Assessing Alcohol Consumption Effect On Epithelial Ovarian Cancer Mediated By DNA Methylation Based On A High-dimensional Causal Inference Test

Objective:As the country's fifth most common cause of cancer mortality in women,epithelial ovarian cancer(EOC)is the leading cause of gynecologic cancer death in the United States.The enormous physical,societal,and economic burdens associated with EOC along with the current lack of success in the early diagnosis of this disease,underscore the urgent need of studies aimed toward understanding the molecular basis of EOC susceptibility.Some studies identified that wine consumption is in fact associated with a reduced risk of EOC.Increasing evidence shows that alcohol consumption can induce epigenetic changes,For example,recent alcohol use is associated with widespread changes in DNA methylation in women.DNA methylation alterations are an early step in EOC carcinogenesis and could represent a mechanism of disease.Given the potential role of alcohol in inducing methylation changes associated with EOC,it is clinically important to identify meaningful methylation biomarkers and genes for effective prevention and treatment.We aimed to identify DNA methylation markers acting as potential mediators for alcohol-associated EOC risk.Methods:We implemented a high-dimensional causal inference test and the VanderWeele mediation model to examine CpG sites that mediate the association between alcohol consumption and EOC risk.We modified step 2 in causal inference test(CIT)by adopting de-sparsifying LASSO high-dimensional inference procedure,the modified CIT procedure by incorporating the high-dimensional testing step can be adopted in high-dimensional data generated in epigenetics.The data were based on 196 cases and 202 controls from the Mayo Clinic Ovarian Cancer Case-Control Study.DNA methylation data were measured on 396 study participants' leukocyte-derived DNA.Results:(1)In CIT,step 1 results show that alcohol use is associated with EOC status,OR=0.336,95% CI[0.169,0.651],P=0.001.Step 2 results show that 3,055 CpGs are associated with EOC status conditional on alcohol use.Step 3 results indicat that 61 CpGs are associated with alcohol use conditional on EOC status.Step 4 results show that EOC status is independent of alcohol consumption conditional on 2 CpGs.In summary,implementation of the CIT test revealed 2 CpG sites(cg09358725,cg11016563),which represent potential methylation-mediated relationship between alcohol consumption and EOC risk.(2)Implementation of the VanderWeele mediation model further revealed that these 2 CpGs were the key mediators.Alcohol consumption can lead to changes of 2 CpGs methylation levels,which consequently influence the EOC status.(3)Cg11016563 is located in gene TRPC6.Overexpression of TRPC6 was reported in ovarian cancer cells.The overexpression of this gene might be due to the low methylation level in CpG island region located within TRPC6 gene promoter region.(4)Cg09358725 is located in gene LMO2,which may be associated with EOC status.Conclusions:(1)Our analysis identified 2 CpG sites,which represent potential methylation-mediated relationship between alcohol consumption and EOC risk for the first time.These findings suggest two CpGs may serve as novel biomarkers for EOC susceptibility.(2)The overexpression of gene TRPC6 in ovarian cancer cells might be due to the low methylation level in TRPC6.Gene LMO2,which may be associated with EOC status.(3)Our research highlighted the importance of the causal inference procedure in analyzing the potential epigenetic markers-mediated relationship between environmental factors and disease risk.(4)Our analyses suggested that the modified CIT procedure by incorporating the high-dimensional testing step can be adopted in high-dimensional data generated in epigenetics to improve causal inference analysis.