Confirmation of multiple seizure susceptibility QTLs on chromosome 15 in C57BL/6J and DBA/2J inbred mice

Abstract

To confirm seizure susceptibility (SZS) quantitative trait loci (QTLs) on chromosome (chr) 15 identified previously using C57BL/6J (B6) and DBA/2J (D2) mice and to refine their genomic map position, we studied a set of three congenic strains in which overlapping segments of chr 15 from D2 were transferred onto the B6 background. We measured thresholds for generalized electroshock seizure (GEST) and maximal electroshock seizure (MEST) in congenic strains and B6-like littermates and also tested their responses to kainic acid (KA) and pentylenetetrazol (PTZ). Results document that MEST is significantly lower in strains 15M and 15D, which harbor medial and distal (telomeric) segments of chr 15 (respectively) from D2, compared with strain 15P, which harbors the proximal (acromeric) segment of chr 15 from D2, and with control littermates. Congenic strains 15P and 15M exhibited greater KA SZS compared with strain 15D and B6-like controls. All congenic strains were similar to controls with regard to PTZ SZS. Taken together, results suggest there are multiple SZS QTLs on chr 15 and that two QTLs harbor gene variants that affect MEST and KA SZS independently. The MEST QTL is refined to a 19 Mb region flanked by rs13482630 and D15Mit159. This interval contains 350 genes, 183 of which reside in areas where the polymorphism rate between B6 and D2 is high. The KA QTL interval spans a 65 Mb region flanked by markers D15Mit13 and rs31271969. It harbors 83 genes in highly polymorphic areas, 310 genes in all. Complete dissection of these loci will lead to identification of genetic variants that influence SZS in mice and provide a better understanding of seizure biology.

Fig. 1.

Schematic diagram of chr 15 showing regions of D2 genomic introgression (gray blocks) onto the B6 background (black blocks) for three chr 15 congenic strains (15P, 15M, and 15D). The intersection of gray and black blocks represents “breakpoint” intervals for each strain where there is no genotype information. Marker positions are taken from the database at http://uswest.ensembl.org/Mus_musculus/Info/Index.

Fig. 2.

Scatter plot of maximal electroshock seizure (MEST, A) and generalized electroshock seizure (GEST, B) values in chr 15 congenic strains and B6-like littermate controls (n = 15 per group). The solid lines show the mean MEST value. For MEST, strains 15M and 15D are significantly different than B6 controls (P < 0.001) and strain 15P (P < 0.001) (ANOVA/Tukey). For GEST, comparison of median values revealed that strain 15P was significantly different from strains 15M and 15D (P < 0.001, Kruskal-Wallis ANOVA and Holm-Bonferroni adjusted pair-wise Wilcoxon rank sum).

Fig. 3.

Box-plot of the distribution of kainic acid (KA) seizure endpoint responses in chr 15 congenic strains and B6-like littermate controls (n = 12 per group). The horizontal line inside each box represents the median response value, and the plus sign represents the mean response value. The top line and bottom line of each box represent the 75th percentile and 25th percentile, respectively. The line above extends to the largest value within 1.5 × IQR (interquartile range) of the 75th percentile, where IQR is the 75th percentile minus the 25th percentile, and the line below extends to the smallest observation within 1.5 × IQR of the 25th percentile. The 2 circles above the line on Clonus 15P are outliers that are >1.5 × IQR above the 75th percentile.

Fig. 4.

Kaplan-Meier plots for KA seizure endpoints in chr 15 congenic strains and B6-like littermate controls (n = 12 per group). Colored lines represent strain trajectories for achieving each endpoint. Statistical comparisons were conducted using Holm-Bonferroni-adjusted log-rank tests. A: clonus: all congenic strains are significantly different from B6-like controls (P < 0.001); there are no differences between any of the other groups (P = 1.000). B: generalized seizure: all congenic strains are significantly different from B6-like controls (P < 0.001 for 15M and 15P, P = 0.002 for 15D); 15D is significantly different from other congenic strains (P = 0.024 compared with 15P, P < 0.001 compared with 15M); there is no significant difference between 15M and 15P (P = 0.161). C: status epilepticus: 15P and 15M are significantly different from B6-like controls (P < 0.001 for both) and from 15D (P < 0.001 compared with 15P, P = 0.001 compared with 15M). There is no significant difference between 15D and B6-like controls (P = 0.485) or between 15P and 15M (P = 0.648).

Fig. 5.

Haplotype block patterns for B6 and D2 mice in the KA (top) and MEST (bottom) quantitative trait locus (QTL) intervals on chr 15. White blocks are regions of the genome that are identical by descent between the strains. Gray blocks are regions that have higher polymorphism rate. Genomic location (in Mb) is shown on the x-axis.