Exploratory locomotion, a predictor of addiction vulnerability, is oligogenic in rats selected for this phenotype Zhifeng Zhoua,1, Peter Blandinob,c,1, Qiaoping Yuana, Pei-Hong Shena, Colin A. Hodgkinsona, Matti Virkkunend, Stanley J. Watsonb,c, Huda Akilb,c,2, and David Goldmana,2 aLaboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20852; bMolecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109; cDepartment of Psychiatry, University of Michigan, Ann Arbor, MI 48109; and dDepartment of Psychiatry, University of Helsinki, 00014 Helsinki, Finland Contributed by Huda Akil, May 10, 2019 (sent for review November 30, 2018; reviewed by Elissa J. Chesler and Robert Hitzemann) Artificially selected model organisms can reveal hidden features of resilience, the search for genetic components that influence re- the genetic architecture of the complex disorders that they model. sponse to novelty and vulnerability to addiction has proven Addictions are disease phenotypes caused by different intermedi- challenging, with only a fraction of the genetic variance in ad- ate phenotypes and pathways and thereby are potentially highly diction liability accounted for by genome-wide association stud- polygenic. High responder (bHR) and low responder (bLR) rat lines ies (GWAS). This may be due to multiple confounding factors have been selectively bred (b) for exploratory locomotion (EL), a including genetic heterogeneity, small effect sizes of each gene/ behavioral phenotype correlated with novelty-seeking, impulsive locus, and phenotypic heterogeneity resulting from variable response to reward, and vulnerability to addiction, and is inversely causes for use emergent at different stages of addiction and correlated with spontaneous anxiety and depression-like behav- normal development. The failure to identify loci via GWAS has iors. The rapid response to selection indicates loci of large effect been interpreted to mean that behavioral traits are polygenic and for EL. Using exome sequencing of HR and LR rats, we identified has led to speculation that behavioral diseases are omnigenic and alleles in gene-coding regions that segregate between the two caused by alleles of infinitesimal effect (5). As well as implicating lines. Quantitative trait locus (QTL) analysis in F2 rats derived from novel genes, addiction GWAS have succeeded in recovering NEUROSCIENCE a bHR × bLR intercross confirmed that these regions harbored several genes previously implicated by study of genes and path- genes affecting EL. The combined effects of the seven genome- ways known to be important in addiction, for example ADH1B wide significant QTLs accounted for approximately one-third of and ALDH2 for alcoholism and CHRNA3 for nicotine addiction the total variance in EL, and two-thirds of the variance attributable (6–8). Cellular and molecular studies of addictions, including to genetic factors, consistent with an oligogenic architecture of EL studies of the transcriptome, have identified diverse changes estimated both from the phenotypic distribution of F2 animals and rapid response to selection. Genetic association in humans linked Significance APBA2, the ortholog of the gene at the center of the strongest QTL, with substance use disorders and related behavioral pheno- While addiction disorders have a genetic basis, the expected types. Our finding is also convergent with molecular and animal number of genes contributing to them is large, hence their behavioral studies implicating Apba2 in locomotion. These results classification as polygenic disorders. Animal models can enable provide multilevel evidence for genes/loci influencing EL. They discovery of genes of larger effect by targeting specific com- shed light on the genetic architecture of oligogenicity in animals ponents of genetic vulnerability. High responder and low re- artificially selected for a phenotype modeling a more complex sponder rats were selectively bred based on exploratory disorder in humans. locomotion (EL) in a novel environment, a predictor of drug- seeking behavior. Using exome sequencing and quantitative addiction | genetics | locomotion | novelty seeking | oligogenic trait locus analysis, we identified seven genome-wide signifi- cant loci accounting for approximately one-third of total vari- ddictions are common, etiologically complex disorders that ance and two-thirds of genetic variance in EL. We found Alead to diverse morbid outcomes and early mortality. En- convergent evidence for a role of APBA2 in humans. Thus, EL is vironmental and genetic factors play important roles in vulner- oligogenic, being strongly influenced by a limited number of ability and resilience to addiction, and the unfolding of the loci of large effect size. addiction process (1, 2). Although addictive disorders, defined in the Diagnostic and Statistical Manual of Mental Disorders, 5th Author contributions: S.J.W., H.A., and D.G. designed research; Z.Z. and P.B. performed Edition (DSM5) as use disorders, are contingent on availability research; P.B., C.A.H., S.J.W., H.A., and D.G. contributed new reagents/analytic tools; Z.Z., and use of an addictive agent, people are differentially vulner- P.B., Q.Y., P.-H.S., M.V., and D.G. analyzed data; and Z.Z., C.A.H., H.A., and D.G. wrote the paper. able at multiple stages of the cycle of addiction, including the Reviewers: E.J.C., The Jackson Laboratory; and R.H., Oregon Health & Science University initial propensity to use, transition from use to abuse, long-term and Portland VA Medical Center. addiction, and relapse (3). Genetic variation influences risks, The authors declare no conflict of interest. responses, and trajectories of addiction. Response to novelty and × This open access article is distributed under Creative Commons Attribution-NonCommercial- novelty seeking play a particularly important role in gene en- NoDerivatives License 4.0 (CC BY-NC-ND). vironment correlation and interaction, because exposure and use Data deposition: Sequences of the bHR/bLR exomes have been deposited in NCBI as a set the stage for addiction. Bioproject (accession no. PRJNA521139). Measured as extraversion, novelty seeking and response to 1Z.Z. and P.B. contributed equally to this work. 2 = novel environments is moderately heritable (h 0.4) in people. 2To whom correspondence may be addressed. Email: [email protected] or David.Goldman@ Addictions themselves are also moderately to highly heritable, as nih.hhs.gov. observed in large samples of twins representative of populations This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (4). However, despite the discovery of causal variants at several 1073/pnas.1820410116/-/DCSupplemental. genes involved in drug metabolism, receptor/signaling and stress Published online June 10, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1820410116 PNAS | June 25, 2019 | vol. 116 | no. 26 | 13107–13115 Downloaded by guest on September 26, 2021 associated with neural adaptive processes (9–11). Perturbations Results of molecular components critical for neurotransmission such as Exome Sequencing Identifies Genetic Variants Segregating between glutamatergic, GABAergic, and dopaminergic systems have been bHR and bLR Rats. Broad sense heritability (H2) of EL was 0.489, recognized (12–15). However, the main successes in the discov- calculated based on variance of this phenotype in bHR and bLR ery of processes involved in addiction have been in under- F0, F1, and F2 offspring (SI Appendix, Fig. S1). Therefore, EL is standing intermediate mechanisms and tertiary consequences, substantially heritable and the bHR and bLR rats are potentially rather than primary causes. a model for identification of loci influencing novelty response. By Animal models enable well-controlled designs to minimize applying Wright’s estimator (23, 24) to the means and variances confounding factors. Animals selectively bred for targeted phe- of locomotor scores in bHR and bLR and variance in F2 off- notypes are of particular value for the identification of alleles spring, we calculated that the minimum number of loci that may naturally occurring in those species (16, 17) because selection contribute to total variance in EL is 15.7 (SI Appendix, Fig. S1). enriches and even genetically fixes alleles that influence the EL in bHR/LR might therefore be regarded as oligogenic, but phenotype. In such models, genetic, phenotypic, and environ- not omnigenic, a hypothesis potentially resolvable by discovery of mental complexity can be reduced, enabling detection of loci loci and measurement of their individual and joint contributions. with relatively large effect sizes. For example, two stop codons We performed exome sequencing in 12 bHR and 12 bLR F0 influencing alcohol preference together account for 6% of the animals that were the product of 18 generations of selection and total variance for this phenotype and were genetically fixed by 37 generations of breeding, identifying genetic variants in the selection in alcohol-preferring (P) and nonpreferring (NP) rats gene-coding regions that segregate between bHR and bLR lines (18). Fixation of the Grm2 stop codon, which is either common (25). The exome sequencing generated adequate coverage (av- or genetically fixed in outbred Wistar rats from different sources, erage 86×, across the 50-Mb exome target) and identified a total leads to uncompensated loss of metabotropic glutamate receptor of 110,172 single nucleotide variants