EFTUD2基因通过mTOR信号通路影响成骨和软骨细胞发育与功能的分子机制研究

Growth is the basic element of human health. Genetic defects and associated syndromes are the hot spots of current research. With the huge progress in DNA sequencing technique especially the advance in next generation sequencing, the possibility for detection of potential genetic defects is ever increasing, and mandibulofacial dysostosis and microcephaly (MFDM, MIM#610536) was luckily in the line. MFDM also known as mandibulofacial dysostosis, Guion-Almeidatype (MFDGA), caused by genetic mutation of elongation factor Tu GTP-binding domain-containing 2 (EFTUD2) gene mutation, usually characterized by a wide spectrum of congenital anomalies, including micrognathia, malar hypoplasia, dysplastic ears with or without conductive hearing loss, microcephaly, limb defects, short stature and so on, while, its pathogenesis is still in exploring.. We used Whole Exon Sequencing technique to detect the potential genetic defects in a Chinese children and found a novel EFTUD2 gene mutation c.1030_1031delTG (p.Trp344fs*2). We then used Zebrafish as a study model, we first studied the expression profile and found that EFTUD2 gene was widely expressed in zebrafish at both early and adult stages; transient disruption of the gene resulted in a 6-fold increase in larval deformity, while rescue significantly decreased the incidence of deformity. Dysplasia in multiple bones or even otolith loss was present, and the deformities of the upper jaw and jaw bones observed in adult heterozygous zebrafish were similar to those recorded in human survivors. . We down regulated the expression of EFTUD2 gene in human calvarial osteoblasts (HCOs) and articular chondrocytes (HC-a) through specific short hairpin RNAs (shRNA) , we found that the expression of the ALP, COL1A1, OPN and SOX9 genes associated with cell differentiation was inhibited, the normal proliferation of the HCO and HC-a was disrupted, calcium deposition significantly and consistently decreased without evident calcific nodules in the shRNA-treated groups. We then further sequenced the mRNA using RNA_Seq technique in the above groups and found significantly increased splicing of EIF4B, RPS6KB2, MLST8, STRADB and so on, while all these genes are the key elements of mTOR pathway which have robust evidences involving osteogenesis. . Based on the above evidence, We will further generate EFTUD2 over-expression zebrafish, p.T rp344fs * 2 mutant zebrafish, and analyze the characteristics of phenotype changes in bone growth, development, and study the corresponding changes of mTOR pathways and the other key genes involving bone development after EFTUD2 abnormal regulation both in zebrafish and HCO, HCA cells, and finally we will explore the mTOR pathway negative regulatory factor PTEN intervention effects on above Zebrafish and cell lines, unveil the bone malformations mechanism caused by the EFTUD2 defects and to provide the clinical clues for potential intervention.

生长相关遗传缺陷及其综合征是研究热点，下颌面骨发育不全并小头畸形综合征（MFDM）是其中之一，致病机制不详。我们首次发现MFDM致病基因EFTUD2新发突变（p.Trp344fs*2），该基因短时敲减和长时敲除斑马鱼模型纯合子早期死亡，杂合子颅面等骨畸形；EFTUD2基因下调导致人颅骨成骨(HCO)和关节软骨(HCA)细胞增殖受显著抑制，凋亡增加，钙化结节生成受抑；mRNA剪切差异分析发现骨发育枢纽分子mTOR通路相关基因RPS6KB2、MLST8、STRADB等可变剪切显著增加。我们进一步构建EFTUD2过表达、p.Trp344fs*2突变体斑马鱼，分析骨骼生长、发育等表型特征，研究EFTUD2调控对斑马鱼和HCO、HCA细胞mTOR通路及其他骨发育调控关键基因的影响，探究mTOR通路负性调控因子PTEN干预作用，较完整揭示EFTUD2缺陷致骨发育畸形机制，为临床提供潜在的干预线索。

生长是儿童健康的基本要素，遗传缺陷及其导致的关联综合征是研究的热点，下颌面骨发育不全并小头畸形综合征（MFDM）是其中之一，致病机制不详。我们首次发现MFDM致病基因EFTUD2新发突变（p.Trp344fs*2），围绕EFTUD2基因通过mTOR信号通路影响成骨和软骨细胞发育与功能的分子，我们先后构建EFTUD2基因短时敲减、过表达、稳定敲除斑马鱼，观察生理表型变化，系统研究了EFTUD2基因突变对骨骼发育、听力形成的影响，并采用骨骼细胞进行了验证，分析了骨细胞分化发育调控经典通路基因及mTOR信号通路基因的相应时空表达变化特征，与斑马鱼的早期总体死亡率、畸形率、骨骼发育变化间的关联性；初步阐明了EFTUD2基因变异致骨骼发育的机制以及人工挽救干预的潜在效果。本研究项目共发表SCI论文共4篇（总IF 23.265），培养毕业博士研究生1名、硕士研究生1名。

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