Gnao1 G184S mutant mice die perinatally
In light of the de novo mutants of GNAO1 recently reported in early infantile epileptiform encephaolopathy (Nakamura et al. 2013), we reexamined previously described mice carrying the G184S Gnao1 mutant gain-of-function allele (tm2Rneu—MGI:5508226) (Goldenstein et al. 2009). Heterozygous mutant mice are viable but obtained in reduced frequencies, while homozygotes are essentially not viable. From het × het crosses of Gnao1 G184S mutants on the 129 strain background, we obtained at weaning: 20 wt, 19 het, and only 2 homozygous mutants (5 %, 1 male and 1 female). Both homozygous and heterozygous mutants were underrepresented (Fig. S1A Chi squared χ
2 df(2) = 16.02 p < 0.001). Similarly, after back-crossing four times onto a C57BL/6J background (N4 B6), no homozygotes survived to weaning out of fifty-five offspring from het x het crosses and hets were substantially underrepresented (21 het vs 34 wt; expected value for hets is 2 × WT or 68; Fig. S1B Chi squared χ
2 df(2) = 45, p < 0.0001). Sex did not influence genotype distributions on either background (data not shown).
To understand the loss of viability of mice carrying the Gnao1 G184S allele, timed pregnancies were performed for N7 B6 het x het crosses and embryos were collected. At E18.5, genotypes did not differ from the expected Mendelian ratio (Fig. S1C), indicating that the loss of viability occurred after E18.5 but before 2 weeks of age. With close monitoring of litters, there were very few viable homozygous offspring and hets were also underrepresented (Fig. S1D, Chi squared χ
2 df(2) = 8.76 p < 0.02). Many homozygotes and some hets were found dead but there were no obvious anatomical abnormalities. Nissl staining performed on brains of E18.5 embryos revealed no major defects in brain structure (Fig. S2A–d). Gαo protein expression in the brains of hets at 8–12 weeks of age was found to be normal, indicating that the mutation was not significantly disrupting protein levels (Fig. S2e–f).
+/G184S mice experience sudden death
Gnao1 G184S B6 hets that survived to weaning (~30 % of expected) experience lethality starting at ~80 days (11–12 weeks) with ~50 % dead by 25 weeks of age (Fig. 1a). This was due to the Gnao1 mutant allele rather than to strain background as wild-type littermates survived to greater than 1 year (data not shown). Interestingly, there appeared to be heterogeneity in survival as the ~50 % that survived to 25 weeks showed little further premature lethality.
To understand the cause of death, mice were monitored three times weekly in daylight hours over 2 months. There were no evident signs of illness, weight loss, distress, or seizures prior to death. However, using continuous IR video recording of B6 female Gnao1 G184S mutant mice over ~900 mouse-days, we captured two deaths. Females were chosen for these studies due to the ability to group hour up to four mice for these long-term studies. In one instance, there was an apparent seizure with rearing and wild jumping (Fig. 1c and video supplement) observed during the night prior to the mouse being found dead in the cage. The second death showed no evident seizure.
+/G184S mice have EEG abnormalities
We also assessed video EEGs of B6 females (Fig. 1d, e). Mutant mice (age ~24 weeks) had a substantially elevated frequency of interictal epileptiform discharges (IEDs; 363, 296 and 460 IEDs/h for hets versus 2 and 24 IEDs/h for controls—unpaired t test t = 7.37 df = 2.2, p < 0.02). The IEDs seemed to occur more frequently during electrographic sleep than during wakefulness. Frequently, they occurred in runs of 1–3 Hz, but over the 4–8 day monitoring period, none met criteria for frank electrographic seizures due to the short duration the discharges (<10 s) nor were any overt seizures seen on video over this time period.
+/G184S mice are sensitized to PTZ kindling
To provide an independent and earlier assessment of the susceptibility to epileptogenesis in the mutant mice, we performed a PTZ kindling model in 8–12 week old mice (see “Materials and Methods”). Female B6 Gnao1
+/G184S mutants were more sensitive to PTZ kindling than were controls (Fig. 2a Gehan–Breslow–Wilcoxon df(1) = 5.74, p < 0.02). This was independent of gender (similar to spontaneous death) as mutant males also showed enhanced PTZ sensitization (Fig. 2b, Gehan–Breslow–Wilcoxon df(1) = 6.88, p < 0.01).
To determine whether the cause of seizure susceptibility in our mice might be due to loss of function of Gnao1 as suggested for humans in EIEE17 (Nakamura et al. 2013), we evaluated Gnao1 knockout mice. The homozygotes show early lethality (Valenzuela et al. 1997) and we were unable to generate sufficient numbers to undertake studies. The heterozygous knockout (Gnao1
+/−) mice showed no enhancement in sensitivity to PTZ kindling (Fig. 2c). The wild-type mice did show earlier seizure onset than the WT controls for the Gnao1 G184S mutants. This was unexpected. The most plausible explanation is that the two lines were developed from separate colonies and the backgrounds may be somewhat different despite both being C57Bl/6. The lack of increased kindling in the Gnao1
+/− mice combined with the normal level of expression of Gαo in our Gnao1 G184S mutants suggests that loss of function in our mutants does not explain our results (see “Discussion”).
Both spontaneous death and increased sensitivity to epileptogenesis are strain dependent
Interestingly, Gnao1 G184S heterozygous mutant mice on a 129 background (N6 129S1/SvImJ) live a relatively normal lifespan with 100 % surviving > 1 year (Fig. 3a). This contrasts strongly with the ~50 % of mutants on the B6 background that die by 25–40 weeks (Fig. 3a). Paralleling the strain-dependent nature of the adult lethality, Gnao1 G184S het mice on the 129 background also showed no difference in susceptibility to PTZ kindling compared to wild-type littermates (Fig. 3b). This suggests that one or more modifier loci that differ between B6 and 129 mice affects the penetrance of the lethality and seizure susceptibility phenotypes of the Gnao1 G184S mutants. To determine whether the protective or susceptible alleles were dominant, we tested B6/129 F1 mice. No deaths were observed up to 47 weeks in F1 mice regardless of their gender (n = 16 per genotype Fig. 3c). Thus, 129 alleles appear to provide a dominant protective effect against the spontaneous death phenotype of the Gnao1
+/G184S mutation or, conversely, the C57BL/6J background effect is recessive. Similarly, Gnao1 mutant mice on the B6/129 F1 background are protected against PTZ kindling (Fig. 3d, Gehan–Breslow–Wilcoxon df(1) = 7.9, p < 0.01). Thus, a single copy of 129 alleles throughout the genome is sufficient for protection in kindling as well as for spontaneous lethality.
Mapping modifier loci
Evaluating F2 offspring from intercrosses of B6/129 F1 mice carrying the Gnao1 G184S mutant allele showed that only 4 out of 60 heterozygous mutant offspring died before one year of age. Genome-wide SNP analysis was done but the small number of mice with the lethal phenotype was insufficient to obtain statistically significant LOD scores (data not shown). Given that 25 % of the F2 mice should be homozygous for B6 alleles at any given locus, 15 mice should have died if there had been a single high-penetrance modifier region; this suggests that there may be more than one modifier.
As an alternative approach to define genetic loci underlying the strain differences, we did a preliminary genome scan on 18 progeny of a single N6 male Gnao1 G184S mutation carrier. This analysis was based on the hypothesis that protective 129 alleles should be enriched in the long-lived group. Out of the 11 male and 7 female N7 B6 Gnao1
+/G184S mice from Fig. 3a, six were short-lived (i.e., died prior to the survival curve leveling off at ~25 weeks of age) and 12 were long-lived (i.e., alive past 25-week cutoff). The distribution of 129 (heterozygous) alleles across the genome was assessed by Illumina genotyping (see Supplemental Methods with confirmation markers defined in Table S1). The percent of short-lived and long-lived mice carrying 129 alleles at the indicated chromosomal location is shown in Fig. 4a, b. As expected, all mice retained 129 alleles on Chr 8 (i.e., those linked to the Gnao1 locus). The only region that preferentially retained 129 alleles in the long-lived cohort was on Chr17 at a point after 39 Mb through the end of the chromosome (Fig. 4a, b). Details of other retained regions on Chr 5 and Chr16 are presented in Fig. S3a, b, respectively, and Table S2. This initial analysis was not sufficiently powered to statistically validate the modifier due to the corrections needed for multiple comparisons.
To both confirm and reduce the size of the potential modifier region, we obtained recombinant mice that maintained 129 alleles at 47 and 62 Mb on Chr17 but were homozygous for B6 alleles at 41 and 70 Mb allowing us to reduce the size of the putative modifier region by half (see details of markers used in Table S1). In an independent experiment with 44 mice, we tested the ability of the (Chr17: 41–70 Mb) subregion to afford protection. This approach of identification followed by validation is supported by the previous literature (Williams and Haines 2011). The presence of 129 alleles in the Chr17: 41–70 Mb subregion protected Gnao1 G184S het mice from spontaneous death in the independent cohort (Fig. 4c Gehan–Breslow–Wilcoxon df(1) = 4.9, p < 0.05). The other retained 129 regions (e.g., Chr5 and Chr16 in Fig. 4a) did not influence viability within this cohort (data not shown).
Chr 17 locus also modifies PTZ susceptibility
We next tested the hypothesis that the Chr17 spontaneous death modifier protected against enhanced PTZ susceptibility. In this case, experiments were restricted to females due to their enhanced rate of kindling. The presence of 129 alleles within the Chr 17: 41–70 Mb modifier region also protected B6 Gnao1 mutant hets from kindling (Fig. 5a, Gehan–Breslow–Wilcoxon, df(1) = 4.28 p < 0.05). To determine whether the effect of this Chr17: 41–70 Mb locus to protect against kindling depended on the presence of the Gnao1 G184S mutant allele, we assessed its effect in Gnao1 WT mice. As expected, mice without the Gnao1 mutation showed markedly delayed kindling (half-max at injection 7, Fig. 5b vs. than 1 or 2 for the Gnao1 mutants, Fig. 5a). There may be a small effect of 129 alleles in the Chr17: 41–70 Mb region on kindling of Gnao1 WT mice but there was no statistically significant protection. The striking effect of this region in the presence of the Gnao1 mutant prompted us to name this Chr17 locus Mogs1 (modifier of G protein-induced PTZ susceptibility 1) in parallel with the Moe1 and Moe2 loci which have been previously described for protection against spontaneous seizures induced by sodium channel mutants (Bergren et al. 2005, 2009).