Setup Of Methylation-Specific Quantitative PCR System For ID4 Gene And Its Application In Detection Of Acute Leukemia Minimal Residual Disease

Acute leukemia is caused by genetic and epigenetic mechanisms involving tumor suppressors and oncogenes. Hypermethylation in CpG islands in various tumor suppressors leads to relative gene silencing, which plays a vital role in the onset and development of acute leukemia, and hypermethylation becomes one of the characteristics of acute leukemia cells. Inhibitor of DNA binding 4 (ID4), a member of helix-loop-helix (HLH) protein family, was screened out by Professor Yu Li via the method of RLGS, and demonstrated for the first time to be a methylation-regulated tumor suppressor. Due to the technology of methylation-specific PCR (MSP), ID4 gene methylation was ulteriorly found to be highly related to the progress of acute leukemia. The study on ID4 gene methylation remains at qualitative analysis stage currently, and it has not been studied both home and abroad for the application of quantitative MSP in analyzing the relationship between acute leukemia progressing and methylation density. Setup of quantitative methylation-specific PCR (qMSP) system for ID4 gene theoretically improves the specificity and sensitivity of methylation detection assays, and might play an important role in clinical practice, such as acute leukemia minimal residual disease (MRD) detection.This study established the method of qMSP system for ID4 gene, and discussed its application in MRD detection via analysis on tumor load and methylation density in acute leukemia patients. Firstly, it was demonstrated in cell lines with MSP and qMSP: 1) hematological malignancy cell lines were hypermethylated cell lines according to ID4 gene promoter. K562 was found to be methylation-positive, but the density of methylation was lower than other four cell lines. The cell lines 293-t and 293 ID4 was completely non-methylated. 2) ID4 gene promoter in both acute lymphoblastic and myeloid leukemia was methylation-positive, indicating a broad coverage of ID4 methylation in acute leukemia subtypes. 3) results of qMSP were in accordance with those of MSP, suggesting the credibility of qMSP. Secondly, a complete qMSP system for ID4 was established, including design of ID4 methylation-specific primers and probe; choice of inner reference and controls; setup of standard curves for target gene and inner reference, and choice of appropriate quantitative assay. It was demonstrated that this qMSP method had both good specificity and high sensitivity. Finally, using the above qMSP method, we found in acute leukemia bone marrow samples: 1) Drug resistant and refractory clones and easy relapse and refractory subtypes in acute leukemia tended to have high methylation positive rate. 2) percentage of methylated reference (PMR) , an indicator of the density of methylation, seemed to be related to body tumor load, and individual variation was obvious at initial treatment. 3) among serial samples, PMR value had significant correlation with tumor load. Furthermore, PMR might predict leukemia relapse. It was also found to have similar changing trend to other genetic biomarkers for MRD. In conclusion, this study successfully established the system and method of qMSP for ID4 gene, and demonstrated wide existence of ID4 promotor methylation among various acute leukemia subtypes. This methylation-specific quantitative PCR system had high specificity and sensitivity. The quantitative indicator of methylation density, PMR value, might reflect the change of tumor load in acute leukemia patients. Different clones of leukemia cells had different PMR values, and dynamic monitoring of PMR might predict leukemia relapse and become a new MRD biomarker. This study preliminarily laid the laboratory foundation for the application of qMSP in MRD detection.