<i>In Vivo</i> Modelling of <i>ATP1A3</i> G316S-Induced Ataxia in <i>C</i>. <i>elegans</i> Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects

<div><p>The NIH Undiagnosed Diseases Program admitted a male patient with unclassifiable late-onset ataxia-like symptoms. Exome sequencing revealed a heterozygous <i>de novo</i> mutation converting glycine 316 to serine in <i>ATP1A3</i>, which might cause disease. <i>ATP1A3</i> encodes the Na⁺/K⁺ ATPase pump α3-subunit. Using CRISPR/Cas9-mediated homologous recombination for genome editing, we modelled this putative disease-causing allele in <i>Caenorhabditis elegans</i>, recreating the patient amino acid change in <i>eat-6</i>, the orthologue of <i>ATP1A3</i>. The impact of the mutation on <i>eat-6</i> function at the neuromuscular junction was examined using two behavioural assays: rate of pharyngeal pumping and sensitivity to aldicarb, a drug that causes paralysis over time via the inhibition of acetylcholinesterase. The patient allele decreased pumping rates and caused hypersensitivity to aldicarb. Animals heterozygous for the allele exhibited similar defects, whereas loss of function mutations in <i>eat-6</i> were recessive. These results indicate that the mutation is dominant and impairs the neuromuscular function. Thus, we conclude that the <i>de novo</i> G316S mutation in <i>ATP1A3</i> likely causes or contributes to patient symptoms. More broadly, we conclude that, for conserved genes, it is possible to rapidly and easily model human diseases in <i>C</i>. <i>elegans</i> using CRIPSR/Cas9 genome editing.</p></div

The dominant negative effects of G304S increase over time.

<p>(A) <i>eat-6(rt252)</i> heterozygous animals had decreased pumping rates as young adults. Pumping defects caused by <i>ad467</i> were recessive. However, (B) both <i>eat-6(rt251)</i> and <i>eat-6(rt252)</i> mutants had reduced pumping rates when aged to 8 days of adulthood (median, flanking quartiles in Tukey format; 3 trials, *: Mann-Whitney U-test (two-tailed) p-value<0.05; n.s.: not significant, p-value>0.05).</p

EAT-6 G304S has a dominant negative effect.

<p>(A) Animals heterozygous for the G304S mutation were hypersensitive to 1mM aldicarb, whereas the reference <i>ad467</i> allele was recessive (3 trials ± SEM, *: log-rank p-value<0.05). (B) Animals with heterozygous <i>eat-6</i> complete loss of function alleles did not exhibit neuromuscular defects (2 trials ± SEM; n.s.:not significant, p-value>0.05).</p

Supplemental Material for Sorkaç et al., 2018

Raw Data for Table 1 in the article entitled:<div><br></div><div>"LIN-12/Notch regulates GABA signaling at the <em>C. elegans</em>neuromuscular junction"</div