Epigenetic Biomarkers and Diagnosticshttp://dx.doi.org/10.1016/B978-0-12-801899-6.00008-5 Copyright © 2016 Elsevier Inc. All rights reserved.155

C H A P T E R

8The Role of Methylation-Specific PCR and Associated Techniques

in Clinical DiagnosticsFang Zhao1,2, Bharati Bapat1,2,3

1Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada;

3Department of Pathology, University Health Network, Toronto, ON, Canada

O U T L I N E

1. Overview of DNA Methylation as an Epigenetic Marker 156

2. DNA Methylation in Diagnostics 156

3. Restriction Enzyme Digestion for Analyzing DNA Methylation 158

4. Methylation-Specific PCR and Associated Techniques 158

5. Sodium Bisulfite Conversion 158

6. Methylation-Specific PCR 158

7. Methylation-Sensitive Dot Blot Assay 162

8. MethyLight 162

9. Technical Specifications for MethyLight 163

10. Applications of MethyLight Technique 163

11. Multiplex MethyLight 164

12. Methylation-Sensitive Multiplex Ligation-Dependent Probe Amplification 167

13. Pyrosequencing 167

14. High-Resolution Melting Analysis 168

15. Emerging Applications 168 15.1 Digital MethyLight 168 15.2 A Novel Methylation Marker:

5-Hydroxymethylation 169

16. Future of DNA Methylation in Clinical Applications 169

List of Abbreviations 170

Acknowledgment and Funding Support 170

References 170

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS156

1. OVERVIEW OF DNA METHYLATION AS AN EPIGENETIC MARKER

Epigenetics was originally defined as “The causal interactions between genes and their products, which bring the phenotype into being” by Conrad Waddington in 1942 [1]. The definition of epigenetics has changed over time as it has been found to be implicated in a wide variety of biological processes. The cur-rent definition for epigenetics could be summa-rized as “the study of heritable changes in gene expression that occur independent of changes in the primary DNA sequence” [2]. Epigenetics includes a variety of different phenomena such as histone modifications, RNA interfer-ence through microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), as well as DNA methylation [2].

Among these, histone modifications are quite diverse in range including acetylation, meth-ylation, and ubiquitination. These modifica-tions play key roles in a variety of functional pathways including transcriptional activation/repression, DNA repair, genomic structural change between euchromatin and heterochro-matin, and apoptosis [3,4].

MicroRNAs primarily serve to regulate gene translation through the Drosha/Dicer/RISC pathway [5]. lncRNAs may inhibit gene tran-scription through blocking access points for transcription factors or facilitate transcription through recruiting transcription factors [6].

On the other hand, DNA methylation is heri-table, causes changes to gene expression, and is involved in cell differentiation. DNA meth-ylation is a post-replication modification. It is predominantly but not exclusively found in the fifth carbon of the pyrimidine ring of cyto-sine nucleotides occurring usually in the palin-dromic sequence of 5′-CpG-3′ (CpG). CpG-rich regions, known as CpG islands, are located in the promoter regions of around 60% of genes. CpG islands are usually unmethylated in

normal cells except for certain genes involved in the inactivation of the paternal X-chromosome [7], testis-specific genes in somatic tissues [8], genomic imprinting [9], and cell-type specific expression [10].

DNA methylation is involved in the control of gene expression. Unmethylated promoter CpG islands are usually associated with euchro-matin structure and activate gene transcription. Conversely, promoter region CpG island meth-ylation is associated with heterochromatin structure and gene silencing. Contrary to pro-moter methylation, gene body methylation is commonly associated with active gene expres-sion. Although, the functional roles of gene body methylation are still poorly understood, a recent study described a global remethylation event following treatment with the demethyl-ating agent 5-aza-2′-deoxycytidine (5-aza-CdR) that resulted in increased CpG methylation of the gene body. It was found that CpG methyla-tion in the gene body directly increases gene expression [11]. These findings demonstrate the potential impact of demethylating drugs in a clinical setting. This will be discussed in a later section.

Aberrant methylation of CpG islands has been found to be associated with many disor-ders including cancer. These well-characterized epigenetic features form the foundation of MSP (methylation-specific polymerase chain reaction (PCR)) and its associated techniques.

2. DNA METHYLATION IN DIAGNOSTICS

In many cancers, a phenomenon of global hypomethylation combined with hypermethyl-ation of the promoter regions of tumor suppres-sor genes is often observed [12]. Usually several genes are silenced through promoter hypermeth-ylation in proliferating tumors cells. Tumor-specific DNA methylation changes are used in cancer diagnostics for both disease detection

2. DNA METHyLATION IN DIAgNOSTICS 157

and classification through various DNA meth-ylation analysis techniques. DNA hypermethyl-ation can be measured in tissue samples such as surgically resected fresh or archival tumors and biopsies, or from body fluids collected noninva-sively (or less invasively) such as saliva, urine, and serum.

Methylation biomarkers have already been integrated into clinical applications for the diagnosis of certain cancers. One such exam-ple is the detection of Septin 9 (SEPT9) meth-ylation as a diagnostic marker in colorectal cancer (CRC, MIM #114500). SEPT9 methyla-tion in serum samples is detected with high sensitivity (90%) and specificity (88%) for all stages of CRC [13]. Compared with the cur-rent standard screening modalities for CRC detection including fetal-occult blood test (FOBT) and internal imaging through colo-noscopy, circulating DNA methylation-based test is an approach both more sensitive than FOBT and far less invasive than colonoscopy for CRC diagnosis [14]. In addition to SEPT9, hypermethylation of several gene clusters, termed as the CpG island methylator pheno-type (CIMP), is able to differentiate subgroups of CRC characterized by pathological, clini-cal, and molecular features. CIMP positive CRC tumors show distinct association with survival and chemotherapeutic response [14]. Hypermethylation of CDKN2A, MLH1, and TMEFF2 genes are representative examples of CRC CIMPs. However, a consensus panel of markers has not yet been applied in clinical setting [15,16].

In prostate cancer (PCa, MIM #176807), a major limitation for accurate diagnosis is that up to 25% of all prostate biopsies are false neg-atives, due to the fact that less than 1% of the prostate gland is biopsied and occult cancer may be missed simply due to random chance. It has been observed that many cancers, including prostate tumors, display a “field effect” (some-times also called “field cancerization effect”) such that tumor-adjacent tissue, although

normal in appearance, shows a methylation profile similar to that of the tumor. Thus, despite histologically normal biopsy cores, if an occult tumor were present, the “adjacent normal” core will show aberrant DNA meth-ylation [17]. Based on this field effect, a three-gene panel consisting of adenomatous polyposis coli (APC), glutathione S-transferase P (GSTP1), and Ras- association domain family 1 isoform A (RASSF1A) methylation has been approved as a diagnostic biomarker panel to discriminate false-negative biopsies in PCa. Such a methyla-tion detection-based test may reduce the rate of false-negative biopsies and improve the overall efficiency of diagnosis.

DNA methylation of GSTP1 and APC is associated with aggressive breast cancer (MIM #114480) development and can serve as a screen-ing test to identify high-risk breast cancers. These markers can be detected in circulating tumor cells isolated from blood samples which may offer a noninvasive prognostic marker for breast cancer in the near future [18].

For non-cancer diseases, some genes are epigenetically silenced through DNA meth-ylation in a “parent-of-origin” manner, known as genomic imprinting. Mammalian imprinted genes remain methylated/silenced through-out development [19]. For example, insulin-like growth factor 2 (IGF-2) is only expressed from the paternal allele and the H19 gene is only expressed from the maternal allele. Improper imprinting could result in two active copies or two inactive copies of the gene, and consequently lead to severe devel-opmental disabilities, cancer, or other disor-ders [19]. Prader–Willi (PWS; MIM #176270, ORPHA739) and Angelman syndromes (AS; MIM #105830, ORPHA72) are examples of such disorders caused by improper imprinting on chromosome 15.

In this chapter, DNA methylation detection using MSP and its associated techniques will be discussed in relation to diagnosis and prognosis of cancers and other diseases.

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS158

3. RESTRICTION ENZYME DIGESTION FOR ANALYZING

DNA METHYLATION

Restriction enzymes that are sensitive to methylation status of cytosines at the enzyme recognition sequence and are unable to digest methylated DNA can be used to detect distinct DNA methylation patterns. Enzyme pairs such as HpaII–MspI can be used to discriminate meth-ylation status of CpG-containing sequences. For example, both HpaII and MspI target the same (CCGG) sequence during digestion. However, HpaII is methylation sensitive and digestion is inhibited if the cytosine nucleotides are methyl-ated. While MspI, an isoschizomer of HpaII, can cleave the CCGG site regardless of methylation status. This difference in methylation sensitivity for the pair of enzymes allows for methylated and unmethylated CpGs to be distinguished in downstream applications such as methylation-sensitive multiplex ligation-dependent probe analysis (MS-MLPA), as described later.

4. METHYLATION-SPECIFIC PCR AND ASSOCIATED TECHNIQUES

MSP relies on PCR to amplify specifically methylated or unmethylated alleles. Since con-ventional PCR is unable to differentiate between methylated and unmethylated cytosines, prior to MSP, template DNA must first undergo sodium bisulfite treatment in a process known as bisulfite conversion or bisulfite modification to differentiate methylated cytosines [20].

5. SODIUM BISULFITE CONVERSION

Bisulfite conversion deaminates unmethyl-ated cytosines to uracil. Methylated cytosines are resistant to the deamination process and are thus protected during bisulfite conversion. This unique characteristic of bisulfite-converted

DNA, which was first discovered in 1970 by Shapiro et al. [21], provides the basis to distin-guish between methylated and unmethylated CpG sites. Bisulfite conversion allows for down-stream applications of a variety of techniques to determine the methylation status at selected/specific CpG sites and/or general methylation pattern of a particular region of the genome.

However, bisulfite conversion is a harsh pro-cess and known to cause fragmentation of DNA which may be a limitation. Furthermore, since bisulfite conversion is assumed to convert all unmethylated cytosines to uracil, it is necessary to have a methylation-independent control post-bisulfite conversion, to validate the success of the process [22]. One such example is ALU-C4 (ALU) repeats, which do not harbor any CpGs, and therefore do not incur any methylation, and are ideal to be used as internal control [22]. Following bisulfite conversion, DNA samples are analyzed using locus-specific or genome-wide techniques.

6. METHYLATION-SPECIFIC PCR

In MSP, bisulfite-converted DNA is amplified using PCR with primers specifically designed to target either methylated or unmethylated CpGs. Following MSP, amplicons/bands are visualized on agarose, boric acid, or nondenaturing poly-acrylamide gels that are specific to either methyl-ated or unmethylated alleles. These bands are then assessed through densitometry analysis. Due to the nature of this technique, standard MSP is unable to quantify the number of methylated alleles if both methylated and unmethylated alleles are present in the DNA sample. Depending on the primer location, both methylated and unmethylated DNA-specific primers would produce bands of dif-ferent sizes and can be easily distinguished by gel electrophoresis. DNA extracted from low- quality and/or limited quantity tissues such as from for-malin-fixed paraffin-embedded (FFPE) samples following bisulfite conversion tends to be damaged and/or fragmented, which may limit MSP ampli-fication restricted to short amplicons (Table 1).

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TABLE 1 The Advantages, Limitations, and Clinical Applications of Methylation-Specific PCR and Associated Assays

Assay Advantages Limitations Clinical applications References

Methylation-specific PCR (standard MSP)

Methylation specific Not quantitative Analysis of X chromosomal inactivation and disorders linked to X-chromosomal inactivation such as fragile X syndrome, PWS, and AS

[20,24,26–30]

Detection down to 0.1% of methylated alleles

Cannot determine methylation pattern if methylated and unmethylated alleles are present

Bisulfite conversion dependent

Cannot detect damaged/fragmented DNA

Nested MSP Rescues damaged/fragmented DNA for MSP analysis

Same as MSP other than damaged/fragmented DNA

[20]

Methylation-specific dot blot assay

Methylation specific Takes longer than more advanced techniques

Detection of methylation of genes in prostate cancer (PCa)

[31,32]

More sensitive than standard MSP

Semi-quantitative

Does not require high-tech equipment

Methylation-specific qPCR (MethyLight (singleplex))

Methylation specific Not apt for screening of large number of CpG sites

Assessment of methylation of specific CpG sites. May assess up to 8–12 CpG sites in the same assay. Promoter methylation of genes associated with PCa, bladder cancer, breast cancer

[33,37–43]

10 times more sensitive than standard MSP

Primer/probe must contain 8–12 CpG sites

Quantitative Bisulfite conversion dependent

Multiplex MethyLight Methylation specific Gene combinations and target CpG sites are limited by primer probe combination

Detection of methylation of genes in CRC, PCa, and cervical cancer

[36,44,45]

Can detect methylation in as little as 370 pg of input DNA

Bisulfite conversion dependent

Quantitative

High throughput compared with singleplex MethyLight

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Assay Advantages Limitations Clinical applications References

Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA)

Detect changes in copy number of a gene

Can only detect copy number variations in regions targeted by probes

Diagnosis of PWS and AS, Lynch syndrome; tumor diagnosis and prognosis when used in combination of other methods

[46–50]

DNA methylation and point mutations

Mutations or polymorphism in the target sequence may be detected as copy number variation leading to false negatives

Up to 50 different loci could be analyzed in the same MLPA reaction

Relies on enzyme digestion: restricted to CGCG sites

Bisulfite conversion independent

Cannot be used for unknown SNPs

Can be used on fragmented DNA 150 bp long

Detection in as small as 20 ng of input DNA

Pyrosequencing Fast compared to other techniques

Bisulfite conversion dependent for methylation detection

Diagnosis of PCa, CRC, lung cancer prognosis; cervical cancer prognosis; bladder cancer recurrence screening; soft tissue sarcoma prognosis; bladder cancer

[51–58]

Can detect SNPs, insertions/deletions, copy number variation, DNA methylation

Only short stretches of DNA can be analyzed

Quantitative Not suitable for discovery of new markers

High throughput

Methylation-specific high-resolution melting analysis

Quantitative Limited by available dyes Diagnosis of PWS and AS, nasopharyngeal carcinoma

[59–63]

Can quantitate methylation status of all CpGs in a sample

Not locus specific

Digital MethyLight Quantitative Requires time-consuming optimization

CRC detection [64–66]

Up to three times more sensitive than MethyLight

Relatively more expensive

Able to measure methylation from a single molecule of DNA template

Low noise

AS, Angelman syndrome; CRC, colorectal cancer; PCa, prostate cancer; PCR, polymerase chain reaction; PWS, Prader-Willi syndrome; qPCR, quantitative PCR.

TABLE 1 The Advantages, Limitations, and Clinical Applications of Methylation-Specific PCR and Associated Assays—cont’d

6. METHyLATION-SPECIfIC PCR 161

Nested MSP (MN-MSP) [20] was devel-oped to overcome limitations of standard MSP described above. MN-MSP involves an initial round of amplification after bisulfite conver-sion, using primers that are independent of the methylation status for the gene of interest (GOI) followed by amplification using methylation-specific primers. The advantage of this two-step process is that the first round of amplification may rescue fragmented input DNA. Although with modern and improved bisulfite conver-sion kits, this process may not be necessary. Cur-rent bisulfite conversion kits are able to convert >99% of all unmethylated cytosines and result in a DNA recovery of >80% [23].

Applications of standard MSP technique include assessment of X-chromosomal inactiva-tion, diagnosis of fragile X syndrome (FXS), and diagnosis of PWS and AS.

X-chromosomal inactivation acts through DNA methylation and heterochromatin-depen-dent gene silencing of one of the two X-chro-mosomes randomly in females. Analysis of X-chromosomal inactivation patterns is widely used as a diagnostic tool for assessment of X-linked disorders in clinical settings. Tradition-ally, X-chromosomal inactivation was evaluated by methylation-sensitive restriction enzyme-based digestion analysis to assess differential methylation of genes between the active and the inactive X-chromosomes. MSP can also be used to determine the methylation levels of X- inactivation which could be used for DNA samples that are unsuitable for restriction enzyme digestion analysis [24].

FXS (MIM #300624, ORPHA908), also known as Martin–Bell syndrome, is a genetic disorder that is the most common cause of autism by a single gene [25]. It is associated with a lack of expression of the fragile X mental retardation protein (FMRP). Initially, expansion of trinu-cleotide CGG repeats in the FMR-1 gene, which codes for FMRP, was thought to cause FXS. Sub-sequently, abnormal DNA methylation of a CpG island upstream of the CGG repeats was also

found to be involved in the disease pathogen-esis. Patients with these two alterations show silencing of FMR-1 and FXS [26]. Historically, screening of FXS was performed by Southern blot analysis of DNA digested with methylation-sensitive restriction enzymes. However, South-ern blotting is a slow, labor-intensive procedure to detect mutations of FMR-1. MSP is now rou-tinely used as a diagnostic tool for FXS for the detection of both CGG repeat expansion and FMR-1 methylation.

The MSP test for FXS takes advantage of the length of CGG repeats and methylation status of promoter and also CGG repeats, between nor-mal (unmethylated promoter and unmethylated CGG repeats, CGG repeats less than 52), premu-tation (unmethylated promoter and unmethyl-ated CGG repeats, CGG repeats 52–200), and full mutation of FMR-1 alleles (de novo methylated promoter and de novo methylated CGG repeats, CGG repeats greater than 200) [27]. Separate sets of primers are designed to be specific for either methylated or unmethylated promoter sequences as well as methylated and unmethylated CGG repeat sequences. The PCR products are visual-ized on agarose gel postamplification. Compar-ing the methylation status of the promoter and the size of the CGG repeat amplicon, this semi-quantitative assay can determine the 11 distinct patterns encountered in nonaffected, carrier, and affected individuals [26]. The most current diag-nosis strategy for fragile X includes methylation-specific fluorescent PCR which incorporates fluorescent primers complementary to bisulfite-converted methylated or unmethylated DNA. The PCR products are analyzed by capillary electrophoresis. The mutation status of FMR1 is determined by fluorescent peak sizes and pat-terns on the GeneScan electropherogram and can accurately predict the CGG repeat lengths of all normal and premutation samples [28].

MSP-based diagnosis test of FXS is quicker and more cost-effective compared to Southern blot-based tests and may be the only method used in the near future [29].

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS162

PWS and AS are neurological disorders with distinct symptoms but share a common cause rooted in genomic imprinting defects of chromosome 15q11-q13 through either the loss of maternally inherited genes with pater-nal imprinting in AS or the loss of paternally inherited genes with maternal imprinting in PWS. Traditionally both PWS and AS are diag-nosed using fluorescence in situ hybridization (FISH) DNA polymorphism analysis for the detection of chromosome 15q11-q13 deletion and DNA analysis with microsatellite markers for the detection of uniparental disomy (UPD). However, there are limitations to both methods as FISH cannot discover UPD, and microsatel-lite analysis requires parental blood samples. Furthermore, neither method will be able to detect rare imprinting mutations. MSP analysis of DNA methylation of the small nuclear ribo-nucleoprotein-associated polypeptide N gene (SNRPN) or at locus PW71 can detect PWS that is concordant with FISH and DNA microsatellite analysis, indicating specificity for chromosomal deletion in 15q11-q13 and UPD as well as PWS caused by imprinting mutations. Therefore MSP analysis of SNRPN is an efficient screening test for PWS [30].

Due to the limitations of standard MSP, diagnosis and prognosis of cancers are usually assessed through more advanced techniques such as MethyLight, pyrosequencing, and methylation-specific high-resolution melting analysis.

7. METHYLATION-SENSITIVE DOT BLOT ASSAY

Methylation-sensitive dot blot assays (MS-DBAs) were developed in 2005 in order to over-come limitations of standard MSP. MS-DBA is a semiquantitative technique and decreases the false-positive rate of MSP.

In MS-DBA, PCR primers are designed to flank CpG sites of interest but do not contain

CpGs themselves and consequently are meth-ylation independent. After PCR amplification, the PCR product is dotted and cross-linked to a nylon membrane. Internal probes labeled with digoxigenin-11-dUTP (DIG) are designed to tar-get either methylated alleles (complementary to CpGs) or unmethylated alleles (complemen-tary to UpGs). Probes are allowed to bind to the target templates and any excess/unbound amounts are washed away. Anti-DIG antibody is applied followed by chemiluminescent sub-strate application. Data can be analyzed visually or by densitometry, yielding a semiquantitative result [31]. MS-DBA can be completed without the need for high-tech equipment and is practi-cal for use in developing countries with limited resources.

A recent publication has shown that MS-DBA performed following MSP assay can accurately detect methylation of GSTP1 and RASSF1A in paraffin-embedded radical prostatectomy samples that is more sensitive than standard MSP [32].

Despite the advantages of MS-DBA over stan-dard MSP, it requires optimization and can be time-consuming compared to more advanced techniques.

8. METHYLIGHT

One such technique is MethyLight or quan-titative real-time MSP. MethyLight was first developed in 1999 [33]. MethyLight is based on standard MSP but is able to provide quan-titative analysis using fluorescence-based quantitative PCR (qPCR) technology due to methylation-specific primers and TaqMan™ fluorescent probes used in the amplification of bisulfite-converted DNA. Since the distinguish-ing method is through qPCR amplification, high sensitivity, at up to a single-nucleotide resolution, may be achieved while maintaining high specificity. MethyLight is at least 10-fold more sensitive than standard MSP [33] and is

16310. APPLICATIONS Of METHyLIgHT TECHNIQUE

capable of detecting very low frequencies of hypermethylated alleles. In MethyLight, SssI (a CpG-specific methylase) treated DNA also known as universal methylated DNA, in which every CpG in the entire genome is methylated, is used as a positive control to produce the per-centage of methylated reference (PMR). PMRs are used to assess methylation levels of a spe-cific gene when comparing against universal methylated DNA. Singleplex MethyLight is capable of detecting 1 methylated allele among 10,000 unmethylated alleles or a detection fre-quency of 0.01%. Due to its high sensitivity and specificity, MethyLight is ideal for the detection of DNA methylation biomarkers for disease diagnosis and prognosis.

Similar to standard MSP, primers are designed to specifically anneal with either meth-ylated or unmethylated CpGs. With addition of the fluorescent probe, another layer of specific-ity is added which allows improved detection of methylated alleles. The ratio of methylated alleles among total number of alleles is then assessed.

9. TECHNICAL SPECIFICATIONS FOR METHYLIGHT

The target sequences for primers and probes for MethyLight assay should be in a CpG island. The primers should each contain two CpG dinucleotides minimum. The 3′ end should end in a G or CG to promote clamping. The melting temperature (Tm) of the primers should be between 60° and 65° and ±2° of each other. The primers should be ≥20 base pairs (bp) to ensure specificity. The probe should contain at least 3 CpG dinucleotides that are biased toward the center with no CpGs within 3 bp of either end of the probe to allow for cleavage of the quencher. The Tm of the probe should be 10° higher than the primers. The probe should be ≤30 bp. The probe and primers should be able to cover 8–12 CpGs.

The amplicon length should be ≤130 bp to pre-vent secondary structures. There are many online tools that are able to aid in the design of primers and probes for MethyLight. We have successfully used Primer3 [34], OligoCalc [35], and online DNA tools from companies such as IDT DNA and Life Technologies in our Methy-Light studies.

In order to quantify methylated alleles, a reference gene is used to calculate the PMR. As mentioned previously, ALU is an ideal reference gene due to its genome-wide dis-tribution and methylation-independent char-acteristics. Universal methylated DNA is used both as positive control and for gener-ating the standard curve for each assay. For singleplex MethyLight, the standard curve is prepared using 5 ng of universal methylated DNA which is serially diluted 1:25 for ALU (4 points) and 1:5 for GOI (5 points). For mul-tiplex MethyLight, described in the next sec-tion, the dilution ratio for the standard curve depends on the detection level of your GOI and can range from 1:3 to 1:5 serial dilutions starting with 5 ng of universal methylated DNA. It is important to determine the lowest detection level of all GOI included in the mul-tiplex panel. Positive control consists of 0.5 ng of universal methylated DNA [36]. PMR is cal-culated according to the formula of Eads et al. [33] as follows: [(GOI/ALU)sample/(GOI/ALU)universal methylated DNA] × 100.

Standard curves and samples should be run in duplicate, positive control in triplicate. Sam-ples with high deviation between duplicates should be rerun in independent assays.

10. APPLICATIONS OF METHYLIGHT TECHNIQUE

MethyLight has been used to assess the diag-nostic, prognostic, and predictive potential of biomarkers for various cancers. For example, analysis of methylation status of repetitive

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS164

elements such as long interspersed element (LINE-1) and Sat2 by MethyLight showed that LINE-1 hypermethylation in circulating white blood cells was significantly associated with early diagnosis of pancreatic cancer (MIM #260350) [37].

Our group has extensive experience with MethyLight technology. Using DNA extracted from archival FFPE radical prostatectomy tis-sues samples of two large PCa patient cohorts consisting of several hundred patients, we have discovered and validated promising tumor-specific methylation biomarkers (APC, TGFβ2, HOXD3, KLK10, and RASSF1A) associated with PCa progression and/or biochemical recurrence [38–41].

In addition to tissue samples, MethyLight has also been used to analyze DNA methylation of noninvasive body fluids such as blood and urine. Representative examples include circulating cell-free DNA assessed by MethyLight, which showed that RASSF1A and/or APC methyla-tion in pretherapeutic serum samples of breast cancer patients was linked with poor prognosis for breast cancer [42]. A panel of six DNA meth-ylation biomarkers (EOMES, HOXA9, POU4F2, TWIST1, VIM, and ZHF154) obtained from urinary sediment DNA were found to be sig-nificantly correlated with recurrence of bladder cancer (Table 2) [43].

11. MULTIPLEX METHYLIGHT

Multiplex MethyLight can simultaneously analyze methylation of multiple genes in the same sample. The number of genes constituting the panel is only limited by the number of emis-sion detectors of the qPCR machine.

Multiplex MethyLight was the technique first published by He Q. et al. in 2010 in the investigation of a panel of biomarkers (ALX4, SEPT9, and TMEFF2) for screening of CRC from cell-free DNA in peripheral blood samples or fresh tissue samples [44]. The

specificity for detection of the selected panel of biomarkers was 87% for primary tissues and 90% for peripheral blood samples. How-ever, the reference gene used in this study was ACTB which is a less stable and/or less reproducible measure of bisulfite-converted template DNA, especially in the context of analyzing tumor DNA where amplification or deletions of single genes may occur. To address this issue, standard MethyLight assays now include short interspersed ALU element as an internal control as previously mentioned, since it is methylation independent and also less sensitive to copy number variations due to its genome-wide distribution [22].

We recently developed a multiplex Methy-Light assay using ALU as the reference gene with a sensitivity limit for methylation detec-tion of APC, HOXD3, and TGFβ2 at an esti-mated 370 pg of universal methylated DNA and an estimated 1.6 pg for ALU. In addition, multiplex MethyLight assay was able to dis-criminate between fully methylated alleles and unmethylated alleles with 100% specificity [36]. Multiplex MethyLight is highly accurate and reproducible when compared with single-plex MethyLight. Furthermore, in our study, multiplex MethyLight has successfully been applied to measure DNA methylation in FFPE tissue, fresh frozen tissue, tissue biopsy, and urine samples.

Multiplex MethyLight further reduces the requirement for quantity of initial input DNA when analyzing multiple genes, making it ideal for samples with low DNA yields such as saliva, urine, hair, or valuable clinical samples with limited quantity such as punch core biopsies. Since multiplex MethyLight is efficient and cost-effective, it is very likely that in the near future this particular technique will become more prevalent for locus-specific DNA methylation analysis.

Multiplex MethyLight has been used to detect methylation in PCa, CRC, and cervical cancer (MIM #603956) [36,44,45].

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TABLE 2 Methylation Biomarkers of Various Cancers

Disease Genes analyzedBiomaterials analyzed Specificity and sensitivity Techniques used References

Colorectal cancer (CRC)

SEPT9 Plasma 90% sensitivity and 88% specificity MethyLight [14]

CDKN2A/p14 Tissue Adenoma from FAP-Pts (41%, 13/32), multiple adenoma Pts (69%, 20/29), MSI-H CRC Pts (86%, 12/14), MSS/MSI-L CRC Pts (88%, 14/16)

MSP [15]

Tissue MSS CRC (12%, 3/24), MSI CRC (39%, 17/28)

MSP [15]

Tissue CRC (32%, 61/188) MSP, MethyLight [15]

CDKN2A/P16 Tissue Adenomas (34%, 14/41) MSP [15]

Stool Pts with adenomas (31%, 9/29), HD (16%, 3/19)

MSP [15]

Serum CRC pts (71%, 12/17), HD (0%, 0/10) MSP [15]

MLH1 Tissue MSS (0%, 0/25) CRC, MSI CRC (39%, 11/28)

MSP [15]

Peripheral blood CRC Pts (13.4%, 35/262) MSP [15]

Serum CRC Pts (39%, 19/49), HD (2%, 1/41) MethyLight [15]

TMEFF2 (TPEF/HPP1) Plasma CRC Pts (65%, 87/133), HD (31%, 56/179)

MethyLight [15]

ALX4, SEPT9, and TMEFF2

Tissue 84% sensitivity and 87% specificity Multiplex MethyLight

[44]

Peripheral blood 81% sensitivity and 90% specificity Multiplex MethyLight

[44]

IGFBP3, miR137 Tissue 95.5% sensitivity and 90.5% specificity Pyrosequencing [54]

THBD-M Plasma 71% sensitivity and 80% specificity Digital MethyLight [66]

Breast cancer APC Serum 29% sensitivity MethyLight [18]

GSTP1 Serum 18% sensitivity MethyLight [18]

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Disease Genes analyzedBiomaterials analyzed Specificity and sensitivity Techniques used References

Prostate cancer HOXD3 Tissue 61.8% sensitivity and 86.9% specificity MethyLight [38,40]

APC Tissue 82.4% sensitivity and 95.2% specificity MethyLight [40]

TGFb2 Tissue 42.4% sensitivity and 95.5% specificity MethyLight [40]

RASSF1A Tissue 92% sensitivity and 85.6% specificity MethyLight [40]

RARβ2 Ejaculate 100% sensitivity and 15% specificity Pyrosequencing [53]

Bladder cancer EOMES Urine 94% sensitivity and 39% specificity MethyLight [43]

HOXA9 Urine 92% sensitivity and 38% specificity MethyLight [43]

POU4F2 Urine 87% sensitivity and 47% specificity MethyLight [43]

TWIST1 Urine 89% sensitivity and 28% specificity MethyLight [43]

VIM Urine 90% sensitivity and 43% specificity MethyLight [43]

ZHF154 Urine 93% sensitivity and 47% specificity MethyLight [43]

SOX1, IRAK3, L1-MET Urine 93% sensitivity and 94% specificity Pyrosequencing [58]

Nasopharyngeal carcinoma

RASSF1A, WIF1, DAPK1, RARβ2

Tissue 95.8% sensitivity and 67.4% specificity MS-HRM [63]

Glioblastoma MGMT Tissue 80% sensitivity and 67% specificity MS-MLPA [49]

Lynch syndrome MLH1 Tissue 96% sensitivity and 66% specificity MS-MLPA [50]

Urothelial cancer SOX1, TJP2, MYOD, HOXA9_1, HOXA9_2, VAMP8, CASP8, SPP1, IFNG, CAPG, HLADPA1, RIPK3

Tissue 94.3% sensitivity and 97.8% specificity Pyrosequencing [55]

MS-HRM, methylation-specific high-resolution melting; MS-MLPA, methylation-sensitive multiplex ligation-dependent probe amplification; MSP, methylation-specific polymerase chain reaction.

TABLE 2 Methylation Biomarkers of Various Cancers—cont’d

13. PyROSEQUENCINg 167

12. METHYLATION-SENSITIVE MULTIPLEX LIGATION-

DEPENDENT PROBE AMPLIFICATION

MS-MLPA is a multiplex PCR-based tech-nique that can detect changes in gene copy number status, DNA methylation, and point mutations, simultaneously.

Standard MLPA technique requires two hemiprobes which target adjacent sequences in the same stretch of the template DNA. Both probes contain a PCR primer-binding region. One of the probes also contains a nonhybridizing “stuffer sequence” at the 3′ end to give the PCR product of desired length. The MLPA probe mix is allowed to hybridize with the denatured tem-plate DNA overnight. The annealed hemiprobes are then enzymatically ligated. The ligation product is amplified by PCR using the primer pair specific to the primer-binding regions of the two probes. Due to differences in length of the “stuffer sequences,” the amplification prod-ucts can be separated, identified, and quantified by capillary electrophoresis. Up to 50 different probes can be amplified in a single MLPA reac-tion for 50 different loci [46].

MS-MLPA uses the endonuclease HhaI to digest unmethylated CpGs after probe hybrid-ization. The MLPA probes target sequences that are 50–100 nucleotides in length which means it can detect methylation in highly fragmented DNA samples such as bisulfite-converted DNA or DNA extracted from FFPE tissue samples. MLPA is a highly sensitive technique with input DNA being as low as 20 ng and is capa-ble of detecting small copy number variations such as heterozygous deletion (2 copies to 1 copy per cell) or duplication (2–3 copies per cell) in a sample. MS-MLPA does not require bisulfite conversion of template DNA and thus does not have the limitations associated with the bisulfite conversion process such as DNA fragmentation and/or incomplete con-version. However, MLPA is limited in detecting

only copy number changes of the regions tar-geted by MLPA probes. In addition, a mutation or polymorphism in the target sequence may be detected as a change in copy number and potentially lead to a false-negative result. Fur-thermore, MS-MLPA relies on HhaI digestion which means only CpGs in the target sequence of HhaI (CGCG) will be identified. Thus, MLPA is ideal for the detection of deletions/insertions and known point mutations but is not suitable for detection of unknown single nucleotide polymorphisms (SNPs) [47].

MS-MLPA can be used to diagnose PWS and AS for the three major classes of genetic defects (See previous sections: 6. Methylation-Specific PCR; Table 1) [48].

In addition, MS-MLPA is used for tumor diagnosis and prognosis either alone or in com-bination with other techniques such as MSP. For example, MS-MLPA targeting MGMT methyla-tion is able to detect glioblastoma (MIM #137800) progression after chemotherapy with 80% diag-nostic accuracy [49], while assessment of MLH1 hypermethylation by MS-MLPA showed 96% sensitivity and 66% specificity in ruling out Lynch syndrome (MIM #120435) in individuals with a family history of CRC [50].

13. PYROSEQUENCING

Pyrosequencing is a reliable, fast, and high-throughput technique that can analyze up to 96 bisulfite-converted DNA samples in approxi-mately 4 h. It is based on sequential addition and incorporation of nucleotides that can be quantitated through conversion of naturally released pyrophosphate into a light signal in real time [51]. The released pyrophosphate is used in a sulfurylase reaction releasing ATP that is subsequently used by luciferase to pro-duce light. Pyrosequencing has a wide range of applications including detection of SNPs, insertion/deletions, gene copy number, and DNA methylation.

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS168

Bisulfite-converted pyrosequencing, first developed in 2003 by Colella et al. [52], has been successfully applied in the detection of DNA methylation biomarkers for diagnosis and prognosis of cancer. For example, a recent report described a noninvasive test for PCa diagnosis using bisulfite-converted pyrose-quencing to detect DNA methylation in body fluids such as ejaculate to predict PCa with RARβ2 promoter methylation [53]. Quanti-tative bisulfite pyrosequencing was used to identify methylation of IGFBP3 and MIR137 to stratify CRC patients with 95.5% sensitivity and 90.5% specificity [54].

A limitation of pyrosequencing is that only short stretches of DNA can be analyzed. It is primarily used as a validation step for DNA methylation biomarkers and is not recom-mended for discovery of new biomarkers. For example, quantitative pyrosequencing was used to validate 12 differentially methylated loci from urothelial bladder cancer patients (MIM #109800). The 12 loci combined showed 94.3% sensitivity and 97.8% specificity as diag-nostic markers [55]. Also, in a recent study by Sandoval et al. pyrosequencing was used to validate a panel of DNA methylation bio-markers selected from 10,000 CpGs obtained through DNA methylation microarray in the prognosis of non-small cell lung cancer (MIM #211980). It was found that p16INK4a, CDH13, RASSF1A, and APC hypermethylation is asso-ciated with early recurrence of stage I lung cancer [56].

Other examples of applications of pyrose-quencing include the identification of a panel of 32 genes to be associated with prognosis of cervical squamous cell carcinoma. In particular, VIM gene methylation in cervical squamous cell carcinoma was shown to predict a favor-able prognosis [57]. Also, a three-marker panel (SOX1, IRAK3, and L1-MET) was validated using pyrosequencing that can predict between bladder cancer recurrence with sensitivity of 93% and specificity of 94% [58]. This technique is described in detail in the following section.

14. HIGH-RESOLUTION MELTING ANALYSIS

High-resolution melting analysis (HRM) is a qPCR-based technique based on the Tm differ-ence between purine and pyrimidine bases that can quantify the methylation status of all CpG sites in a sample. Following bisulfite conversion, unmethylated cytosines are converted to uracils. Due to differences in Tm between cytosines and thymines, a melting profile can be compiled with slowly increasing temperatures and by the incor-poration of an intercalating fluorescent dye. The melting profiles of unmethylated and methylated variants of the same area of amplification will be significantly different due to the differences in GC content. This difference in the melting pro-file is used to assess the methylation status of the template DNA by comparing against the native DNA that has not been bisulfite converted. HRM will assess the entire amplified region rather than only a few selected CpG sites.

The melting curve analysis of DNA methyl-ation was first developed in 2001 [59]. At that time, the sensitivity of the technique was limited by the availability of intercalating dyes. HRM for DNA methylation was developed in 2003 using the LCGreen dye [60]. LCGreen PCR HRM can discriminate between heterozygous and homo-zygous sequence variants. Methylation-specific HRM (MS-HRM) was developed in 2008 using methylated- and unmethylated-specific prim-ers to amplify bisulfite-converted DNA in a PCR [61]. Although HRM is not locus specific, it has practical applications for diagnosis of diseases such as PWS and AS [62], and nasopharyngeal carcinoma (MIM #607107) [63] based on screen-ing of methylated markers.

15. EMERGING APPLICATIONS

15.1 Digital MethyLight

Bisulfite-converted DNA samples analyzed using standard MSP techniques are limited

16916. fUTURE Of DNA METHyLATION IN CLINICAL APPLICATIONS

by their throughput capacity, sensitivity of methylation detection, and resolving power. To address these issues, digital PCR technol-ogy is applied in MethyLight assays. Digital PCR employs distribution/dilution of DNA samples into multiple reaction chambers which can amplify signals from a single molecule of template DNA and, at the same time, decreases nonspecific noise [64]. Due to distribution of a sample over multiple PCR reaction wells to concentrations below that of a single-tem-plate molecule per well, specific and sensitive amplification is achievable for single-template molecules. Digital bisulfite genomic sequenc-ing is able to omit the cloning step in bisulfite sequencing. Using SYBR Green, positive wells are visualized and positive PCR products can be sequenced directly after amplification. In addition, if the sample contains heterogeneous populations of DNA methylation patterns, tem-plates may be sequenced separately to obtain individual DNA methylation patterns of the entire population.

Digital MethyLight is considerably more sensitive than traditional MethyLight assays with up to three times the sensitivity [65]. Although digital MethyLight was established in 2008, there have been very few publica-tions using this method. Some potential dis-advantages of digital MethyLight may be that optimization is required to find the most effec-tive dilution. Also it may be too expensive to sequence all positive wells for an individual sample.

One study published in 2012 used digital MethyLight in a genome-wide discovery and validation of blood-based biomarkers for CRC detection. Stringent multi-step filtering criteria were achieved with digital MethyLight per-formed following MethyLight and array profil-ing of CRC tumors. Two candidate methylation biomarkers (THBD and C9orf50) were identified and validated in as little as 50 μL of sera/plasma compared with conventional SEPT9 assays that use 4–5 mL of plasma [66].

15.2 A Novel Methylation Marker: 5-Hydroxymethylation

DNA hydroxymethylation is a recently discovered modification on CpG dinucleo-tides and involves the addition of a hydroxyl group on 5-methylcytosines (5mCs) to produce 5-hydroxymethylcytosine (5hmC). Although its biological role has not been fully elucidated, it has been speculated that 5hmC is an intermedi-ate during the process of demethylation medi-ated through the TET family of proteins [67].

Conventional techniques using bisulfite-con-verted DNA cannot distinguish between CpG methylation and hydroxymethylation since both are protected from bisulfite conversion. Emerg-ing MS-PCR applications are focusing on selec-tive enrichment of 5hmC for epigenetic analysis. Therefore, future developments will provide new epigenetic biomarkers based on hydroxy-methylation of specific genes.

16. FUTURE OF DNA METHYLATION

IN CLINICAL APPLICATIONS

The future of DNA methylation-dependent diagnosis will involve panels of multiple bio-markers for screening and prognosis of specific diseases including cancers. Many recent pub-lications describe development of biomarker screening panels for DNA methylation as well as other epigenetic markers, including miRNA, lncRNA, and histone modifications [68–70]. The panels of epigenetic biomarkers may offer more sensitive detection and accurate prognosis of diseases, as well as the discovery of potential therapeutic targets.

Since DNA methylation is a reversible phe-nomenon, it provides a potentially valuable target for the treatment of methylation-depen-dent diseases. Currently there are few stud-ies investigating epigenetic modifications as potential therapeutic targets. Re-expression of

8. METHYL SPECIFIC PCR AND ITS APPLICATION IN CLINICAL DIAGNOSTICS170

methylation-silenced tumor suppressor genes by inhibiting DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) has emerged as an effective strategy against cancer [71]. In fact 5-aza-CdR, a drug which produces irrevers-ible inactivation of DNA methyltransferases, has shown interesting antineoplastic activity in patients with leukemia, myelodysplastic syndrome, and non-small cell lung cancer [72]. Importantly, MSP can be used as a monitoring strategy for alterations in methylation levels fol-lowing demethylation treatments.

In the past decade, it has become very apparent that epigenetic alterations are heav-ily involved in disease onset and progression. Specifically, aberrant DNA methylation of genes serves as biomarkers for screening of epigenetic diseases and many types of cancers through potentially noninvasive means such as cheek swabs, urine, and blood.

Emerging initiatives are now focused on not only discovery and validation of novel bio-markers, but also elucidation of the underlying mechanism of these diagnostic and prognos-tic epigenetic changes as well as exploration of their potential for therapeutic targets. DNA methylation investigation/assessment through MSP and its associated techniques have played a key role in this regard with the discovery of many clinically relevant diagnostic, prognos-tic, and predictive biomarkers. With continued advancement in technology, DNA methylation as both biomarkers and therapeutic targets will become even more important in the future for health care.

LIST OF ABBREVIATIONS

5-aza-CdR 5-Aza-2′-deoxycytidine5hmC 5-Hydroxymethylcytosine5mC 5-MethylcytosineAS Angelman syndromeBp Base pairsCIMP CpG island methylator phenotypeCpG 5′-Cytosine-guanine-3′

CRC Colorectal cancerFFPE Formalin-fixed paraffin-embeddedFISH Fluorescence in situ hybridizationFMRP Fragile X mental retardation proteinFOBT Fetal-occult blood testFXS Fragile X syndromeGOI Gene of interestHRM High-resolution melting analysislncRNAs Long noncoding RNAmiRNA MicroRNAMN-MSP Nested MSPMS-DBA Methylation-sensitive dot blot assayMS-MLPA Methylation-sensitive multiplex ligation-depen-

dent probe amplificationMSP Methylation-specific PCRPCa Prostate cancerPCR Polymerase chain reactionPMR Percentage of methylated referencePWS Prader–Willi syndromeqPCR Quantitative PCRRISC RNA-induced silencing complexSNP Single-nucleotide polymorphismTm Melting temperature

Acknowledgment and Funding SupportThe authors would like to acknowledge support from Ontario Institute of Cancer Research (OICR) Personalized Medicine Research Fund #10Nov-412 and Prostate Cancer Canada #2011-700; PCC TAG #2014-01 1417 (to B. Bapat). F. Zhao is supported by Ontario Student Opportunity Trust Funds award.

We would like to thank Andrea Savio for critical reading of the manuscript.

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