The Research Of DAPK Gene Aberrant Methylation In Multiple Myeloma

[Objective] The purpose of this experiment is to study methylation state of the area of the DAPK gene promoter and its mRNA expression in the patients with multiple myeloma (multiple myeloma, MM), and to discuss their interaction and relation in occurrence and development of MM. It provides certain molecular biology basis for generally understanding the pathogenesis of MM, and provides corresponding laboratory data for the further diagnosis and targeted therapy to MM.[Methods] There were54cases bone marrow samples of multiple myeloma patients which collected from the Second Affiliated Hospital and the First Affiliated Hospital of Kunming Medical University from January2009to June2011, Among these Samples,35cases are male and19cases are female, the age from38to79years old and median age is64years;27cases of newly diagnosed patients,27cases of referral patients. In Druie-Salmon stage,2patients were in group A of stage Ⅰ,16patients were in group A of stage Ⅱ,4patients were in group B of stage Ⅱ,19patients were in group A of stage Ⅲ, and13patients were in group B of stage Ⅲ; According to classification of abnormal globulins, the number of patients with the type of IgG is26, the number of patients with the type of IgA is14, the number of patients with the type of non-secretion is9, and the number of patients with the type of light chain is5. The control group are made up of40cases of bone marrow specimens of patients with non-hematologic malignancies. Among these samples,15cases are male and25cases are female, the age from19to68years old and median age is42years; In these patients, the number of patients with iron deficiency anemia is28, the number of patients with megaloblastic anemia is2, the number of patients with anaphylactioid purpura is1, and the number of patients with idiopathic thrombocytopenic purpura is9. The first step is to study methylation of the area of DAPK gene promoter in bone marrow of patients with MM by methylation-specific polymerase chain reaction, and to sequence and analysis cytosine methylation of amplification products by automatic sequencing technology. The second step is to detect the expression of mRNA in DAPK gene by real-time quantitative polymerase chain reaction. The results are analyzed in statistics by software package of SPSS11.5.[Result]1.The results of methylation of the area of DAPK gene promoter in patients with MM show that:①In54patients of MM,10cases were detected methylation of the area of DAPK gene promoter, and the rate of methylation positive was18.52%; In40cases of control group, no case was detected methylation of the area of DAPK gene promoter, and the rate of methylation positive was0.00%; The difference was statistically significant between the group of patients with MM and the control group(P<0.05).②The rate of methylation positive in the area of DAPK gene promoter of patients whose abnormal globulins with the type of IgG, IgA, non-secretion and light chain were respectively30.77%,7.14%,20.00%and0.00%.The difference was statistically significant between the patients with MM of the type of IgG, and the control group(P<0.0056).Compared with the patients of control group, there were no statistically significant difference in patients with MM of the type of IgA and light chain(P>0.0056). In patients with MM of the type of non-secretion, there were not methylation of the area of DAPK gene promoter detected; There were no statistically significant difference in compare in pairs of the type of IgG, IgA, non-secretion and light chain in patients with MM.③There were no statistically significant difference between the rate of methylation positive of the area of DAPK gene promoter in patients of those groups which were the compare of male patients and female, the compare of patients aged≥65years and aged<65years, the compare of patients with Ⅰ+Ⅱ stage and those with Ⅲ stage, the compare of groups A and B, and the compare of groups of the diagnosis group and referral group (P>0.05).2. The products of methylation-specific polymerase chain reaction was sequenced by using automated sequencing technology, the results showed that: Among the area of DAPK gene promoter region has10places change C to T, which confirmed the existence of methylation.3. DAPK gene mRNA expression in patients of multiple myeloma:①There were no statistically significant difference that the expression of patients with MM below control group's(1.09±1.35Fold vs1.26±1.38Fold);②The expression of methylation below non-methylation's in patients with MM(0.76±1.09Fold vs1.18±1.28Fold), compared with control group, there were no statistically significant difference between the group of methylation and the unmethylation's(P>0.0167);③The expression of first diagnosis group below the return visit group'in patients with MM(0.98±1.19Fold vs1.17±1.72Fold), there were no statistically significant difference among first diagnosis group, the return visit group and control group (P>0.0167).[Conclusion]1. The rate of methylation positive of the area of DAPK gene promoter in patients with MM was significantly higher than the control group's, so patients with the type of IgG was, and the above pointed out that methylation of the area of DAPK gene promoter had a certain effect in the pathogenesis of MM.2. The rate of methylation positive of the area of DAPK gene promoter was irrelevant with staging, grouping and treatment or not of patients with MM.3. The expression of mRNA of DAPK gene in patients with MM below the control group's. On the expression of mRNA of DAPK gene, the group of methylation below the groups of unmethylation and control group. There were the same rate of methylation positive of the area of DAPK gene promoter between the group of newly diagnosed patients and the return visit patients', and the expression of mRNA of DAPK gene in the group of newly diagnosed patients below the return visit patients' and the control group's, but there were no statistically significant difference among these and there would need to expand the sample size to further study.