title: Combined Whole Methylome and Genomewide Association Study Implicates CNTN4 in Alcohol Use.
abstract: BACKGROUND
  Methylome-wide association (MWAS) studies present a new way to advance the search for biological correlates for alcohol use. A challenge with methylation studies of alcohol involves the causal direction of significant methylation-alcohol associations. One way to address this issue is to combine MWAS data with genomewide association study (GWAS) data.
  
  
  METHODS
  Here, we combined MWAS and GWAS results for alcohol use from 619 individuals. Our MWAS data were generated by next-generation sequencing of the methylated genomic DNA fraction, producing over 60 million reads per subject to interrogate methylation levels at ~27 million autosomal CpG sites in the human genome. Our GWAS included 5,571,786 single nucleotide polymorphisms (SNPs) imputed with 1000 Genomes.
  
  
  RESULTS
  When combining the MWAS and GWAS data, our top finding was a region in an intron of CNTN4 (p = 2.55 × 10(-8) ), located between chr3: 2,555,403 and 2,555,524, encompassing SNPs rs1382874 and rs1382875. This finding was then replicated in an independent sample of 730 individuals. We used bisulfite pyrosequencing to measure methylation and found significant association with regular alcohol use in the same direction as the MWAS (p = 0.021). Rs1382874 and rs1382875 were genotyped and found to be associated in the same direction as the GWAS (p = 0.008 and p = 0.009). After integrating the MWAS and GWAS findings from the replication sample, we replicated our combined analysis finding (p = 0.0017) in CNTN4.
  
  
  CONCLUSIONS
  Through combining methylation and SNP data, we have identified CNTN4 as a risk factor for regular alcohol use.
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Shaunna L
authors.initials: SL
authors.lastname: Clark
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Karolina A
authors.initials: KA
authors.lastname: Aberg
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Srilaxmi
authors.initials: S
authors.lastname: Nerella
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Kumar
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Joseph L
authors.initials: JL
authors.lastname: McClay
authors.affiliation: Department of Biostatistics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Wenan
authors.initials: W
authors.lastname: Chen
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Linying Y
authors.initials: LY
authors.lastname: Xie
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Aki
authors.initials: A
authors.lastname: Harada
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Shabalin
authors.affiliation: Department of Biostatistics, School of Medicine, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Guimin
authors.initials: G
authors.lastname: Gao
authors.affiliation: Psychiatric and Neurodevelopmental Genetics Unit, Massachusetts General Hospital, Boston, Massachusetts.|Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, Massachusetts.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Bergen
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Hultman
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Patrik K E
authors.initials: PK
authors.lastname: Magnusson
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.|Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Sullivan
authors.affiliation: Center for Biomarker Research and Precision Medicine, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia.
authors.forename: Edwin J C G
authors.initials: EJ
authors.lastname: van den Oord
mesh_terms: D000328:Adult; D000368:Aged; D000428:Alcohol Drinking / Q000235:genetics*; D058966:Contactins / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D055106:Genome-Wide Association Study / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D008297:Male; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
chemical_list: C098304:CNTN4 protein, human; D058966:Contactins
keywords: CNTN4; Genomewide Association Study; Methylation; Next-Generation Sequencing