Feasibility And Efficacy Of Blood Promoter Methylation Of Sox10 Gene In Diagnosis Of Intestinal Neuronal Dysplasia

Objective To explore the efficacy of blood promoter methylation of Sox10 gene in diagnosis of intestinal neuronal dysplasia(IND)and to seek a non-invasive genetic diagnosis method based on peripheral blood for diagnosis of IND.Methods The blood were collected from 24 enteric nervous system(ENS)anomalies(15 Hirschsprung disease(HD)and 9 IND)and 15 controls(the colon trauma cases).In addition,colon specimens were taken from each group.The IND group was based on barium enema(BE),anorectal manometry(ARM)and histopathology(including immunology).The blood promoter methylation of the Sox10 gene were detected by Bisulfite Genomic Sequence(BSP)technology and its expression level in colon by immunohistochemical staining,qRT-PCR and western blot and the correlation between them was analyzed.The diagnostic efficacy of Sox10 methylation level in peripheral blood was evaluated by receiver operating characteristics(ROC)curve.Results In the results of methylation sequencing,neither the control group,HD group nor IND group,we found the blood promoter methylation level was positively correlated with that of colon tissues(r=0.74).The blood promoter methylation level at the 32 th locus of Sox10 was 100%(90%-100%;95%CI:91%-98%)in the control,80%(70%-90%;95%CI:65%-90%)in HD and 60%(50%-80%;95%CI:52%-82%)in IND.ROC curve indicated area under curve(AUC)of Sox10 methylation in diagnosis of Hirschsprung disease was 0.818,with a cut-off value of 85% and low diagnostic sensitivity,and AUC of Sox10 methylation in IND was 0.907,with a cut-off value of 85%.The diagnostic sensitivity and specificity were 88.9% and 93.3% respectively.Immunohistochemical staining showed that Sox10 was mainly expressed in glial cells and a small amount in intestinal neurons in the colon of the control group.QRT-PCR and western blot showed that the expression of Sox10 protein in the colon of the control group,the HD spasmodic segment and the IND lesion group increased,and the difference was statistically significant(P<0.05),and was positively correlated with the number of Sox10 positive cells.The blood promoter methylation level was negatively correlated with its expression level in colon(qRT-PCR and western blot)(r=-0.88).Conclusion The promoter methylation level of the Sox10 gene in peripheral blood might be used as a non-invasive genetic diagnosis method for diagnosis of IND,which provides a new diagnostic idea and reference indicator for the clinical diagnosis of IND.