DESCRIPTION:
Chromosome 5 cis-eQTL transcripts showing peak association with the peak behavioral QTL EPM marker for EPM behaviors during spontaneous OXY withdrawal (rs33209545; chromosome 5: 59,833,642 bp). We mapped the genetic basis of OXY behavioral sensitivity and spontaneous withdrawal in a B6J×B6NJ‐F2 reduced complexity cross (RCC). Upon identifying a major distal chromosome 1 QTL for oxycodone locomotion and withdrawal, we implemented an efficient fine mapping strategy using recombinant lines within the QTL interval, and resolved a 2.45‐Mb region. We then sourced a historical striatal expression QTL dataset from the same genetic cross to home in on functionally plausible candidate genes. Finally, we conducted immunoblot analysis of eQTL genes within a recombinant line capturing the behavioral QTL, providing further support for the candidacy of Atp1a2 and Kcnj9. The results provide multiple positional loci and highly plausible candidate genes underlying opioid behavioral sensitivity and withdrawal. 425 F2 mice (213 SAL, 212 OXY) were phenotyped in the OXY locomotor/CPP protocol during Weeks 1 and 2 (Fig.2A). F2 mice were trained in a two‐chamber unbiased CPP protocol over nine days (D), with initial preference (D1, SAL i.p.), two alternating pairings of OXY (1.25 mg/kg, i.p.) and SAL (i.p.), separated by 48h (D2‐D5), a consolidation period (D6‐D7), a drug‐free CPP test following SAL (i.p.) (D8), and a drug‐induced CPP test (D9) following either SAL (i.p.) in the SAL group or OXY (1.25 mg/kg, i.p.) in the OXY group. A subset (118 SAL, 118 OXY) went on to receive a high-dose, multi-week OXY regimen over Weeks 3 and 4 (Fig.2A). For week 3, mice were injected once daily with either SAL (i.p.) or OXY (20mg/kg, i.p.) on Monday through Thursday at 1600h. Sixteen hours later on Friday, the majority of these 236 F2 mice (160F2 mice total: 77F, 83M) were tested for baseline nociceptive sensitivity on the 52.5°C hot plate assay and then assessed for OXY‐induced antinociception (5mg/kg, i.p.) at 30min post‐OXY (see Data S1). The remaining 76 F2 mice (37F, 39M) received the same OXY regimen during Week 3 but were not exposed to hot plate testing. For Week 4, we continued injecting all 236 F2 mice (118 SAL, 118 OXY) once daily with SAL (i.p.) or OXY (20mg/kg, i.p.) for four days. On Day 5, 16h post‐injection, mice were tested for anxiety‐like behavior in the elevated plus maze (EPM). Striatum punches were collected from 23 OXY-trained F2 mice at 24 h after behavioral testing on the EPM. Libraries were prepared according to Illumina's detailed instructions accompanying the TruSeq® Stranded mRNA LT Kit (Part# RS-122-2101). The purified DNA was captured on an Illumina flow cell for cluster generation and sample libraries were sequenced at 23 samples per lane over 5 lanes (technical replicates) on the Illumina HiSeq 4000 machine, yielding an average of 69.4 million total reads per sample. We aligned FASTQ files to the mm38 genome via TopHat (Trapnell et al. 2012) and used the Ensembl sequence and genome annotation. We used featureCounts for read alignment. Exons with low expression (less than 10 reads total across all 115 count files) were removed. A a cis-eQTL was liberally defined as any transcript with a genome-wide significant association between expression and a polymorphic marker that was within 70 Mb of a SNP, given the large linkage disequilibrium in a lowly recombinant F2 cross and given that this was the largest distance between any two SNP markers. Analysis was conducted using limma with default TMM normalization and VOOM transformation (Law et al. 2014; Ritchie et al. 2015). To account for the presence of replicates of each sample in the data, we used the duplicateCorrelation() function to estimate the within-sample correlation which we then included in the lmFit() function. An ANOVA test was conducted for gene expression, with Genotype as a fixed effect and Sex as a covariate. Gene-level tests were conducted using the Likelihood Ratio test. A false discovery rate of 5% was employed as the cut-off for statistical significance (Benjamini & Hochberg 1995). From supplementary table S6.
LABEL:
Striatal candidate genes assoc. chr5 rs33209545 EPM marker OXY withdrawal B6JxB6NJ F2 cross_pvalue
SCORE TYPE:
P-Value
THRESHOLD:
<= 0.5
GENES IN THRESHOLD:
5
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DATE UPDATED:
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AUTHORS:
TITLE:
JOURNAL:
ABSTRACT:
Genes in threshold: 5
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