目的：探索半侧颜面短小畸形的高危致病变异，并通过斑马鱼模型对候选基因致病性进行验证。

方法：对12个半侧颜面短小畸形先证者及其无症状父母进行全外显子组测序，测序结果经过频率比对、功能注释和致病性预测筛选高危致病变异。通过原位杂交实验建立候选基因在斑马鱼胚胎的时空表达谱。运用CRISPR/Cas9 基因编辑技术敲除候选基因，进一步对斑马鱼颌面发育情况进行观察。通过阿尔新兰染色实验对突变体胚胎颌面软骨发育情况进行检测。通过免疫荧光染色探究胚胎细胞的增殖和凋亡情况。采用标记基因对特定时期斑马鱼胚胎进行原位杂交染色，探究候选基因在颌面软骨发育过程中的作用。

结果：CDC27, MUC6, MUC16, FAM136A和XKR3可能是半侧颜面短小畸形的高危致病基因。在斑马鱼中利用CRISPR/Cas9敲除cdc27可使胚胎出现颌面软骨发育畸形以及小头、小眼、脊柱畸形和心腔水肿的表型。cdc27编码的Cdc27功能缺失可引起神经嵴细胞的增殖减少并阻碍神经嵴细胞向软骨细胞的分化过程。

结论：CDC27某些位点的非同义变异可能是人类半侧颜面短小畸形的致病原因。斑马鱼胚胎发育过程中Cdc27功能缺失导致神经嵴细胞的增殖减少并阻碍其向软骨细胞分化，最终引起颌面软骨和脊柱的发育畸形。

Objective

To investigate the high-risk pathogenic variation of hemifacial microsomia (HFM), and verify the pathogenicity of candidate genes through zebrafish model.

Methods

Whole exome sequencing was conducted in 12 probands with HFM and their asymptomatic parents. The sequencing results were screened for high-risk pathogenic genes through frequency comparison, functional annotation and pathogenicity prediction. The temporal and spatial expression profiles of candidate genes in zebrafish were established by in situ hybridization (ISH). CRISPR / cas9 technique was used to knock out the expression of candidate genes to further observe the maxillofacial development of zebrafish. The maxillofacial cartilage of zebrafish embryos was stained by alcian blue staining technique. The proliferation and apoptosis of embryonic cells were observed by immunofluorescence staining. ISH was used to detect the role of candidate genes in the development of maxillofacial cartilage.

Results

CDC27, MUC6, MUC16, FAM136A and XKR3 may be high-risk pathogenic genes of HFM. Knockout of cdc27 by CRISPR/Cas9 in zebrafish can lead to the phenotype of maxillofacial cartilage malformation, small head, small eye, spinal deformity and cardiac edema. The loss of Cdc27 function encoded by cdc27 can reduce the proliferation of neural crest cells and hinder the differentiation of neural crest cells into chondrocytes.

Conclusion

The nonsynonymous variation of CDC27 may be the cause of HFM. During zebrafish embryonic development, the Cdc27 function loss leads to the reduction of the proliferation of neural crest cells and hinders their differentiation into chondrocytes, which eventually leads to the developmental deformity of maxillofacial cartilage and spine.