Analysis of morphine responses in mice reveals a QTL on Chromosome 7

In this study we identified a quantitative trait locus (QTL) on mouse Chromosome 7 associated with locomotor activity and rearing post morphine treatment. This QTL was revealed after correcting for the effects of another QTL peak on Chromosome 10 using composite interval mapping. The positional candidate genes are Syt9 and Ppfibp2. Several other genes within the interval are linked to neural processes, locomotor activity, and the defensive response to harmful stimuli.


Introduction
Responses to drugs of abuse vary among individuals and are genetically influenced (Mistry et al., 2014). Such drugs stimulate the brain reward pathway (Gardner, 2011). Analysis of genetic variation in the behavioral and neurobiological processes can reveal molecular mechanisms that regulate or mediate the response to drugs, and neurobiological changes associated with chronic drug use. Many of the genes and signaling processes involved in the reward pathway in humans are conserved in mice (Adinoff, 2004), and mouse genetic methods provide an efficient means of identifying these genes and loci.
Quantitative trait loci (QTL) analysis of recombinant inbred mouse strains (RIS) integrates phenotype and genotype data and is a widely used approach for studying the genetic basis of drug effects and addiction susceptibility (Spence et al., 2005). The BXD RIS, derived from C57BL/6J (B) and DBA/2J (D), are a wellestablished genetic reference population used in behavioral neuroscience studies for mapping complex traits associated with drug use (Crabbe et al., 1996;Dickson et al., 2016;Gora-Maslak et al., 1991;Peirce et al., 2004;Plomin et al., 1991).
In the present study we reanalyzed data from high throughput behavioral phenotyping of morphine-treated BXD RIS mice (Philip et al., 2010). Our purpose was to identify additional morphine response related genetic loci beyond those initially reported. We employed composite interval mapping for two behavioral phenotypes, rearing and locomotion in response to morphine, and identified a QTL on mouse Chromosome 7 that was masked by a major QTL on Chromosome 10.

Methods
Experimental protocols have been described elsewhere (Philip et al., 2010). BXD data were generated in the laboratory of Dr. Charles D. Blaha at the University of Memphis and obtained from GeneNetwork.org.
QTL mapping QTL mapping was performed using GeneNetwork 1.0 with a composite interval mapping function using 2000 permutations. This is a forward regression approach in which a single locus with a major effect is included in a mapping model that scans for additional additive effects and interactions with the major locus. The technique will miss higher order interactions among loci that are not detectable as main effects, but is effective when a large, consistently observed major effect locus is present. Strain mean scores were Winsorized when statistical outliers were present (Wilcox, 2005). We first determined which marker had the highest LRS value, using a marker regression analysis and then performed composite interval mapping, controlling for the Chromosome 10 SNP rs3721803, one of the 3 markers with the highest LRS scores.

Bioinformatics
The MGI database (http://www.informatics.jax.org/) was used to find information about SNPs and strain polymorphisms occurring within the Chromosome 7 interval (queried -June 20, 2016). This database now includes variants detected in the sequencing of 17 mouse genomes (Keane et al., 2011). The 3 SNPs located at the Chromosome 7 QTL peak (rs13479451, rs3724540, rs6386601) are at ~114.5 Mb in GeneNetwork and ~107.6 Mb in the MGI and NCBI databases (http://www.ncbi.nlm.nih.gov/gene). In GeneNetwork, the relevant QTL interval on Chromosome 7 was 110-125 Mb whereas this region corresponds to 103-118 Mb in the MGI database. As finer mapping methods are developed, the exact interval location as reported here, may change. DAVID version 6.8 (https://david.ncifcrf.gov/) was used to obtain functional annotations and pathway information for the genes within the interval and GeneWeaver 1.0 (http://www.geneweaver.org/) to identify other drug-related phenotypes associated with the candidate genes. We remapped these data for each time point after morphine injection (up to180 min) using composite interval mapping to identify QTLs potentially masked by this large QTL. This revealed a second significant QTL on Chromosome 7 for locomotion (LRS= 18.0,114.7 Mb; Figure 1C) and, in an overlapping position, for rearing (LRS=16.5, 114.3 Mb). We also observed a time dependent decrease of the peak LRS values for both traits after the 60-75 minute time interval post-morphine injection. A suggestive QTL for the rearing trait data was already apparent on Chromosome 7 from the whole genome scan, but this was not the case for the locomotion trait data (Philip et al., 2010). There are 3 SNPs mapped at the location of the LRS peak and two positional candidates: Synaptogamin 9 (Syt9: rs13479451, rs3724540) and PPFIA binding protein 2 (Ppfibp2, rs6386601). Both of these genes differ between the parental strains (MGI database query -June 20, 2016). Syt9 functions in vesicle traffic and Ca2+ triggered exocytosis (Ncbi, 2016b) and Ppfibp2 is involved in the regulation and development of neuronal synapses (Ncbi, 2016a). These two candidates are also differentially expressed in the striatum of the brains of mice from strains with distinct opioid sensitivity (Korostynski et al., 2006; GeneWeaver: Syt 9, geneset #86830; Ppfibp2, geneset #86906).

Conclusions
Genetic analysis of the BXD RIS resulted in the detection of an additional locus for morphine induced locomotor activity and rearing on Chromosome 7 which may be associated with activation of the reward system pathway in response to morphine treatment. Whether this effect is opioid-specific or also occurs with other classes of drugs is not yet clear. Alternatively, this QTL may also be related to general locomotor activity or nociception, a central nervous system response to potentially harmful stimuli. The best gene candidates within the QTL interval, besides the positional candidates Syt9 and Ppfibp2, are Calca and Cckbr (sensory perception of pain) and Pde3b (morphine addiction).
Author contributions WEC assisted in interpreting the results and writing the manuscript; ED performed the functional annotation analysis; EJC provided the original data and assisted with interpreting the results and writing the manuscript; AD conceived the study, supervised the analyses, and wrote the manuscript. All authors approved the final submitted draft.

Competing interests
No competing interests were disclosed.

Grant information
BioScience Project is a 501(c)(3) research and teaching organization. EJC was supported by grants from NIAAA (AA018776) for GeneWeaver and NIDA (DA037927) for addiction genetics.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
F1000Research afforded by both GeneNetwork and the BXD recombinant inbred mice. Specifically, they used composite interval mapping to reanalyze publicly available phenotypic and genotypic data in the BXDs to identify a QTL on chromosome 7 that had been masked by a major QTL on chromosome 10. Next, they employed a bioinformatics approach to prioritize among candidate genes within the QTL interval. This resulted in the identification of promising candidate genes that may be associated with drug-abuse phenotypes.
One step that would strengthen the manuscript would be to use QTLminer in GeneNetwork to examine cis -regulation of the genes located within the QTL support interval (or even just the most promising candidate genes) and to see if any of those genes were expression QTLs in BXD mice.
In addition, the authors raise an interesting question when they ask whether the chromosome 7 QTL is opioid-specific, or if it also occurs with other classes of drugs. They could use GeneNetwork to search for genetic correlations between the expression levels of their top candidate genes and drug-related traits.
Minor Points: On the GeneNetwork website, Trait ID 11851 is defined as the number of beam breaks after morphine injection, but is described as "distance traveled" and "locomotion" in the Figure 1 legend of the manuscript.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed.

Competing Interests:
Author Response 19 Oct 2016 , anna delprato Reviewer comment: "One step that would strengthen the manuscript would be to use QTLminer in GeneNetwork to examine cis-regulation of the genes located within the QTL support interval (or even just the most promising candidate genes) and to see if any of those genes were expression QTLs in BXD mice." Of the candidate genes there is a marginally significant cis-eQTL for Syt9 ( LRS =18) but this is with one probe and in the striatum dataset only. There are no cis-eQTL for the other gene candidates.
Reviewer comment: "In addition, the authors raise an interesting question when they ask whether the chromosome 7 QTL is opioid-specific, or if it also occurs with other classes of drugs. They could use GeneNetwork to search for genetic correlations between the expression levels of their top candidate genes and drug-related traits." Correlation analyses do not rule out or support association with other drugs. The few significant correlations that we found were based on less than 10 common strains.
No competing interests were disclosed. Competing Interests: