PDF(Pubmed)
Abstract：
Hemifacial microsomia (HFM) is a rare congenital genetic syndrome primarily affecting the first and second pharyngeal arches, leading to defects in the mandible, external ear, and middle ear. The pathogenic genes remain largely unidentified. Whole-exome sequencing (WES) was conducted on 12 HFM probands and their unaffected biological parents. Predictive structural analysis of the target gene was conducted using PSIPRED (v3.3) and SWISS-MODEL, while STRING facilitated protein-to-protein interaction predictions. CRISPR/Cas9 was applied for gene knockout in zebrafish. In situ hybridization (ISH) was employed to examine the spatiotemporal expression of the target gene and neural crest cell (NCC) markers. Immunofluorescence with PH3 and TUNEL assays were used to assess cell proliferation and apoptosis. RNA sequencing was performed on mutant and control embryos, with rescue experiments involving target mRNA injections and specific gene knockouts. CDC27 was identified as a novel candidate gene for HFM, with four nonsynonymous de novo variants detected in three unrelated probands. Structural predictions indicated significant alterations in the secondary and tertiary structures of CDC27. cdc27 knockout in zebrafish resulted in craniofacial malformation, spine deformity, and cardiac edema, mirroring typical HFM phenotypes. Abnormalities in somatic cell apoptosis, reduced NCC proliferation in pharyngeal arches, and chondrocyte differentiation issues were observed in cdc27-/- mutants. cdc27 mRNA injections and cdkn1a or tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia, while sox9a mRNA administration partially restored the defective phenotypes. Our findings suggest a functional link between CDC27 and HFM, primarily through the inhibition of CNCC proliferation and disruption of pharyngeal chondrocyte differentiation.
摘要：
HFM是一种罕见的先天性遗传综合征，主要影响第一和第二咽弓，导致下颌骨缺损,外耳,中耳。致病基因在很大程度上仍未被识别。对12个HFM先证者及其未受影响的生物亲本进行全外显子组测序（WES）。使用PSIPRED（v3.3）和SWISS-MODEL对靶基因进行了预测性结构分析，而STRING促进了蛋白质与蛋白质相互作用的预测。CRISPR/Cas9用于斑马鱼的基因敲除。采用原位杂交（ISH）检查靶基因和神经c细胞（NCC）标记的时空表达。使用PH3和TUNEL测定的免疫荧光来评估细胞增殖和凋亡。对突变和对照胚胎进行RNA测序，包括靶mRNA注射和特异性基因敲除的拯救实验。CDC27被鉴定为HFM的新候选基因，在三个不相关的先证中检测到四个非同义从头变异。结构预测表明CDC27的二级和三级结构发生了显着变化。斑马鱼cdc27基因敲除导致颅面畸形，脊柱畸形,心脏水肿,反映典型的HFM表型。体细胞凋亡异常，减少咽弓的NCC增殖，在cdc27-/-突变体中观察到软骨细胞分化问题。cdc27mRNA注射和cdkn1a或tp53基因敲除可显着挽救咽弓软骨发育不良，而sox9amRNA的给药部分恢复了有缺陷的表型。我们的研究结果表明CDC27和HFM之间存在功能联系，主要通过抑制CNCC增殖和破坏咽软骨细胞分化。
