Confirmation of an epilepsy modifier locus on mouse chromosome 11 and candidate gene analysis by RNA-Seq

Abstract

Epilepsy is a neurological disorder affecting approximately 1% of the worldwide population. Mutations in voltage-gated sodium channels have been identified in several monogenic epilepsy syndromes. Over 800 mutations have been identified in the voltage-gated sodium channel genes SCN1A and SCN2A in human epilepsies, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In GEFS+ families, affected members with the same mutation often display variability in clinical severity of the disease. This suggests that additional genes modify the effect of the primary mutation, resulting in the variable clinical presentation. The Scn2a(Q54) transgenic mouse model has an epilepsy phenotype that varies depending on the genetic strain background. Scn2a(Q54) mice congenic on the C57BL/6J strain exhibit delayed seizure onset and improved survival compared to (C57BL/6J × SJL/J)F1.Q54 mice. Two modifier loci of Scn2a(Q54) seizure susceptibility were mapped and designated Moe1 (modifier of epilepsy) on chromosome (chr) 11 and Moe2 on chr 19. To confirm Moe1 and refine its position, we generated interval-specific congenic lines carrying C57BL/6J-derived chr 11 alleles on the SJL/J strain and refined the map position to 89-104 Mb. We then used RNA-Seq for candidate analysis in the modifier region. C57BL/6J and SJL/J male and female brain RNAs were sequenced, revealing numerous significant transcriptome differences and coding single-nucleotide polymorphisms. Additional consideration of gene function and expression suggested several strong candidate modifier genes, including two voltage-gated calcium channel subunits, Cacna1g and Cacnb1, and the proline and acidic amino acid-rich basic leucine zipper transcription factor, Hlf.

Figure 1. Fine mapping of Moe1 on chr11 with interval specific congenic lines

A. ISC lines were generated with varying B6-derived chr 11 segments (colors) on an SJL background (white). Microsatellite markers used for ISC generation are identified along the chromosome. From the original mapping, the Moe1 peak was at D11Mit289. B. ISC lines were crossed with B6.Q54, generating F1.Q54 offspring. F1.Q54 mice will be heterozygous or homozygous for B6 alleles in Moe1. C. Seizure frequency in ISC males. Average number of seizures in 60 minutes for each genotype group is shown. Significant differences between groups were determined by ANOVA with Fishers PLSD post-hoc tests. D. Seizure frequency in ISC females. Average number of seizures in 60 minutes for each group is shown. Significant differences between groups were determined by ANOVA with Fishers PLSD post-hoc tests. Data are represented as mean ± SEM. Significant P-values determined by post-hoc Fishers PLSD are indicated in the figure.

Figure 2. Expression of Hlf-002 transcript in B6 and SJL brain

The RNA-Seq FPKM values and confidence interval for each group is shown. Boxes represent the range of values and whiskers represent the 95% confidence interval calculated by Cuffdiff. Q-values are false discovery rate-adjusted p-values. (Hlf-002=ENSMUST00000043658).

Figure 3. Differential expression of Cacna1g alternatively spliced transcripts in females

Inclusion/exclusion of cassette exons 14 and 26 and alternate splicing of exon 25 are shown for the five transcripts that exhibited significant differences between B6 and SJL females. The location of alternatively spliced exons are shown on the Cacna1g channel protein (bottom). FPKM values and confidence intervals for B6 and SJL females are shown. Q-values are false discovery rate-adjusted p-values.

Figure 4. Alternative splicing and differential expression of Cacnbl

A. Cacnb1 alternatively spliced transcripts are shown. Dark shaded boxes represent coding regions and light shaded boxes are non-coding regions. B. Respective FPKM values and confidence intervals for B6 and SJL female transcripts are shown (*q< 0.02). Q-values are false discovery rate-adjusted p-values.