Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility

Abstract

Objective: Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants.

Methods: We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression.

Results: We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity.

Significance: New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

Keywords: Gabra2; Collaborative Cross; SUDEP; epileptogenesis; seizure susceptibility.

Figure 1.. Seizure thresholds and severity highly depend upon genetic background.

(A) Schematic of flurothyl-induced seizures. MS = myoclonic seizure; GS = generalized seizure; SUDEP = sudden unexpected death in epilepsy. (B) MS threshold (MST) and (C) GS threshold (GST) of 35 CC strains as well as B6J and DBA/2J. The order of strains was ranked from the most resistant (highest threshold) to the most susceptible (lowest threshold), n=3–10. Arrows denote parental strains used for generating F2 mapping population. Data are presented as mean ± SEM and analyzed using one-way ANOVA with post hoc Bonferroni’s multiple comparisons test. *p<0.05, **p<0.01 and ***p<0.001 compared to DBA/2J. (D) Seizure threshold and (E) sum of maximum behavioral seizure score after PTZ injection (40 mg/kg, i.p.), n=5. (F) Latency to onset of generalized seizures induced by repeated low dose of KA (5 mg/kg, i.p. every 30 min), n=4–5. Data are presented as mean ± SEM and analyzed using one-way ANOVA with post hoc Tukey’s multiple comparisons test. *p<0.05, **p<0.01 and ***p<0.001.

Figure 2.. Correlation of myoclonic and generalized seizure thresholds and identification of seizure propagation resistant CC strains.

(A) Correlation of MST and GST (R²=0.824, p<0.001 without CC032 and CC058). (B) MST-GST difference of 35 CC strains as well as B6J and DBA/2J, n=3–10. Data are presented as mean ± SEM and analyzed using one-way ANOVA with post hoc Tukey’s multiple comparisons test. ***p<0.001 compared to other strains.

Figure 3.. Identification of strains resistant to epileptogenesis.

(A) MST and (B) GST during 8-day flurothyl kindling. The order of the CC strains are ranked based on the value of kindling slope of myoclonic (A) and generalized (B) seizures, respectively. (C) Kindling slopes of MST and GST (R²=0.325, p<0.001) are correlated across strains except CC032. (D) Maximum behavioral seizure score during 30 min after each injection of PTZ (35 mg/kg, i.p.) every other day over 10-trial PTZ kindling (n=5). Data are presented as mean ± SEM and analyzed using two-way ANOVA with post hoc Tukey’s multiple comparisons test, **p<0.01.

Figure 4.. Mortality of SUDEP susceptible CC strains.

Survival curve of CC003 (n=9), CC008 (n=10), CC009 (n=8), CC029 (n=6) and B6J (n=7) during 8-day flurothyl kindling. Data are analyzed using Log-rank (Mantel-Cox) test, n=6–10, **p<0.01 and ***p<0.001 compared to B6J; and ^##p<0.01 compared to young CC009 (2–3 month).

Figure 5.. QTL mapping for myoclonic (slate-blue) and generalized (violet-red) seizure threshold using F2 crosses from B6J and CC027.

Chromosomes 1 through 19 are represented numerically on the x-axis, and the y-axis represents the LOD score. The relative width of the space allotted for each chromosome reflects the relative length of each chromosome. A magnification of each myoclonic seizure susceptibility (Mss) and generalized seizure susceptibility (Gss) locus is shown. Solid colored horizontal bar indicates the significance threshold at p = 0.05.

Figure 6.. Identification of genetics variants from QTL.

(A) Mss2 QTL region on chromosome 5 and list of 21 protein coding genes found within the interval. * denotes genes that were measurably expressed in the hippocampi. (B) Genes in the QTL region with significant differential (p<0.05, t-test) expression in the hippocampus of B6J and CC027 mice. Data are presented as median with the lower and upper hinges corresponding to 25–75 percentiles. (C) Gabra2 with an intronic base pair deletion located next to a splice acceptor site preceding exon 5. This deletion is only found in B6J mice.