantage of this technique is its high relative sensitivity, ease of design, and low complexity of the reaction.

In bisulphite sequencing, sequencing of bisulfite converted DNA is carried out. General sequencing has its own limitations. Moreover, since the sequence information of the target DNA is known, it does not make sense to sequence the target DNA again for methylation purpose.

In this paper we study the methylation status of E-cadherin in tumor tissue samples of colorectal and head and neck cancer of 15 subjects. E-cadherin (120 kDa; chromosome 16q) is a super family of calcium-mediated membrane glycoproteins and forms the key functional component of adherence junctions

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between epithelial cells and also functions as an invasion/tumor suppressor protein [11]. In recent years, a large number of studies have revealed that E-cadherin function is frequently inactivated during the development of human carcinomas, including those of the breast, colon, prostate, stomach, liver, esophagus, skin, kidney, and lung [12]. It is matter of detail that to capture complete methylation information, one has to design methylation sensitive primers on all CpG islands, a task which can be highly cumbersome and time taking. To partially overcome this issue we describe a novel method of mapping complete methylation information by a process called methyl sequencing. Using a part of E-Cadherin gene as template DNA, we demonstrate the proof-of-concept of simplified methyl sequencing method.

II. MATERIALS AND METHODS

A. DNA extraction and sodium bisulphite conversion: Genomic DNA from formalin fixed tissue (Apollo

Specialty Hospital, Chennai) was extracted by following standard phenol/chloroform method [13]. Purified extracted genomic DNA was bisulphite treated to convert unmethylated cytosine to uracil [14]. For the preparation of 100% methylated DNA as positive control, a Placental DNA sample was treated with SssI methylase (New England Biolabs, USA) that methylates all cytosine residues of CpG dinucleotide in the genome.

B. Methylation Specific PCR: Following methylation specific primers were designed for

E-cadherin promoter. For methylation positive samples: Ecad–M Forward: 5’TTA GGT TAG AGG GTT ATC GCG T3’; Ecad-M Reverse: 5’TAA CTA AAA ATT CAC CTA CCG AC3’; and for methylation negative samples: Ecad–U Forward: 5’TAA TTT TAG GTT AGA GGG TTA TTG T3’; Ecad-U Reverse: 5’CAC AAC CAA TCA ACA ACA CA3’ [15]. PCR was carried out using the 2X Master Mix (Fermentas) with 0.2 µM concentration of each primer. PCR amplicons of 116 bp for Ecad–M and 97 bp for Ecad-U were obtained. Thermal cycler (PTC-150, MJ Research) protocol giving optimal results was: for Ecad-M 95 °C for 3 min; 10 cycles with 95 °C for 30 sec, 62 °C for 30 sec, 72 °C for 30 sec and 25 cycles with 95 °C for 30 sec, 60 °C for 30 sec, 72 °C for 30 sec; and for Ecad-U 95 °C for 3 min; 10cycles with 95 °C for 30 sec, 60 °C for 30 sec, 72 °C for 30 sec and 25 cycles with 95 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec. PCR products were electrophoresed on 12% Native PAGE and visualized using silver staining.

C. Methyl Sequencing: Modified Sanger sequencing reaction was performed using

DNA Sequencing System Kit (Promega) on following template DNA designed to contain 11 CpG spots: 5’CGA TCG TAT TCG GCG TTT GTT TTC GTT CGG CGT TTT CGG TTA GTT ATG GGT TTT TGG AGT CGT AGT TTT TCG GCG TTG TTG TTG TTG TTG TAG GTA TTT3’. Fluorescein labeled primer Flu-5’AAA TAC CTA CAA CAA CAA CAA CAA C3’ was used to sequence the template DNA. The sequencing reaction were performed at 95 °C for 2 min, 35

cycles of 95 °C for 30 sec, 42 °C for 30 sec and 72 °C for 60 sec. PCR sequencing reaction is electrophoresed using 6% denaturing PAGE in 89 mM Tris, 89 mM boric acid, 2 mM ethylenediamine tetra acetic acid (EDTA) at field strength of 10 V/cm for 180 min and analysed by fluorescence excitation scanning using home built methyl detector apparatus.

D. Confocal Fluorescence Excitation and Detection: Custom made confocal laser epifluorescence excitation and

detection apparatus used in this measurement is built around Nikon TE2000 microscope. The details of the apparatus are given elsewhere [16]. In brief, it consists of a 473 nm laser for excitation, motorized XY stage having 100 mm span for scanning the DNA gel, and sensitive PMT detector. Electrophoresised DNA band in acrylamide gel are scanned across confocal laser excitation. Fluorescence emission from DNA band is recorded as a function of band separation. The apparatus can detect ~100 femtomole of fluorescein at 100 msec integration.

III. RESULTS

A. MSP Analysis of E-Cadherin Gene: MSP was used to detect the promoter methylation of

E-cadherin in colorectal cancer and Head and Neck cancer [17-18]. Nine colorectal cancer tissues and six head and neck cancer tissue samples were analyzed. Methylation was observed in 7 samples of colorectal cancer in a sample volume of 9. Two samples tested positive with both methylated and unmethylated primers. These samples are designated as partially methylated. Methylation was observed in 3 samples of head and neck cancer in a sample volume of 6 (Fig. 1).

a)

b)

c)

Figure 1. MSP Analysis of E-cadherin in colorectal and Head and Neck Cancer. a) Methylation positive sample exhibiting 116 bases amplicons, b)

Methylation negative samples exhibiting 97 base amplicons, c) Hemi-methylation samples giving 116 and 97 bases amplicons. Control dsDNA

ladder of 50base pairs used.

B. Design of generic Methyation Detection: Generic methylation map which is independent of

methylation in primer region can be obtained in following two steps. A. Amplify the bisulphite converted DNA without depending on status of methylation. For this, a region on the DNA target without CpG motif but close to the target gene upstream and downstream sequences are located. Both forward and reverse primers are designed in this region. B. Sequencing

of target region enclosed by the methyl independent primers was performed using restricted Sanger Method. In that, only dideoxy guanine is introduced in place of all dideoxy bases. It therefore results in random termination only at guanine positions. These positions correspond to methylated cytosine of parent strand. PCR product can be read using standard sequencing gel. In this work, we have used custom built sequencer based on fluorescence detection.

(a)

(b)

Figure 2. Methyl Sequencing of target DNA using Modified Sanger sequencing with fluorescently tagged primer. a) Represents detection of

known oligo bands of indicated length, as control/ calibration of the method. b) detection of PCR amplicons of Number of methyl sequencing bands

corresponds to number of methylated cytosines in the target DNA

TABLE I. PROMOTER METHYLATION STATUS OF E-CADHERIN

CANCER TISSUE TYPE

No. of Samples Analysed

Methylated Unmethylated Positive For Both

Colorectal

9

5

2

2

Head & Neck

6

3

3

0

C. Methyl Sequencing: An apparatus to scan DNA band through fluorescence

measurement was constructed. To calibrate the system prior to use, known length of oligos with fluorescine tag at 5’ end is run in the gel and visualized. Fig. 2a demonstrates detection of bands of different length in sequencing gel. It further demonstrates the ability of the apparatus to resolve less than 100 femtomole of DNA (fluorophore). All the eleven peaks in fig. 2b correspond to the methylation sites obtained by sequencing the template DNA and detection using custom built fluorescent apparatus.

IV. DISCUSSION We have analysed the methylation status of colorectal and

head & neck cancer tissue sample DNA. Conventional methyl specific detection reveal E-cadherin gene is methylated to 78% in colorectal cancer and 50% in Head & Neck cancer (Table I). Interestingly, partially methylated amplicons are seen in the products of colorectal cancer. Since only in the advanced stage of cancer all cells of the tissue will be affected, it is possible that tissue samples may comprise of both normal and cancerous cells. This may explain occurrence of positive result with both methylated and unmethylated primers. Since the epigenetic is a temporal process, it is most likely that extent of methylation of target gene is progressive in nature than complete [19]. This may also partially explain the observation of conflicting results (Table I). In other words, the premise that all CpG sites in the CpG Island of a gene are methylated need not be true. This can be known only when methyl sequencing of the gene is accomplished.

There is increasing interest in exploring methylation information on gene target as markers of disease. It is noted that bioinformatics tools have revealed presence of CpG islands spread over entire gene, representing probable hotspots for methylation [20]. However, current experimental methodology of methylation mapping is either cumbersome and expensive requiring capital equipment for detail study or simple but very narrow in giving methylation status of primer length alone. The work presented here addresses this problem in a novel way. Firstly, a protocol is developed to amplify the target gene using methylation independent primers. Secondly, exploiting the presence of sequence information of target gene, a modified Sanger sequencing procedure is adapted to extract information on methylated cytosine positions. To account for the low-yield in the sequencing PCR product and to accurately resolve the DNA band, PCR primers are fluorescently tagged. Calibration of our methodology using known concentration of fluorescently tagged ssDNA (Fig. 2a) stand reveal that DNA band of concentration less than hundred femtomoles can be resolved. The method reported here is generic one which can be extended to map genomic DNA. For instance, to gain more insights into the disease, methylation status of different exon of a target gene can be mapped. In this way, it can be useful in the development of DNA markers. It is noted that in the era of personalized medicines, DNA methylation markers have profound applications in diagnostics and therapeutics.

In brief, we have provided proof-of-concept for development of a novel methylation mapping protocol which

can have potential high-throughput and high-fidelity application. The use of custom-made DNA sequencing apparatus tuned to detect methylation bands provides a way to rapid and low-cost alternative to DNA methylation sequencing.

ACKNOWLEDGMENT We thank Dr.Pravin Raj Solamon for his help in bisulphite

modification and Dr.Mitra Ghosh, Apollo Specialty Hospital, Chennai, for providing cancer tissue samples. This work was supported by Department of Science and Technology, Government of India Grant (Grant No. DST/TSG/PT/2006/63).

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