In Vivo Modelling of ATP1A3 G316S-Induced Ataxia in C. elegans Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects.
In Vivo Modelling of ATP1A3 G316S-Induced Ataxia in C. elegans Using CRISPR/Cas9-Mediated Homologous Recombination Reveals Dominant Loss of Function Defects.
复制标题
DOI：
10.1371/journal.pone.0167963
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Hart AC
中科院分区：
文献类型：
--
作者：
Sorkaç A;Alcantara IC;Hart AC
美国国立卫生研究院未确诊疾病项目收治了一名患有无法分类的迟发性共济失调样症状的男性患者。外显子组测序揭示了 ATP1A3 中的杂合从头突变将甘氨酸 316 转变为丝氨酸，这可能导致疾病。 ATP1A3 编码 Na+/K+ ATP 酶泵 α3 亚基。利用 CRISPR/Cas9 介导的同源重组进行基因组编辑，我们对秀丽隐杆线虫中这一假定的致病等位基因进行了建模，重现了 ATP1A3 的直系同源 eat-6 中患者氨基酸的变化。使用两种行为分析来检查突变对神经肌肉接头处 eat-6 功能的影响：咽部泵送速率和对涕灭威的敏感性，涕灭威是一种通过抑制乙酰胆碱酯酶随时间推移导致麻痹的药物。患者等位基因降低了泵血率并导致对涕灭威过敏。等位基因杂合的动物表现出类似的缺陷，而 eat-6 的功能丧失突变是隐性的。这些结果表明该突变占主导地位并损害神经肌肉功能。因此，我们得出结论，ATP1A3 中的从头 G316S 突变可能导致或促成患者症状。更广泛地说，我们得出的结论是，对于保守基因，可以使用 CRIPSR/Cas9 基因组编辑快速、轻松地对线虫中的人类疾病进行建模。
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.
登录
查看更多内容
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
4.7
作者：
Lakso, M;Vartiainen, S;Wong, G
通讯作者：
Wong, G
影响因子：
3.5
作者：
Doi, Motomichi;Iwasaki, Kouichi
通讯作者：
Iwasaki, Kouichi
DOI：
10.1006/bbrc.1998.8981
发表时间：
1998-07-30
影响因子：
3.1
作者：
Shima, Y;Tada, Y;Ohta, H
通讯作者：
Ohta, H
影响因子：
2.5
作者：
Bottger, Pernille;Tracz, Zuzanna;Lykke-Hartmann, Karin
通讯作者：
Lykke-Hartmann, Karin