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. 2021 Jan 1;22(1):85-91.
doi: 10.31557/APJCP.2021.22.1.85.

Aberrant Methylation of Tumour Suppressor Gene ADAM12 in Chronic Lympocytic Leukemia Patients: Application of Methylation Specific-PCR Technique

Amira Mohamad  1 , Rosline Hassan  2 , Azlan Husin  3 , Muhammad Farid Johan  2 , Sarina Sulong  1
Affiliations

Aberrant Methylation of Tumour Suppressor Gene ADAM12 in Chronic Lympocytic Leukemia Patients: Application of Methylation Specific-PCR Technique

Amira Mohamad et al. Asian Pac J Cancer Prev. .

Abstract

Objective: Chronic Lymphocytic Leukemia (CLL) is a common leukemia among Caucasians but rare in Asians population. We postulated that aberrant methylation either hypermethylation or partial methylation might be one of the silencing mechanisms that inactivates the tumour suppressor genes in CLL. This study aimed to compare the methylation status of tumour suppressor gene, ADAM12, among CLL patients and normal individuals. We also evaluated the association between methylation of ADAM12 and clinical and demographic characteristics of the participants.

Methods: A total of 25 CLL patients and 25 normal individuals were recruited in this study. The methylation status of ADAM12 was determined using Methylation-Specific PCR (MSP); whereas, DNA sequencing method was applied for validation of the MSP results.

Results: Among CLL patients, 12 (48%) were partially methylated and 13 (52%) were unmethylated. Meanwhile, 5 (20%) and 20 (80.6%) of healthy individuals were partially methylated and unmethylated, respectively. There was a statistically significant association between the status of methylation at ADAM12 and the presence of CLL (p=0.037).

Conclusion: The aberrant methylation of ADAM12 found in this study using MSP assay may provide new exposure to CLL that may improve the gaps involved in genetic epigenetic study in CLL.

Keywords: ADAM12; Chronic Lympocytic Leukemia; methylation; tumour suppressor gene.

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Figures

Figure 1
Figure 1
Determining the Methylation Status of ADAM12 Using MSP Analysis. Primer sets used for amplification are designated as unmethylated (U) and methylated (M). Blank, reagent blank; Sample P, patient’s DNA; Sample N, normal DNA control. One normal sample and one patient sample showed unmethylated at ADAM12; whereas, another patient’s sample showed partial-methylated at ADAM1
Figure 2
Figure 2
Sequencing Result of Targeted Region of Methylated ADAM12 in Partially Methylated CLL Sample. A) At the methylation sequence detected (MD), thymine (T) marks the presence of unmethylated cytosine (T); whereas, cytosine (C) marks the presence of methylated cytosine (C). At the MD, T appears at unmethylation sites, G appears at the methylation sites, thereby confirming that the patient had partially methylated at ADAM12. (B) Electropherogram picture showed successful formation of thymines and cytosines as a result of complete conversion of unmethylated and methylated cytosines, respectively
Figure 3.
Figure 3.
Sequencing Result of Targeted Region of Unmethylated of ADAM12 in Partially Methylated Patient’s Sample. A) Comparison of unmethylated wild type sequence of ADAM12 with methylated ADAM12 sequence detected in a patient sample. At the unmethylation sequence detected (UMD), thymine (T) marks the presence of unmethylated cytosine (T). B) Electropherogram picture shows successful formation of thymines however, there are some sites that show mixture of cytosine and thymine
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