Association Of CDKN2A/2B Methylation With Artery Calcification In Patients With Ischemic Stroke

Part ? Association of CDKN2A/2B methylation with carotid artery calcification Objectives:Although cyclin-dependent kinase inhibitor 2A/2B(CDKN2A/2B)have been associated with both atherosclerosis and artery calcification,the underlying mechanisms remained largely unknown.Considering that the expressions of CDKN2A/2B could be regulated by DNA methylation,this study aimed to evaluate whether carotid artery calcification(CarAC)is related to methylation levels of CDKN2A/2B in patients with ischemic stroke.Methods:Consecutive patients with ischemic stroke were screened from Nanjing Stroke Registry Program between July 2012 and September 2013.Data on demographic and cardiovascular risk factors were collected.CarAC was quantified with Agatston score and calcium volume based on results of computed tomography angiography.DNA were isolated from the peripheral blood samples of 322 ischemic stroke patients.Methylation levels of 36 CpG sites around promoter regions of CDKN2A/2B(24 from CDKN2A,12 from CDKN2B)were examined with BiSulfte Amplicon Sequencing.Baseline characteristics and the methylation levels were compared between patients with and without CarAC.Generalized liner model was performed to explore the association between methylation levels and CarAC.Results:Of the 322 analyzed patients,there were 187(58.1%)patients classified as with and 135(41.9%)as without evident CarAC.CarAC scores presented an extremely left-skewed distribution with a median(interquartile range)of 9.0(0-111.1).The median calcium volume(mm3)was 11.0(0-98.0).Compared with patients without CarAC,those with CarAC were older[66.0(58.0-73.0)vs 57.0(47.0-64.0)years,P<0.001],and had higher prevalences of hypertension(82.9%vs 70.4%,P =0.010)and diabetes mellitus(39.6%vs 26.7%,P = 0.017).The average methylation levels of CDKN2B were higher in patents with CarAC than those without[5.7%(5.0%-6.4%)vs 5.4%(4.7%-5.9%),P = 0.001].After adjustment for potential confounders,methylation levels of CDKN2B were positively correlated with In-transformed calcification scores(P = 0.271 ± 0.120,P = 0.024,Bonferroni corrected P=0.048)in generalized liner model A positive correlation was also detected between average methylation levels of CDKN2B and cube root transformed calcium volumes(p = 0.437 ± 0.140,P = 0.002,Bonferroni corrected P = 0.004).No siginificant association was observed between methylation levels of CDKN2Aand tansformed calcification scores(P = 0.834)or calcium volumes(P = 0.910).Conclusions:Methylation levels of CDKN2B were positively correlated with the severity of CarAC.DNA methylation of CDKN2B may play a potential role in artery calcification.Part ? Association of CDKN2A/2B methylation with aortic arch calcification Objectives:CDKN2A/2B have been proposed as a potential genetic etiology for both atherosclerosis and arterial calcification.DNA methylation,which can influence the expressions of CDKN2A/2B,may be an underlying mechanism for arterial calcification.This study aimed to evaluate whether CDKN2A/2B methylation is associated with aortic arch calcification(AAC)in ischemic stroke patients.Methods:Consecutive patients with ischemic stroke aged 18 years or older were screened from Narjing Stoke Registry Program(NSRP)between July 2012 and September 2013.DNA was isolated from venous blood samples in 322 patients with ischemic stroke.Methylation levels of 36 CpG sites around promoter regions of CDKN2A/2B were examined.AAC was quantified with Agatston score and calcium volume based on results of computed tomography angiography.Generalized liner model was performed to explore the association between methylation levels and AAC.Results:There were 248(77.0%)patients with and 74(23.0%)patients without AAC.AAC scores presented an extremely left-skewed distribution with a median(interquartile range)of 221.5(3.8-803.7).The median calcium volume(mm3)was 195.6(6.8-632.2).Compared with patients without AAC,patients with AAC were older[64.0(58.0-72.0)vs 49.0(43.8-58.3)years,P<0.001],had higher prevalence of hypertension(82.9%vs 70.4%,P = 0.010),and had higher methylation levels of CDKN2B[5.7%(5.0%-6.3%)vs 4.9%(4.5%-5.5%),P<0.001].Using a generalized linear model,positive correlation between methylation levels and In-transformed calcification scores was detected at CDKN2B(P = 0.275 ± 0.116,P = 0.018,Bonferroni corrected P=0.036).And methylation levels of CDKN2B were also positively correlated with cube root transformed calcium volumes((3 = 0.544 ± 0.180,P = 0.003,Bonferroni corrected P = 0.006).No siginificant association was observed between methylation levels of CDKN2A and tansformed calcification scores(P ?0.961)or calcium volumes(P = 0.628).Conclusions:Patients with higher levels of DNA methylation of CDKN2B may bear an increased risk for AAC.Further studies to reveal the underlying mechanisms of this association are warranted for establishing a cause-effect relationship.