Testing genetic associations with addiction phenotypes using moderate-depth whole genome sequencing. S. I. Vrieze1, S. Feng1, X. Zhan1, M. B. Miller2, G. Jun1, M. K. Trost1, A. Tan1, J. Bragg-Gresham1, M. Flickinger1, L. Scott1, A. Locke1, H. M. Kang1, S. Levy3, R. M. Myers3, M. Boehnke1, W. G. Iacono2, M. McGue2, G. R. Abecasis1 1) Biostatistics, University of Michigan, Ann Arbor, MI; 2) Psychology, University of Minnesota, Minneapolis, MN; 3) HudsonAlpha Institute for Biotechnology, Huntsville, AL.

   We report results from genetic association analyses of smoking, alcohol, and drug use and dependence behavior in 1800 individuals of European ancestry with moderate-depth whole genome sequencing from the Minnesota Center for Twin and Family Research. We developed and validated a novel selection method to prioritize pedigrees likely to carry rare disease-associated alleles. The method was applied to the full sample of 7188 individuals from 2400 nuclear family pedigrees, delivering a selected sample of 1480 individuals from 596 pedigrees. To date, DNA samples from 1343 individuals have been sequenced to 10x mean depth, which is estimated to provide >80% power to detect singletons. SNP calling and LD-based genotype refinement is complete on a preliminary data freeze of 686 samples. After quality control filtering, we have identified 21,190,919 autosomal SNPs, including 9,439,824 singletons and 4,117,829 doubletons. Fully 408,879 SNPs were predicted to be nonsynonymous and 18,158 putative loss of function. We evaluated more closely 172 candidate addiction regions tagged by previously-identified loci, which contain 8,090 nonsynonymous and 238 putative loss of function variants. These genes include 11 genes with known nicotine- and alcohol-relevant loci, as well as 161 other candidate addiction genes related to nicotinic receptors, nicotine metabolism, alcohol metabolism, and neurotransmitter systems known to be involved in addictive behaviors. Single nucleotide, CNV, and indel calls on the full sequenced sample will be presented, and will provide detail about the extent of deleterious variation across the genome and within key addiction genes. To test for association we conduct single variant and burden tests across the genome, and present in finer detail results for promising candidate addiction genes. Burden tests include a variable threshold burden test and the sequence kernel association test, separately for nonsynonymous variants and loss of function variants. These experiments will provide a comprehensive evaluation of genetic association in substance use, abuse, and dependence phenotypes, and the family-based design will allow additional traction in the study of very rare variation.

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