Mapping genetic modifiers of survival in a mouse model of Dravet syndrome

Abstract

Epilepsy is a common neurological disorder affecting approximately 1% of the population. Mutations in voltage-gated sodium channels are responsible for several monogenic epilepsy syndromes. More than 800 mutations in the voltage-gated sodium channel SCN1A have been reported in patients with generalized epilepsy with febrile seizures plus and Dravet syndrome. Heterozygous loss-of-function mutations in SCN1A result in Dravet syndrome, a severe infant-onset epileptic encephalopathy characterized by intractable seizures, developmental delays and increased mortality. A common feature of monogenic epilepsies is variable expressivity among individuals with the same mutation, suggesting that genetic modifiers may influence clinical severity. Mice with heterozygous deletion of Scn1a (Scn1a(+/-) ) model a number of Dravet syndrome features, including spontaneous seizures and premature lethality. Phenotype severity in Scn1a(+/-) mice is strongly dependent on strain background. On the 129S6/SvEvTac strain Scn1a(+/-) mice exhibit no overt phenotype, whereas on the (C57BL/6J × 129S6/SvEvTac)F1 strain Scn1a(+/-) mice exhibit spontaneous seizures and early lethality. To systematically identify loci that influence premature lethality in Scn1a(+/-) mice, we performed genome scans on reciprocal backcrosses. Quantitative trait locus mapping revealed modifier loci on mouse chromosomes 5, 7, 8 and 11. RNA-seq analysis of strain-dependent gene expression, regulation and coding sequence variation provided a list of potential functional candidate genes at each locus. Identification of modifier genes that influence survival in Scn1a(+/-) mice will improve our understanding of the pathophysiology of Dravet syndrome and may suggest novel therapeutic strategies for improved treatment of human patients.

Keywords: Dravet syndrome; RNA-seq; epilepsy; epileptic encephalopathy; mouse model; seizures; severe myoclonic epilepsy of infancy; transcriptomics; voltage-gated ion channels; voltage-gated sodium channels.

Figure 1

Generation of mapping backcross progeny and phenotyping. (a) Breeding scheme used to generate backcross progeny. First, 129.Scn1a^+/− males were crossed to B6 females to generate F1.Scn1a^+/− mice. Male F1.Scn1a^+/− mice were then crossed with 129 females to generate 129-N2 offspring, or to B6 females to generate B6-N2 offspring. (b) Survival of Scn1a^+/− backcross progeny. B6-N2 backcross progeny have significantly reduced survival compared to 129-N2 progeny (Log Rank p<0.0001)(n=293 for B6-N2 backcross; n=150 for 129-N2 backcross).

Figure 2

Genome-wide interval mapping for Dravet syndrome modifier loci that influence 12-week survival in the B6-N2 backcross. LOD scores for the combined effects on penetrance and severity are plotted for all mouse chromosomes and show suggestive or significant evidence of linkage to chromosomes 5, 7, and 8. Results shown are from a single QTL genome scan under a two-part model using a maximum likelihood procedure in R/qtl software. Horizontal lines mark significance thresholds for genome-wide significant (p=0.05; solid grey) or suggestive (p=0.25; dashed grey) evidence of linkage based on 10,000 permutations of the data. The X-axes are in centimorgans with chromosome numbers indicated below and tick marks representing the location of SNP markers used for genotyping.

Figure 3

LOD scores for the effect on penetrance (live/die)(blue), severity (age at time of early death)(red) and the combined effect (black) are plotted for chromosomes 5, 7 and 8 and show that the modifier loci exert differential effects on penetrance and severity. Results shown are from a single QTL genome scan under a two-part model using a maximum likelihood procedure in R/qtl software. The horizontal solid grey line marks the significance threshold (p=0.05) for combined effect (LOD(p,mu)) and the dashed grey line marks significance thresholds (p=0.05) for penetrance (LOD(p)) and severity (LOD(mu)). Significance thresholds were determined by 10,000 permutations of the data. The X-axes are in centimorgans with chromosome numbers indicated below and tick marks representing the location of SNP markers used for genotyping.

Figure 4

Effect of alleles at Dsm loci on survival. Genotypes from the marker closest to each significant modifier peak were used to determine the effect of individual loci on survival. Kaplan-Meier plots for each genotype class show the relationship between survival and alleles at modifier loci identified in the B6-N2 backcross on chromosome 5, 7 and 8 (a), and in the 129-N2 backcross on chromosomes 5 and 11 (b). P-values were calculated with the Mantel-Cox log rank test.