Neurogenetics of Stress Response and Reward Sensitivity One system of relevance to the addictions via its effects on anxiety and reward is the oxytocin system. We have recently also been looking for variation at the Oxytocin Receptor gene (OXTR) across species. We screened the regulatory region, first exon and first intron of the rhOXTR gene and identified 17 SNPs. Haplotypes for rhesus macaque were clustered hierarchically using R (www.r-project.org), demonstrating the existence of alternative (yin-yang) haplotpe clades and recombination between markers 7 and 8, potentially suggesting selective pressure for these markers. Using web-based transcription factor binding site (TFBS) prediction algorithms (TESS, U Penn), we found that a number of SNPs within this region resulted in gain-of and loss-of consensus TFBS. Among the SNPs identified at rhOXTR was one conferring an amino acid change in the ligand-binding domain of the receptor. The Ala6Ser SNP appears to confer increased affinity of the receptor for OT, as infant macaques carrying the 6Ser allele exhibit increased behavioral responses to exogenous oxytocin. When given access to ethanol, the peer-reared animals that were carriers of the 6Ser allele exhibited higher levels of self-administration than other groups of study. Since one would expect that 6Ser allele carriers would be more responsive to endogenous oxytocin and, therefore, less anxious than animals with the Ala/Ala genotype, this suggests that the mechanism by which rhOXTR mediates increased alcohol consumption among PR macaques is at least, in part, through effects on reward. We recently sequenced the exomes of rhesus macaque subjects that were selected based on variation in temperament. The major advantage of using Whole Exome Sequencing (WES) is that DNA extracted from whole blood can be used to enrich for putatively functional genetic variation. WES allows for the capture of large insertion/deletions and SNPs, and is best for either non-synonomous or frameshift mutations. Drawbacks to WES include the inability to interrogate sequence sites that are not currently recognized as genes, control/regulatory sequences, structural variants such as translocations and inversions, splice-site variation, and exonic repeats. However, due to exome enrichment and the resulting deep sequencing coverage, the enhanced coding-region interrogation WES provides allows for the sensitive detection of variants at those sites that are targeted. While there are no commercially available reagents for performing WES in nonhuman primates, a human whole exome platform is available. Given that there would likely be more purifying selection in coding regions and, therefore, less interspecific variation, we proposed using a human exome capture and sequencing kit for performing exome sequencing in rhesus macaques that differed in impulsivity and aggression. Exonic sequences were enriched using the Agilent SureSelect all exon capture array targeting 38Mb and were annotated using Ensemble genes, refGene and refSeq xenoRefGene for H. sapiens & M. musculus from UCSC. Sequence alignment was performed using CASAVA, Reclibration & SNP calling using GATK, Annotations, filtering, counts and calculations using custom scripts, and visualizations using R. Missense variants functionalities were inferred using Polyphen. PolyPhen posterior proabability scores associated with false positive rate (FPR) below 5% are characterized as probably damaging and those below 10% are determined to be possibly damaging under the HumDiv classifier model. Focusing on candidate genes relating to risk for alcohol use disorders, we identified a number of non-synonymous SNPs, some of which were predicted to be damaging or potentially damaging by in silico analysis (PolyPhen). Among these were SNPs in the MPDZ, GAL, GABARA6, and CRHR2 genes. The latter 2 genes encode the alpha-6 subunit of the GABA-A receptor and the corticotropin releasing hormone-2 receptor, respectively. Of relevance to the NIAAA mission, variations at these genes have been shown to predict individual differences in alcohol and stress response in rodents and humans. Studies in humans and rodents have demonstrated that variation at the GABRA6 gene predicts individual difference in alcohol response. We, therefore, wanted to determine whether the rhGABRA6 Tyr28Phe polymorphism would predict alcohol response in macaques. Using archived data, we found that animals carrying the 28Phe allele were rated as being more intoxicated. Although little has been done to examine GABRA6 variation as it relates to alcohol abuse or dependence in human subjects, our data also support a role for this SNP in protecting against early stress-induced increases in alcohol intake. Peer reared macaques carrying the 28Phe allele did not exhibit upregulated alcohol self-administration as did their Tyr/Tyr counterparts, suggesting that functional variation at this gene could serve a moderating role in determining alcohol missuse in human subjects. Ethanol is a positive allosteric modulator of GABARA4 and GABARA6 conductivity. GABA-A receptors are ligand-gated channels, comprised of 5 subunits, most commonly 2 alpha, 2 beta and 1 gamma. The GABRA6 gene, which encodes the α6 subunit is confined in its expression to cerebellar granule cells, and, therefore, a role for functional variation at this gene in determining levels of alcohol-induced ataxia and motor coordination is not surprising. Since individuals that are less sensitive to ethanol-induced ataxia and sedation may be more likely to exhibit higher lifetime levels of alcohol abuse, those who carry genetic factors that contribute to differences in alcohol sensitivity may also vary in terms of their vulnerability to the alcohol use disorders. Therefore, GABRA6 variation is held to be an excellent candidate for studies examining genetic basis for alcohol response and potentially addiction risk. However, identification of variation at this gene by WES in the animals sequenced in this study was unexpected, as subjects were selected based on differences in behavioral style and aggression, not on differences in alcohol response or consumption. This being said, recent studies demonstrating a critical role for the cerebellum in behavior, temperament, and social cognition might suggest that variation at this gene could also be linked to non-alcohol related behavioral and psychiatric conditions. Of interest, studies have shown that GABRA6 variation contributes to attachment style and to risk for bipolar disorder. We plan to examine whether other behavioral phenotypes, such as stress response, social cognition, mirroring behavior, or social attachment are predicted by GABRA6 Tyr28Phe genotype. Whole Exome and Whole Genome Sequencing of Domestic Animals At its most basic, domestication is a suite of heritable traits affecting behavior. There are intriguing phenotypic commonalities among domesticates. Most important among these traits is the ability to coexist with humans. The systems that likely permitted early domestication range from those involving fear and impulse control to those involving reward and sociality. At some point in the domestication of some species of animals, there may have also been selection for social cognition, as many domestic animal species are good at looking to humans for cues and for reading human emotions. We are exploring a draft whole genome sequence of a domestic cat, a wild Asian leopard cat (Prionailurus bengalensis) and interspecies hybrid offspring differing in their levels of tameness (ALC X DC). Using WES, we are also exploring genetic variation in two selected fox lines that are distinguished by marked differences in reactivity and temperament (tame vs. aggressive).
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