A Study On The Role Of DRD5, MNX1Aberrant Methylation In Diagnosis Of Type2Diabetic Macrovascular Complications

Objective To study the role of methylation of gene Dopamine receptor D5(DRD5) and Motor neuron and pancreas homeobox1(MNX1) in the pathogenesis oftype2diabetic macrovascular complications and to explore the feasibility of applyingit to diagnosis of these complications.Methods Genome-wide methylation analysis using methylation microarraystechnique was done9patients, randomly3from groups of diabetes with nocomplication (DNC)，diabetes with coronary heart disease (DC) and diabetes withstroke (DS). From all the significant differential methylation genes, DRD5andMNX1, one is a calcium signaling pathway related gene and the other a pancreasdevelopment related gene, were selected.Eight normal control subjects (NC) and24type2diabetes patients, age30-68years old,19male and13female; who werecomposed of three groups:8patients with no complication,8with diabetic coronaryheart disease and8with diabetic stroke, were recruited in this study. For verificationfor methylation level in all the candidates in this study using technique of MSP,RT-PCR and ELISA.Results About gene DRD5: by methylation microarrays, hypomethylation ofDRD5was showed in DNC patients; and part hypermethylation in DC patients; but inDS, hypermethylation of DRD5was seen in all the three patients. MSP results displayed hypomethylation of DRD5in both DNC patients and NC subjects andhypermethylation in both DC and DS patients. RT-PCR displayed that the expressionof mRNA of gene DRD5was low in DS patients; and relatively，the expression ofmRNA of DRD5was higher in both DNC patients and NC subjects (P<0.001,P=0.03,respectively), but lower in DC patient(P<0.001). Using ELISA, low level ofDRD5protein expression was detected in the plasma of DS patients，and bycomparison，the protein level was higher in both DNC patients and NC subjects(P=0.002, P=0.017respectively), but lower in DC patients (P=0.011).About gene MNX1: results through both MSP and methylation microarraysshowed that MNX1was hypermethylated in DC patients，and hypomethylated in DSpatients; hypomethylation of MNX1was also seen in NC subjects by MSP. RT-PCRdisplayed that the expression of mRNA of gene MNX1was high in DS patients; andrelatively，the expression of mRNA of MNX1was lower in both DC,DNC patientsand NC subjects (P=0.001, P<0.001, P<0.001respectively). Using ELISA, high levelof MNX1protein expression was detected in the plasma of DS patients，and bycomparison，the protein level was lower in both DC, DNC patients and NC subjects(P=0.029, P=0.002, P=0.001respectively).Conclusion Patients with different type2diabetic macrovascular complicationshow significant methylation level of promoter in gene DRD5and MNX1. Themethylation level of gene promoter regulates the expression of its mRNA. In diabeticwith stroke and coronary heart disease，DRD5is on hypermethylation state. Its lowexpression is associated with diabetic with stroke and coronary heart disease. Indiabetic with stroke，MNX1is on hypomethylation state. Its abnormal high expressionis associated with diabetic with stroke.