Methylation-specific PCR Revealed Aberrant Promoter Gene Methylation of p16, MGMT and SPOCK2 in Diffuse Large B-cell Lymphoma

DNA methylation silences the gene through addition of methyl group. p16, a tumor suppressor gene that inhibits cyclin-dependent kinase, inactivates the Rb protein and blocks G1 phase in a normal cell cycle. A DNA repair gene, MGMT removes alkyl adduct to a cysteine residue within the protein, thus preventing lethal cross-links. p16 and MGMT methylation has been reported to associate with DLBCL. A member of the extracellular chondroitin and heparin sulfate proteoglycans, SPOCK2 functions mainly in extracellular matrix for cell adhesion. Uniquely, SPOCK2 (testican 2) abolishes the inhibition of membrane-type 1-matrix metalloproteinase by other testican family which might enhance the angiogenesis. In this study, we aimed to screen for aberrantly methylated genes which might contribute to the pathogenesis of DLBCL using methylation specific PCR (MSP). p16 methylation was identified in 64 (73%) of 88 samples. On the other hand, SPOCK2 was found to be unmethylated in 30 (34%) samples. Interestingly, MGMT methylation was detected in all cases. We also found an association between p16 methylation status with patients aged >50 years old (p= 0.023). This finding is parallel with an animal study showing that aging increases p16 methylation. No association was found between the methylation of other genes with age. Unmethylation of SPOCK2 might cause testican 2 expressions, which has been suggested to contribute toward malignant behaviour. MGMT was reported to be methylated among cancer patients who smoke, drink and are non-vegetarian. Thus, it is hypothesized that lifestyle might affect MGMT methylation in this study population

MGMT and SPOCK2 promoter methylation in diffuse large B-Cell lymphoma: a study in two tertiary health centres in the East Coast of Malaysia

MGMT (O6-Methylguanine-DNA Methyltransferase) suppresses tumor development by removing alkyl adduct, while SPOCK2 (SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan) abolishes the inhibition of membrane-type matrix metalloproteinases (MT-MMP) which leads to angiogenesis. Hence, MGMT methylation may initiate malignant cells transformation. In contrast, SPOCK2 methylation is hypothesized not to be a common event in diffuse large B-cell lymphoma (DLBCL). In this study, we examined the methylation status of MGMT and SPOCK2 in DLBCL as in Malaysia the information is extremely lacking. A total of 88 formalin-fixed paraffin-embedded tissue of patients diagnosed with DLBCL from the year 2006 to 2013 were retrieved from Hospital Universiti Sains Malaysia, Kelantan and Hospital Tengku Ampuan Afzan, Pahang. Methylation-specific polymerase chain reaction (MSP) was used to examine the methylation status of both genes. Interestingly, methylation of MGMT was detected in all the 88 DLBCL samples, whereas SPOCK2 was found to be methylated in 83 of 88 (94.3%) DLBCL cases. Our study showed a remarkably high percentage of promoter methylation of both MGMT and SPOCK2 genes. Our finding also negates initial expectation that SPOCK2 methylation would be an uncommon event in the majority of DLBCL cases. This study has shown a very high percentage of promoter methylation of MGMT and SPOCK2 in the DLBCL cases studied by MSP, using archival lymphoma tissues. Nonetheless, additional research is needed to quantitatively evaluate MGMT and SPOCK2 methylation, and to analyse gene expression and/or protein expression in order to further understand the role of MGMT and SPOCK2 methylation in the pathogenesis of DLBCL

Pyrosequencing-based quantitative identification of p16 methylation in diffuse large b-cell lymphoma at two centres in the east coast of malaysia

Introduction: Methylation of promoter region of p16 leading to gene silencing has been implicated in a wide range of malignancies including lymphomas. In diffuse large B cell lymphoma (DLBCL) particularly, a varying percentage of epigenetic inactivation of p16 promoter region was observed ranging from 16 - 54%. However, quantitative analysis of p16 promoter methylation in DLBCL has not been extensively studied in Malaysia. Objective: This study aims to quantitatively analyse p16 methylation in DLBCL samples using pyrosequencing technique. Methods: Genomic DNA was extracted from 16 formalin-fixed paraffin-embedded lymphoma tissue blocks from patients diagnosed with DLBCL. Samples were retrieved from Hospital Tengku Ampuan Afzan, Pahang and Hospital Universiti Sains Malaysia, Kelantan. Primers were designed to amplify bisulfite-treated DNA targeting p16 promoter region. Methylation status of 7 CpG sites was determined by pyrosequencing. Results: All the 16 samples studied showed promoter methylation of p16. The range of mean methylation percentage was between 18 to 81%. Conclusion: The present study has successfully measured the level of methylation of p16 in all 7 CpG sites despite the limitation in sample size. Since p16 methylation is a common event in our series of DLBCL cases, it is worth including a larger sample size in future studies to increase the chance of finding a significant correlation with clinical parameters

p16 tumor suppressor gene methylation in diffuse large B cell lymphoma: a study of 88 cases at two hospitals in the East Coast of Malaysia

Introduction: p16 gene plays an important role in the normal cell cycle regulation. Methylation of p16 has been reported to be one of the epigenetic events contributing to the pathogenesis of diffuse large B-cell lymphoma (DLBCL) which occurring at varying frequency. DLBCL is an aggressive and high-grade malignancy which accounts for approximately 30% of all non-Hodgkin lymphoma cases. However, little is known regarding the epigenetic alterations of p16 gene in DLBCL cases in Malaysia. Therefore, the objective of this study was to examine the status of p16 methylation in DLBCL. Methods: A total of 88 formalin-fixed paraffin-embedded DLBCL tissues retrieved from two hospitals located in the east coast of Malaysia, namely Hospital Tengku Ampuan Afzan (HTAA) Pahang and Hospital Universiti Sains Malaysia (HUSM) Kelantan, were chosen for this study. DNA specimens were isolated and subsequently subjected to bisulfite treatment prior to methylation specific-PCR. Two pairs of primers were used to amplify methylated and unmethylated regions of p16 gene. The PCR products were then separated using agarose gel electrophoresis and visualised under UV illumination. SPSS version 12.0 was utilised to perform all statistical analysis. Result: p16 methylation was detected in 65 of 88 (74%) samples. There was a significant association between p16 methylation status and patients aged >50 years old (p=0.04). Conclusion: Our study demonstrated that methylation of p16 tumor suppressor gene in our DLBCL cases is common and significantly increased among patients aged 50 years and above. Aging is known to be an important risk factor in the development of cancers and we speculate that this might be due to the increased transformation of malignant cells in aging cell population. However, this has yet to be confirmed with further research and correlate the findings with clinicopathological parameters

Epigenetic methylation status of P16, MGMT and SPOCK2 in diffuse Large B cell lymphoma

Introduction: Epigenetic methylation has been implicated in the pathogenesis of diffuse large B cell lymphoma (DLBCL). This study investigated the methylation status of p16, MGMT and SPOCK2. Aberrantly methylated p16 and MGMT have been linked to DLBCL, but not SPOCK2. p16 inhibits cyclin-dependent kinase, which results in retinoblastoma phosphorylation and blockage of cell cycle at G1 phase. MGMT removes alkyl adduct at O6-guanine, thus preventing lethal cross-links. SPOCK2, an extracellular chondroitin and heparin sulfate proteoglycans, abolishes the inhibition of membrane-type 1-matrix metalloproteinase which might enhance the angiogenesis. The absence of SPOCK2 methylation was therefore hypothesized in the majority of cases in this study. Methods: Extracted DNA from 88 formalin-fixed paraffin-embedded (FFPE) tissues of DLBCL were subjected to bisulfite conversion followed by methylation-specific PCR (MSP) analysis for p16, MGMT and SPOCK2 methylation. p16 methylation was also quantified in 16 samples through pyrosequencing assay. Results: p16 methylation was observed in 65/88 (74%) samples by MSP. Pyrosequencing detected p16 methylation in all 16 samples ranging from 18% to 81%. MGMT methylation was detected in all 88 (100%) cases. Methylated SPOCK2 was found in 83 (94.3%) samples. There was a significant association between p16 methylation status with patients above 50 years of age (p= 0.04). Conclusions: These preliminary discoveries may serve as a good platform in order to gain a comprehensive overview on the epigenetics contribution in the pathogenesis of DLBCL. Pyrosequencing is a robust tool in detecting and quantifying methylation

Detection of p16INK4a (p16) methylation in diffuse large B-cell lymphoma using methylation-specific PCR

Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of non-Hodgkin lymphoma, mostly involve lymph nodes. It is the most common lymphoma among adult with median age 70 years.p16is a tumour suppressor gene (TSG) which inhibits cyclin-dependent kinase thus inactivatesRb protein and blockade G1 phase. Silence of p16 induced by DNA methylation epigenetically has been reported as one of the factors toward DLBCL occurrence with varying percentages. Loss of p16 protein causes unregulated cell division which may lead to cancer. In this study, we aim to identify the presence of p16 methylation in DLBCL. DLBCL samples were obtained from two study groups; Kelantan and Pahang states. Methylation-specific PCR (MSP) was utilized to detect p16 methylation using specific primers in 39 formalin-fixed paraffin embedded tissues samples. The PCR products were then visualized on 2% agarose gels. 14 of 34 (41%) samples are found to have p16 methylation.There was no p16 methylation detected in five normal thyroid samples. p16 gene methylation has been detected in almost half of our samples. This could provide data of gene methylation in DLBCL among Malaysian. Thus, more studies should be carried out for further investigation

Qualitative detection of p16INK4a tumor suppressor gene methylation in diffuse large b-cell lymphoma using methylation-specific PCR

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy of non-Hodgkin lymphoma, mostly involve lymph nodes. It is the most common lymphoma among adult with median age 70 years. p16 is a tumour suppressor gene (TSG) which inhibits cyclin-dependent kinase thus inactivates Rb protein and blockade G1 phase. Silence of p16 induced by DNA methylation epigenetically has been reported as one of the factors toward DLBCL occurrence with varying percentages. Loss of p16 protein causes unregulated cell division which may lead to cancer. In this study, we aim to identify the presence of P16INK4a gene methylation in DLBCL and to correlate the methylation status with patients’ age and gender. MATERIALS AND METHODS: The samples of DLBCL were retrieved from two study groups; Kelantan and Pahang states. Methylation-specific PCR (MSP) was utilized to detect p16 methylation using specific primers in 88 formalin-fixed paraffin embedded DLBCL tissues including five normal thyroid samples. The PCR products were then visualized on 2% agarose gels under UV illumination. RESULTS AND DISCUSSION: 64 of 88 (73%) samples were found to have p16 methylation. There was no p16 methylation spotted in five normal thyroid samples. We found statistically significant association between p16 methylation and patients’ age (>50 years) with p = 0.023. While, there is no association between p16 methylation and gender observed in this study. CONCLUSION: p16 gene methylation has been detected in most of the samples with 72% of methylation occurred among patients more than 50 years. This could provide data of gene methylation in DLBCL among Malaysian. Thus, more studies should be carried out for further investigation

Optimization of cell-free plasma RNA extraction for downstream application

The growing interest in biomedical studies has brought RNA from biofluids including plasma, as promising candidates for genetics profiling. The precision and reliability of an analysis in downstream application such as NanoString nCounter® MAX Analysis System (NanoString Technologies, Seattle, WA) ) depend on the RNA quality, purity and level. In this project, NanoString nCounter® miRNA panel was chosen due to rapid identification and ability to profile approximately 800 miRNAs per run which requires total RNAs from plasma with a minimum concentration of 33.3 ng/µL with 260/280 and 260/230 ratios of ≥1.8 for optimal results. Unlike tissues and cells, circulating RNAs in plasma are cell-free and are present in small sizes. However, the abundance of proteins and inhibitors in the plasma as possible contaminants could diminish the effectiveness of molecular isolation techniques and pose challenges in RNA isolation and quantification. This could skew data collection and elucidation. Therefore, the main objective is to determine the optimized plasma RNA isolation protocol to overcome problems in RNA quality and purity with regards NanoString nCounter® MAX Analysis System requirement. Several optimization steps were performed, including the addition of one chloroform extraction step with extra washing steps instead of conducting only once following the actual protocol. After conducting these steps, the average 260/280 ratio falls between 1.7 to 1.8, slightly increased compared to the results before optimization which was around 1.4 to 1.6 since these steps of optimization help to remove excess impurities including phenol and salt. Furthermore, increasing the incubation time in certain steps, for instance, after sample homogenization with Qiazol, during 95% ethanol precipitation and after RNase-free water addition have boosted the RNA recovery allowing RNA concentration of 15 ng/µL and above to be obtained. Hence, the optimized plasma RNA isolation protocol was determined since several issues related to plasma RNA concentration and purity were significantly improved by performing the additional steps in the protocol

Fetal down syndrome: determining the risk based on fetal-specific DNA methylation ratio

Non-invasive prenatal diagnosis has becoming popular in determining the risk of genetic abnormalities in unborn babies, particularly Down Syndrome (trisomy 21). In this research, methylated DNA immunoprecipitation (MeDIP)-real time quantitative PCR (qPCR) method based on differentially methylated regions (DMRs) or methylation differences between mother and fetus has been employed. Circulating cell free DNA (ccfDNA) was extracted from 28 plasma specimens of pregnant women recruited from January 2017 to February 2019 in Kuantan, Pahang. Seven out of 28 subjects were grouped as high risk (probable trisomy) cases referring to the nuchal translucency reading. The extracted ccfDNA was then sheared and subjected to MeDIP-qPCR. Three DMRs namely EP1, EP7 and EP10 were chosen from previous research to be analyzed, along with hypermethylated and hypomethylated controls. At the moment, Cq values have been obtained for a number of samples and normalization of the raw data will be carried out as normalization of PCR reactions and normalization based on the real-time qPCR primer’s efficiency. The non-invasive prenatal detection of trisomy 21 will be achieved by determining the methylation ratio of normal and trisomy 21 cases for each tested fetal-specific DMR present in the maternal peripheral blood, followed by further statistical analysis. It is hoped that by the end of this study, the risk of fetal Down syndrome can be confirmed and tabulated for all high risk samples

Methylation status of MGMT and SPOCK2 in diffuse large B-cell lymphoma

MGMT (O6-Methylguanine-DNA Methyltransferase) suppresses tumour development by removing alkyl adduct, while SPOCK2 (SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan) abolishes the inhibition of membrane-type matrix metalloproteinases (MT-MMP) which leads to angiogenesis. Hence, MGMT methylation may initiate malignant cells transformation. In contrast, SPOCK2 methylation is hypothesized not to be a common event in diffuse large B-cell lymphoma (DLBCL). In this study, we examined the methylation status of MGMT and SPOCK2 in DLBCL as in Malaysia the information is extremely lacking. A total of 88 formalin-fixed paraffin-embedded tissue of patients diagnosed with DLBCL from the year 2006 to 2013 were retrieved from Hospital Universiti Sains Malaysia and Hospital Tengku Ampuan Afzan. Methylation-specific PCR (MSP) was used to examine the methylation status of both genes. Interestingly, methylation of MGMT was detected in all the 88 DLBCL samples, whereas SPOCK2 was found to be methylated in 83 of 88 (94.3%) DLBCL cases. Our study showed a remarkably high percentage of promoter methylation of both MGMT and SPOCK2 genes. Our finding also negates initial expectation that SPOCK2 methylation would be an uncommon event in the majority of DLBCL cases. This study has shown a very high percentage of promoter methylation of MGMT and SPOCK2 in the DLBCL cases studied by MSP, using archival lymphoma tissues. Nonetheless, additional research is needed to quantitatively evaluate MGMT and SPOCK2 methylation, and to analyse gene expression and/or protein expression in order to further understand the role of MGMT and SPOCK2 methylation in the pathogenesis of DLBCL