心脏发育候选致病基因KIAA0196斑马鱼品系的建立及初步机制研究

Establishment and preliminary mechanism study of the zebrafish strain of KIAA0196: A candidate pathogenic gene for heart development

目的:探究KIAA0196基因对心脏发育的影响及斑马鱼品系的建立。方法:提取先天性心脏病患者外周血和gDNA,采用拷贝数变异和靶向序列分析筛选出候选致病基因。用CRISPR/Cas9技术建立KIAA0196基因敲除斑马鱼品系。对野生型和突变型斑马鱼进行解剖和组织学染色,观察心脏形态、大小及环化方向等表型。结果:通过CRISPR/Cas9技术成功构建了斑马鱼KIAA0196基因敲除品系。受精后60h,突变个体(杂合子+纯合子)显微镜检查显示心包积液、心脏受压和尾部严重卷曲。突变型斑马鱼与野生型相比,心脏存在心房缩小,心室增大,心肌细胞更加松散等缺陷。结论:KIAA0196基因在心脏发育过程中起重要的调控作用,该基因可能为先天性心脏病的致病候选基因。

Objective:To explore the effects of KIAA0196 gene on cardiac development and the establishment of zebrafish strain. Methods:Peripheral blood and gDNA from patients were extracted.Copy number variation analysis and target sequencing were conducted to screen candidate genes.The KIAA0196 knockout zebrafish was generated by CRISPR/Cas9 to detect whether KIAA0196 deficiency could affect cardiac development.Finally,the wild-type and mutant zebrafish were anatomized and histologically stained to observe the phenotype of heart defects. Results:The KIAA0196 knockout zebrafish strain was successfully constructed using CRISPR/ Cas9 technology.After 60 hours fertilization,microscopic examination of KIAA0196 knockout zebrafish (heterozygote + homozygote) showed pericardial effusion,cardiac compression and severely curly tail.Compared with wild-type zebrafish,the hearts of mutant KIAA0196 zebrafish had cardiac defects including smaller atrium and larger ventricle,and the myocardial cells were looser. Conclusion:KIAA0196 gene plays an important regulatory role in the development of heart.It might be a candidate gene for congenital heart disease.