Abiusi Emanuela, D'Alessandro Manuela, Dieterich Klaus, Quevarec Loic, Turczynski Sandrina, Valfort Aurore-Cecile, Mezin Paulette, Jouk Pierre Simon, Gut Marta, Gut Ivo, Bessereau Jean Louis, Melki Judith
Institut National de la Santé et de la Recherche Médicale (Inserm) UMR-1169, Université Paris Sud, 94276 Le Kremlin Bicêtre, France.
INSERM U-1217, Institut NeuroMyoGène, Univ Lyon, Université Claude Bernard Lyon 1, CNRS UMR-5310, F-69622 Villeurbanne, France.
Hum Mol Genet. 2017 Oct 15;26(20):3989-3994. doi: 10.1093/hmg/ddx288.
Arthrogryposis multiplex congenita (AMC) is a developmental condition characterized by multiple joint contractures resulting from reduced or absent fetal movements. Homozygosity mapping of disease loci combined with whole exome sequencing in a consanguineous family presenting with lethal AMC allowed the identification of a homozygous frameshift deletion in UNC50 gene (c.750_751del:p.Cys251Phefs*4) in the index case. To assess the effect of the mutation, an equivalent mutation in the Caenorhabditis elegans orthologous gene was created using CRISPR/Cas9. We demonstrated that unc-50(kr331) modification caused the loss of acetylcholine receptor (AChR) expression in C. elegans muscle. unc-50(kr331) animals were as resistant to the cholinergic agonist levamisole as unc-50 null mutants suggesting that AChRs were no longer expressed in this animal model. This was confirmed by using a knock-in strain in which a red fluorescent protein was inserted into the AChR locus: no signal was detected in unc-50(kr331) background, suggesting that UNC-50, a protein known to be involved in AChR trafficking, was no longer functional. These data indicate that biallelic mutation in the UNC50 gene underlies AMC through a probable loss of AChR expression at the neuromuscular junction which is essential for the cholinergic transmission during human muscle development.
先天性多发性关节挛缩症(AMC)是一种发育性疾病,其特征是由于胎儿活动减少或缺乏导致多个关节挛缩。在一个患有致死性AMC的近亲家庭中,结合疾病基因座的纯合子定位和全外显子组测序,在索引病例中鉴定出UNC50基因的纯合移码缺失(c.750_751del:p.Cys251Phefs*4)。为了评估该突变的影响,使用CRISPR/Cas9在秀丽隐杆线虫直系同源基因中创建了一个等效突变。我们证明,unc-50(kr331)修饰导致秀丽隐杆线虫肌肉中乙酰胆碱受体(AChR)表达缺失。unc-50(kr331)动物对胆碱能激动剂左旋咪唑的抗性与unc-50基因敲除突变体相同,这表明在该动物模型中AChR不再表达。通过使用一种敲入菌株进行了证实,在该菌株中红色荧光蛋白被插入到AChR基因座中:在unc-50(kr331)背景中未检测到信号,这表明已知参与AChR转运的蛋白质UNC-50不再具有功能。这些数据表明,UNC50基因的双等位基因突变通过神经肌肉接头处AChR表达的可能缺失成为AMC的基础,而AChR表达对于人类肌肉发育过程中的胆碱能传递至关重要。