Experiment
Identifying genetic risk factors for trauma and stress related disorders is a high priority for psychiatric genetics. In the current study, existing and newly collected data were used for collaborative genome-wide mapping of cued fear acquisition and expression in 65 mouse strains from a BXD genetic reference panel.
The BXD panel comprises more than 100 recombinant inbred strains generated from progressive matings of C57BL/6J (B6) and DBA/2J (D2) offspring. The two parental lines show broad differences in many neurobiological and behavioral traits and high levels of sequence variation.
Mice were acclimated for 30 minutes to the training context and trained the following day using 5-paired presentations of a tone-conditioned stimulus and a foot-shock unconditioned stimulus (US). Mice were tested for a cued-fear 24 hours later in a novel context using 10 tone conditioned stimuli (CS) presentations. Behavioral sessions were videotaped (30 frames/sec) under near infrared light and freezing times were scored automatically using VideoFreeze software. Freezing was defined as the absence of all movement except that required for breathing and was scored manually by an observer who was blinded to strain identity. Fear learning during testing (fear expression) was computed as the average percentage of freezing during the first conditioned stimuli.
Genome wide simple interval mapping (SIM) was performed using the genome mapping tools within GeneNetwork (GN: (https://www.genenetwork.org) to identify suggestive and significant QTL (NCBI Build 37). A subset of 3811 informative markers were used within the GN. 5000 permutation tests were performed within the GN to determine the likelihood ratio statistic (LRS) thresholds for suggestive and significant QTL, corresponding to genome-wide probabilities of 0.63 and 0.05, respectively.
Table 1:
A significant QTL for fear acquisition, Facq1 (fear acquisition QTL 1) was identified on Chromosome 10 with a peak LRS score of 33.49 at peak marker rs3686911 (14.12 Mb), p<0.01. Facq1 mapped within a 1.5 LOD support interval spanning 3.13-21.83 Mb. B6 alleles at this locus had an additive effect size of 17.9% increased freezing. Nine suggestive QTL were identified on Chrs 2,4,5,13 and 15 [Fig 1B.]
For fear expression a significant QTL, Fexq1 (fear expression QTL 1) was identified on Chromosome 13 with a peak LRS score of 21.72 nearest peak marker mCV24625340 (84.64 Mb), p<0.05. Fexq1 mapped within a 1.5 LOD support interval spanning 78.26-96.47 Mb. D2 alleles at this locus had an additive effect size of 11.7% increased freezing.
Another highly suggestive QTL for fear expression, Fexq2 (fear expression QTL 2), mapped to Chromosome 13 with a peak LRS score of 20.67 at peak marker rs13482033 (118.39 Mb), p<0.068. Fexq2 mapped within a 1.5 LOD support interval spanning 116.58-119.97 Mb. B6 alleles at this locus had an additive effect size of 11.3%. There were also 4 suggestive QTL identified on Chrs 10 and X for this trait.
Interestingly QTLs Facq1 and Fexq1 on Chrs 10 and 13 respectively, were detected at the suggestive or significant level for both fear acquisition and expression, consistent with the hypothesis that shared or neighboring genes within these loci affect both traits. Composite interval mapping (CIM) for both traits revealed linkage between QTL Facq1 and Fexq1 and revealed a suggestive QTL on Chr 11. Evidence of epistatic interactions between the two QTL were not displayed.
Candidate gene lists were generated from the 1.5 LOD support intervals and narrowed to protein encoding genes. From those results a prioritized list of candidate genes was generated based on the genes involvement in a human neurological disorder or prioritized brain based phenotype. Nineteen genes met both criteria (Figs 1D and 2D.]
QTL Facq1 contained 13 genes that met one or both criteria, several of which have been implicated previously in conditioned fear (Oprm1, Sgk1), mood and anxiety disorders (Oprm1, Esr1, Sgk1), intellectual disability (Hivep2), and learning and memory (Sgk1, Hivep2).
QTL Fexq1 contained 11 genes that met one or both criteria, several of which have been implicated previously in conditioned fear (Mef2c, Pde8b), learning and memory ((Mef2c), and stress response and anxiety disorder risk (Crhbp).
QTL Fexq2 was located in a gene sparse region that contained a single candidate gene, Hcn1 (hyperpolarization-activated cyclic nucleotide-gated channel 1) which has been implicated in neuronal excitability and learning. Behavioral pharmacology confimred that Hcn channels in the basolateral amygdala are required for fear acquisition and expression.
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