Gene methylation is associated with blood lipid profile in ischemic stroke Oksana Pogoryelova 1, Paul Haggarty 1, Carl Counsell 2, Mary Joan Macleod 2 1 Rowett Institute of Nutrition & Health, University of Aberdeen, Aberdeen, UK, 2 University of Aberdeen, Division of Applied Medicine, Aberdeen, UK. Gene methylation is associated with blood lipid profile in ischemic stroke Oksana Pogoryelova 1, Paul Haggarty 1, Carl Counsell 2, Mary Joan Macleod 2 1 Rowett Institute of Nutrition & Health, University of Aberdeen, Aberdeen, UK, 2 University of Aberdeen, Division of Applied Medicine, Aberdeen, UK. Epigenotype is an important link between genotype and phenotype influenced by environmental factors. We demonstrated that PITX2 and LINE1 methylation was association with lipid profiles in LVD (positive association); INS and LINE1 methylation was associated with lipids in SVD strokes (negative association). This epigenotype/phenotype interaction contribute to our understanding of molecular mechanisms underlying pathological mechanisms involved in interindividual blood lipid variation in male stroke patients. Background Results Methods Conclusions Epigenetic mechanisms, such as DNA methylation, regulate tissue specific gene expression. Environmental and life style influences can lead to stable changes in the epigenome that alter the individual susceptibility to a disease or condition. Epigenetic dysregulation has been implicated in a number of conditions including stroke predisposing states: atherosclerosis and dyslipidaemia. Based on the recent findings in epigenetics, we hypothesised that DNA methylation is associated inter individual variability of blood lipid profile in patients with ischemic stroke. Stroke male patients and age matched male controls were tested for associations between DNA methylation in selected genes and blood lipid levels. Stroke patients were classified according to TOAST classification. We included large vessel disease (LVD n=46, mean age= 67.0, SD=9.4), small vessel disease (SVD n=46 mean age=66.5, SD=9.3) and cardioembolic (CE n=46, mean age= 71.3, SD=13.8) strokes and controls (n=60, mean age 70.9, SD=7.8) in the study. Lipid profiles for each group described in Table 1. Table 1. Mean values with standard deviation of blood lipids and lipoprotein cholesterol by stroke subgroup and control group. DNA methylation status was measured in 10 selected genes: SOD3, ALOX15, LINE1, ER-alpha, MTHFR, IL10, TNF-alpha and IFNGR1 - these genes are implicated in aging, atherosclerosis and inflammation processes; PITX2 is involved in synoatrial node development, INS- insulin coding gene. Methylation was determined by pyrosequencing using a PyroMark MD system (Qiagen, Crawley, UK) after bisulphite conversion of lymphocyte DNA using Epitect Bisulphite kits (Qiagen, Crawley, UK) (Figure 1). The control group and CE stroke subgroup showed no significant correlation between gene methylation and blood lipid profile. In LVD group PITX2 methylation showed a negative correlation with total cholesterol (TC) level (Figure A). LINE1 methylation was associated with TC (Figure B) and LDL blood levels (Figure C) (Table 2.) In SVD group we found two positive correlations: HDL was associated with INS methylation and LINE1 (Figure D) with TC/HDL (Figure E) ratio (Table 2.). Figure 1. Pyrogram of SOD3 with 7 CpG sites. Each methylation site within the region of interest was analysed individually. The mean methylation level across all sites within a gene was used in the regression analysis. Cohort TC mean (SD) LDL mean (SD) HDL mean (SD) TC/HDL ratio mean (SD) TG mean (SD) LVD4.8 (1.1)2.8 (0.9)1.1 (0.3)4.1 (1.1)1.9 (0.9) SVD5.0 (1.2)3.0 (1.0)1.2 (0.3)4.1 (1.1)1.7 (0.9) CE4.5 (1.2)2.8 (1.1)1.1 (0.2)4.3 (1.2)1.5 (0.6) Control5.0 (1.1)2.9 (1.0)1.5 (0.4)3.5 (1.0)1.4 (0.6) CohortGene-FactorCoefficient (95%CI)p-valueR2R2 LVD LINE1-TC (-1.073;-0.265) LINE1- LDL-0.842(-1.346;-0.338) PITX2-TC (-1.039;-0.006) SVD LINE1-TC/HDL0.461 (-0.008;0.931) INS-HDL7.9 (2.400;13.400) Table 2 Statistical analysis of gene methylation and blood lipid levels. AB C D E A - PITX2 methylation plotted against TC levels in LVD cohort B- LINE1 methylation plotted against TC levels in LVD cohort C -LINE1 methylation plotted against LDL levels in LVD cohort D- LINE1 methylation plotted against TC/HDL ratio in SVD cohort E- INS methylation plotted against HDL levels in SVD cohort