title: In Vivo Modelling of ATP1A3 G316S-Induced Ataxia in C. elegans Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects.
abstract: The NIH Undiagnosed Diseases Program admitted a male patient with unclassifiable late-onset ataxia-like symptoms. Exome sequencing revealed a heterozygous de novo mutation converting glycine 316 to serine in ATP1A3, which might cause disease. ATP1A3 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 Caenorhabditis elegans, recreating the patient amino acid change in eat-6, the orthologue of ATP1A3. The impact of the mutation on eat-6 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 eat-6 were recessive. These results indicate that the mutation is dominant and impairs the neuromuscular function. Thus, we conclude that the de novo G316S mutation in ATP1A3 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 C. elegans using CRIPSR/Cas9 genome editing.
authors.affiliation: Department of Neuroscience, Brown University, Providence, RI, United States of America.
authors.forename: Altar
authors.initials: A
authors.lastname: Sorkaç
authors.affiliation: Department of Neuroscience, Brown University, Providence, RI, United States of America.
authors.forename: Ivan C
authors.initials: IC
authors.lastname: Alcantara
authors.affiliation: Department of Neuroscience, Brown University, Providence, RI, United States of America.
authors.forename: Anne C
authors.identifier: http://orcid.org/0000-0001-7239-4350
authors.initials: AC
authors.lastname: Hart
mesh_terms: D000483:Alleles; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005799:Genes, Dominant; D006579:Heterozygote; D059765:Homologous Recombination; D006801:Humans; D009154:Mutation; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
chemical_list: C488756:ATP1A3 protein, human; D000254:Sodium-Potassium-Exchanging ATPase