Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice

Abstract

De novo heterozygous missense mutations in the gene encoding translation elongation factor eEF1A2 have recently been found to give rise to neurodevelopmental disorders. Children with mutations in this gene have developmental delay, epilepsy, intellectual disability and often autism; the most frequently occurring mutation is G70S. It has been known for many years that complete loss of eEF1A2 in mice causes motor neuron degeneration and early death; on the other hand heterozygous null mice are apparently normal. We have used CRISPR/Cas9 gene editing in the mouse to mutate the gene encoding eEF1A2, obtaining a high frequency of biallelic mutations. Whilst many of the resulting founder (F0) mice developed motor neuron degeneration, others displayed phenotypes consistent with a severe neurodevelopmental disorder, including sudden unexplained deaths and audiogenic seizures. The presence of G70S protein was not sufficient to protect mice from neurodegeneration in G70S/- mice, showing that the mutant protein is essentially non-functional.

Figure 1. CRISPR/Cas9 experimental design and results of genotyping of F0 animals.

(A) Diagrammatic representation of mouse Eef1a2 gene showing the position of the G70S mutation in exon 3 of Eef1a2. (B) Sequence of Eef1a2 in the region targeted in the CRISPR/Cas9 experiment. Underlined sequence indicates the ssODN repair template, lower case indicates intron sequence, upper case is coding sequence. Letter in bold red indicates the site of the G70S mutation (changed to A in repair template). *Indicates PAM site (protospacer adjacent motif), targeted by the gRNA, that was mutated in the template to GTG to prevent further targeting. (C) Sequences of alleles recovered in all mice analysed, organised according to the location of the mutation. The number on the left corresponds to the mouse code number shown in Supplementary Table 1. Colour coding indicates the nature of the mutation.

Figure 2. Body weights of F0 mice from weaning.

Mouse weights between postnatal day 17 and 30. Upper panel shows data expressed as one dot per mouse per measurement; mouse genotype is indicated by colour of dot. The lower panel shows mean (dot) and standard error at different timepoints for each genotype. Since there was only one WT mouse in the F0 litters, additional body weights for genetically matched control mice were included. Note that the only G70S/G70S mouse is not included in this analysis as it had been culled before weighing began. One-way ANOVA performed on body weights from P23 and, separately, P25, showed that the body weights of mice with biallelic mutations were significantly different from those of +/− and wildtype mice (P < 0.0001 for both P23 and P25). Tukey’s multiple comparison test subsequently showed that the G70S/− and −/− weights were significantly different from wildtype and +/−, on both P23 and P25. Body weights for G70S/− and −/−, and for +/− and +/+ were not significantly different.

Figure 3. Neurodegeneration in the spinal cords of mice with mutations in eEF1A2.

H&E stained sections of of spinal cord taken from G70S/− (left) and null (right) mutant mice, with age matched control section on far right. Normal neurons are indicated by arrows with dotted lines. Dashed line arrows indicate neurons showing nuclear vacuolation and solid arrows indicate degenerating neurons with marked cytoplasmic vacuolation.

Figure 4. Expression analysis of mice with mutations in eEF1A2.

(A) Representative western blot showing eEF1A2 expression in protein extracts from brains of mutant founder mice from the CRISPR gene targeting experiment. The negative control was a brain extract from a wasted mouse (denoted −/−) and the loading control was GAPDH. The blot images are cropped for clarity and a larger area is shown in the Supplementary Data File. (B) Quantification of eEF1A2 expression in brain samples from mice with key genotypes. Controls were aged matched wild-type mice that were offspring of the F0+/− heterozygous mice. Expression levels are derived from three repeats of the Western analysis, normalised to GAPDH and expressed relative to the average obtained from three independent wild-type controls. **Indicates p < 0.01, n.s means not significant. (C) MnlI digests of RT-PCR products from brain RNA of mice of different genotypes. Predicted band sizes are shown to the right of the gel. The absence of bands at 73 bp and 39 bp in the G70S/− and G70S/G70S mice suggests that no wild-type allele is being expressed.