Balkan Journal of Medical Genetics

ALTERATIONS OF COPY NUMBER OF METHYLATION PATTERN IN MISMATCH REPAIR GENES BY METHYLATION SPECIFIC-MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION IN CASES OF COLON CANCER
Onrat ST1*, Çeken I2, Ellidokuz E3, Kupelioğlu A4
*Corresponding Author: Serap Tutgun Onrat, Department of Medical Genetics, Afyon Kocatepe University Medical Faculty, ANS Arastırma Uygulama Hastanesi, Morfoloji Binası, Ozdilek yolu, Afyonkarahisar, 03200, Turkey; Tel.: +90-272-246-3301, Fax: +90-272-246-3300, E-mail: tutgunonrat@ yahoo.com, sonrat@aku.edu.tr
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METHYLATION SPECIFIC-MULTI PLEX LIGATION-DEPENDENT PROBE AMPLIFICATION ASSAY

Methylation Analysis. The MS-MLPA for methylation status in tumor DNA of MLH1, MSH2, MSH6 promoter regions used the SALSA ME011 kit and protocols provided by the manufacturer (MRC-Holland, Amsterdam, The Netherlands). This ME011-A1 MMR probe mix was developed to detect aberrant CpG islands methylation of six MMR genes and includes five probes for MLH1, three probes MSH2, three probes for MHSH6, three probes for MSH3, one probe for MLH3, three probes for PMS2 and three probes specific for the MGMT promoter region. The MGMT promoter plays a role in removing O(6)-alkylguanine, which is the major mutagenic and carcinogenic lesion induced by alkylating mutagens. The MLH1 gene is located at chromosome 3p22.1, MLH3 at chromosome 14q24.3, MSH2 at 2p21, MSH3 at 05q14.1, MSH6 gene at 2p16, PMS2 at 7p22 and the MGMT gene is located at chromosome 10q26. The kit includes five probe pairs for MLH1 (with the respective HhaI sites located at –638, –402, –251/–245, –8 and +220 relative to the initiating ATG; GenBank accession number U26559), three probe pairs for MSH2 (HhaI positions at –264, –200 and +133/ +148 relative to ATG; Gen- Bank accession number AB006445) and three probe pairs for MSH6 (HhaI positions at –293, –100/–143 and –64 relative to ATG; Gen Bank accession number U73732). Normal DNA specimens derived from lymphocytes from healthy controls were included in every assay. For each MLPA reaction, 150 ng of DNA was used. Approximately 25 ng of genomic DNA in 5 μL of TE buffer [10 mM Tris-HCl (pH 8.5) and 1 mM EDTA] was denatured for 10 min. at 98°C. The SALSA MLPA buffer (1.5 mL) and MS-MLPA probes (1 fmol each and 1.5 μL vol) were then added, and after incubation for 1 min. at 95°C, were allowed to hybridize to their respective targets for 16 hours at 60°C. The mixture was then diluted at room temperature with H2O and 3 μL Ligase buffer A to a final volume of 20 μL and divided equally into two tubes. While at 49°C, a mixture of 0.25 μL Ligase-65 (MRC-Holland), 5 U HhaI (Vavantis Technologies, Subang Jaya, Malaysia) and 1.5 μL Ligase buffer B in a total volume of 10 μL was added to one tube. For the second tube, the HhaI was replaced by H2O. Simultaneous ligation and digestion was performed for 30 min. at 49°C, followed by 5 min. at 98°C. The ligation products were polymerase chain reaction (PCR)-amplified by addition of 5 μL of this ligation mixture to 20 μL PCR mixture containing PCR buffer, dNTPs, SALSA polymerase and PCR primers (one unlabeled and one D4-labeled) at 60°C as described by Schouten et al. [20]. The obtained PCR products were separated by capillary gel electrophoresis in an ABI 310 Genetic Analyzer Capillary, (Applied Biosystems, Foster City, CA, USA), and analyzed using GeneMapper ID V1.8 Software (Applied Biosystems). Information on both methylation status (10-100% methylated) and copy number (number of methylated alleles) was obtained by comparing results of undigested and HhaI-digested samples using the Coffalyser V9.4 Software (MRC-Holland, http:// www.mrc-holland. com/) [21]. All analyses were performed in duplicate. Methylation Specific-Multiplex Ligation-Dependent Probe Amplification Data Processing. Data analysis was performed in Excel as described by the manufacturer of the MLPA kits (MRC-Holland) [21]. To compensate for differences in PCR efficiency of the individual samples, the fraction of each peak is calculated by dividing the peak value of each probe amplification product by the combined value of the control probes within the sample. For hypermethylation analysis, this ‘relative peak value’ or so-called ‘probe fraction’ of the digested sample is divided by the ‘relative peak value’ of the corresponding undigested sample generating the ‘methylation ratio’. From our dilution experiments, it became clear that the ratio indicates the percentage of methylated sequences. Duplicate experiments were performed for methylation analysis and average ratios were calculated. Copy number analysis can be performed using the MLPA results of the undigested samples. After calculation of the ‘relative peak value’ or so-called ‘probe fraction’, this is divided by the ‘mean probe fraction’ of this fragment within the included reference DNAs, generating the ‘normalized peak value’ or the so-called ‘copy number ratio’. According to the Jeuken et al. [22] MLPA, thresholds to detect gains and losses were set at 1.2 and 0.8, respectively. Methylation dosage ratio was obtained from the following calculation: Dm = (Px/Pctrl)Dig/(Px/Pctrl) Undig, where Dm is the methylation dosage ratio, Px is the peak area of a given probe, Pctrl is the sum of the peak areas of all control probes, Dig stands for HhaIdigested samples, and Undig stands for undigested samples. A dosage ratio of 0.15 or higher, corresponding to 15% of methylated DNA, was interpreted to indicate promoter methylation [23]. Statistical Methods. All data were recorded by using a standard data form and analyzed by using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Quantitative values were compared by using the Student t-test for independent groups, and for categorical data, χ2 and Student t tests were applied. All p values of less than 0.05 (p = 0.05) were considered to indicate statistical significance.

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