The Abnormal Methylation Of Vascular Endothelial Cell Growth Factor Receptor-2(KDR) Gene In Infancy Hemangioma

Objective: To investigate the methylation status of vascular endothelial growth factor receptor(VEGFR, KDR) gene in hemangioma, vascular malformation and normal skin tissues by establishing methylation sensitivity high resolution dissolve curve method (MS- HRM). The role of genetic methylation in proliferating and regressing hemangiomas also will be discussed.Methods: 48 different period hemangioma samples, 15 vascular abnormality samples, and 8 normal skin tissue samples were tested respectively. The DNA of samples were extracted, modificated with sulphite methylation. Then, the DNA of samples was purificated and recycled and prepared for test. Genome-wide methylation DNA (QIAGEN Company, USA) was used as 100% methylation standard sample. It was mixed with 100% of non methylated DNA of healthy pregnant women umbilical cord blood and diluted to 0%, 5%, 25%, 50%, 75%, and 100% methylation DNA solutions. After PCR cycle and MS- HRM dissolve curve detection, the standard curve of methylation DNA was drawn out. At the same time, the repeatability and sensitivity of it were evaluated. The methylation levels of KDR in all samples were tested by the methylation sensitivity high resolution dissolve curve method (MS-HRM).Results: 100%, 75%,50%, 25%, 5%, 0% methylation standard curves were made successfully, from right to left arrangement.three replicated detections were done. KDR gene methylation were detected positively in 32 hemangioma specimens(66.67%), including 21 proliferate hemangiomas samples (21/24 , 87.50 %) and 11 involuting hemangiomas(11/24,45.83%).In proliferate hemangiomas,different degree methylation were shown.1 case ranged from 0 % to 5%;11 cases ranged from 25% to 50%;5 cases were 75%;4 cases were 100%. In involuting hemangiomas,the methylation degree also was different.1 case was 75%;8 cases ranged from 25% to 50%;2 cases ranged from 0% to 5%.There was significant difference between proliferate hemangiomas and involuting hemangiomas in KDR methylation(χ2 = 8.889, P < 0.05).The methylation positive rate of vascular malformation samples was 13.33%(2/15,ranged from 0% to 25%). The methylation positive rate of normal wrapping skin tissues was 12.50 %( 1/8, ranged from 0% to 5%). The methylation positive rate in hemangioma was significant higher than that in vascular malformation or normal wrapping skin tissue (P < 0.05).Conclutions:①? Abnormal methylation of KDR expressed in hemangioma which may be associated with hemangioma formation, hyperplasia and degradation;②MS-HRM is a method to detect KDR methylation in hemangioma with simple operation, high sensitivity, low cost, strong repeatability, and established a detection method for further research the abnormal methylation of gene in hemangiomas .