MAP3K4在先天性脊椎发育畸形中的致病作用与机制研究

Congenital vertebral malformation (CVM) has a quite high incidence rate, which could result in severe spinal deformity in severe cases, remaining a worldwide challenge for spine surgeons. For the first time, we find a rate MAP3K4 gene copy number mutation in patients with CVM through comparative genomic hybridization chip analysis, suggesting MAP3K4 could play an essential role in resulting in CVM. Then, CRISPR-Cas9 technique is applied to construct MAP3K4 knockout mice model, which exhibits vertebral malformations via micro-CT analysis. Our preliminary results indicate MAP3K4 mutation could result in CVM. Spine development originated from somitogenesis, which process is regulated by NOTCH pathway. MAPKs is found to regulate NOTCH pathway by activating Notch receptors. Thus, we propose, MAP3K4 could regulate somitogenesis via activation NOTCH pathway, which process could lead to CVM when destroyed by MAP3K4 mutation. In this project, the following aims need to be fulfilled: 1) identify the malformation characteristics of patients with MAP3K4 mutations; 2) identify the phenotypes of Map3k4 mutant mice, prove the pathogenicity of Map3k4 in CVM; 3) clarify the regulation role of MAP3K4 on somitogenesis; 4) explore the molecular mechanism of MAP3K4 regulation of NOTCH pathway. Thus, based on the newly-found mutation and gene knockout mice model, we aim to clarify whether MAP3K4 could influence somitogenesis through regulation of NOTCH signaling pathway. The research results will provide a reference for molecular diagnosis and genetic counseling of CVM.

先天性脊椎畸形(CVM)可导致严重脊柱畸形，是脊柱外科手术面临的世界性挑战。CVM发病率高，但多数患者病因未明。申请人利用比较基因组杂交芯片技术对CVM患者进行全基因组拷贝数变异分析，发现罕见的MAP3K4拷贝数丢失，提示该基因对CVM的致病性；利用CRISPR-Cas9技术成功构建Map3k4基因变异小鼠模型，并成功观察到CVM表型｡NOTCH通路是调控体节形成的关键通路,研究发现MAPKs可以活化Notch受体，然而MAP3K4是否有此作用未有报道｡因此，申请人提出“MAP3K4通过激活NOTCH信号通路调节体节形成进而导致CVM”的课题假说，拟：明确MAP3K4突变患者临床表型规律；鉴定Map3k4突变小鼠表型，确定其致病性；阐明MAP3K4对体节形成的调控作用和机理；探究MAP3K4对NOTCH通路调控分子机制。研究结果将揭示CVM发病机制，为CVM的分子诊断和遗传咨询提供参考｡

背景：先天性脊椎畸形是新生儿重要的发育性疾病，然而，其内在发病机制仍然未明。.研究内容：本研究基于前期临床数据提出MAP3K4可能在先天性脊椎畸形中扮演重要角色，旨在从临床、动物、和细胞水平明确MAP3K4在先天性脊椎畸形中的致病作用与机制。.重要结果和关键数据：动物水平，项目组发现孕期低氧（模拟宫内窘迫）增强Map3k4基因缺陷对CVM的致病性。氧浓度降低至8%时，小鼠胚胎的畸形率均有不同程度的增加，然而Map3k4基因杂合缺失组增加更明显，有统计学差异（卡方检验，p=0.0332）.研究结果基本明确了明确Map3k4基因在小鼠中的致病性。在细胞水平，项目组发现Map3k4通过调节JNK/MAPK信号通路的激活，影响NFATc1和C-fos的转录活性，进一步抑制破骨细胞下游关键基因（NFATc1, C-fos, TRAP, CTSK, CTR, DC-STAMP）的表达,最终影响破骨细胞的分化和骨吸收能力。.科学意义：本项目研究结果在一定程度上解释了Map3k4基因变异与CVM发病的相关性。同时，本项目首次发现环境在Map3k4基因变异导致CVM的关键作用，强调了基因与环境相互作用参与疾病的发生与发展。尤其是对于宫内窘迫的胎儿，如果存在Map3k4基因变异，其出生后发生CVM的风险将大大增加，这将为未来开发新生儿产前先天性发育畸形诊断提供重要的参考。