A newly developed age estimation method based on CpG methylation of teeth-derived DNA using real-time methylation-specific PCR

Abstract Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

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Development and Performance Evaluation of TaqMan Real-time Fluorescence Quantitative Methylation Specific PCR for Detecting Methylation Level of PER2

10.21203/rs.3.rs-844684/v1 ◽

2021 ◽

Author(s):

Huihui Jiang ◽

Xin Yang ◽

Miaomiao Mi ◽

Xiaonan Wei ◽

Hongyuan Wu ◽

...

Keyword(s):

Real Time ◽

Reference Materials ◽

Methylation Level ◽

Gene Promoter ◽

Detection Performance ◽

Clinical Samples ◽

Quantitative Detection ◽

Analytical Sensitivity ◽

Methylation Specific Pcr ◽

Specific Pcr

Abstract Background: PER2 gene methylation is closely related to the occurrence and progress of some cancers, but there is no method to quantitatively detect PER2 methylation in conventional laboratories. So, we established a TaqMan real-time fluorescence quantitative methylation specific PCR (TaqMan real-time FQ-MSP) assay and use it for quantitative detection of PER2 methylation in leukemia patients. Methods: According to the PER2 sequence searched by GenBank, a CpG sequence enrichment region of the PER2 gene promoter was selected, and the methylated and unmethylated target sequences were designed according to the law of bisulfite conversion of DNA to construct PER2 methylation positive and negative reference materials. Specific primers and probe were designed. The reference materials were continuously diluted into gradient samples by 10-fold ratio to evaluate the analytical sensitivity, specificity,accuracy and reproducibility of the method, and the analytical sensitivity of TaqMan real-time FQ-MSP assay was compared with that of the conventional MSP assay. At the same time, the new-established TaqMan real-time FQ-MSP assay and the conventional MSP assay were used to detect the PER2 methylation level of 81 patients with leukemia, and the samples with inconsistent detection results of the two assays were sent to pyromethylation sequencing to evaluate the clinical detection performance .Results: The minimum detection limit of TaqMan real-time FQ-MSP assay for detecting PER2 methylation level established in this study was 6 copies/uL, and the coefficient of variation(CV) of intra-assay and inter-assay was less than 3%. Compared with the conventional MSP assay, it has higher analytical sensitivity. For the samples with inconsistent detection results, the results of pyrosequencing and TaqMan real-time FQ-MSP assay are consistent. Conclusion: TaqMan real-time FQ-MSP assay of PER2 methylation established in this study has high detection performance and can be used for the detection of clinical samples.

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