NDUFA1 retrocopy引起菱脑融合畸形的致病作用及机制研究

Rhombencephalosynapsis (RES) is a rare cerebellar malformation presenting motor and cognitive impairment with an unknown aetiology. So far, there is no useful treatment for RES. In a rare isolated RES case, we had identified NDUFA1 retrocopy as a novel candidate causative variation, which might function in neurodevelopment as an essential component of mitochondrial complex I, by whole exome sequencing. We confirmed that the patient carries only one NDUFA1 retrocopy, which is composed of whole coding sequence region and partial untranslated region as a retrotransposed product of NDUFA1 mRNA, indicating a doubled copy number of NDUFA1 gene. The NDUFA1 retrocopy had been proven with a transposed insertion into intron 29 of VARS gene without an alteration of expression level and splicing pattern of VARS mRNA. Functional study showed a significantly increased expression of NDUFA1 mRNA and a dramatically elevated enzymatic activity of mitochondrial complex I to 3SD higher than reference mean in the patient’s peripheral blood cells, revealing a NDUFA1 gain-of-function mechanism in the pathogenesis of RES. As NDUFA1 is very sensitive to dosages, which is highly conserved between children and adults with little individual difference, we speculated that the elevated NDUFA1 expression level and enzymatic activity caused by NDUFA1 retrocopy would increase energy production, disturb cell apoptosis, and lead to RES finally. To further interpret the involvement of NDUFA1 retrocopy and NDUFA1 gain-of-function in the pathogenesis of RES, we intend to construct NDUFA1/Ndufa1 overexpression cell lines to evaluate the mitochondrial function, ATP level, cell survival and apoptosis of neural cells. Moreover, we also intend to construct Ndufa1 overexpression caused RES mouse model by the efficient PiggyBac transgenic technique to investigate the expression and function of Ndufa1 gene, observe the anatomical changes in the midbrain and hindbrain, and assess motor and cognitive behaviors in terms of both heterozygous and homozygous transgenic model compared with wild-type mice. Our project aims to demonstrate the pathogenetic role of NDUFA1 retrocopy and NDUFA1 gain-of-function mechanism, provide evidence for precise diagnosis and genetic counseling, and establish new platforms of cell and animal model for RES study.

菱脑融合（RES）是罕见小脑发育畸形，机制不明，临床表现为运动障碍，无有效治疗方案。我们前期全外显子组测序首次发现神经发育相关的线粒体呼吸链复合酶I基因NDUFA1 retrocopy为疑似病因。预实验证实患儿携带1个拷贝的NDUFA1 retrocopy，为mRNA逆转录产物，引起NDUFA1拷贝数增加。NDUFA1 retrocopy插入VARS基因内含子，对其mRNA表达和剪接无明显影响，但NDUFA1自身mRNA表达和蛋白功能显著上调。NDUFA1高度保守，对剂量敏感，推测NDUFA1 retrocopy获得功能引起能量增加、细胞凋亡受阻致病。本项目正构建NDUFA1/Ndufa1过表达细胞和小鼠模型，拟分析神经细胞存活和凋亡、线粒体数目和形态以及ATP水平，比对野生、杂合、纯合小鼠中后脑形态和结构、运动和认知功能，以验证推测。本项目将首证RES病因和机制，为精准诊治提供科学依据。

菱脑融合畸形（RES）是罕见的小脑发育畸形，临床表现为运动和认知障碍，其发病机制尚不明了，尚无有效治疗方案。本项目前期全外显子组测序首次发现神经发育相关的线粒体呼吸链复合酶I基因NDUFA1 retrocopy为疑似病因。预实验证实患儿携带1个拷贝的NDUFA1 retrocopy，为mRNA逆转录产物，引起NDUFA1拷贝数增加。NDUFA1 retrocopy插入VARS基因29号内含子，对其mRNA表达和剪接无明显影响，但NDUFA1自身mRNA表达和蛋白功能显著上调。NDUFA1基因高度保守，对剂量敏感，推测NDUFA1 retrocopy获得功能引起能量增加、细胞凋亡受阻致病。.本项目成功构建了NDUFA1/Ndufa1过表达细胞、小鼠和斑马鱼模型，实验结果显示过表达细胞NDUFA1/Ndufa1酶活性升高、细胞存活率降低、凋亡细胞数目无明显差异，但SK-N-SH细胞和N2a细胞ATP浓度以及细胞增殖和迁移能力不完全相符；Ndufa1过表达小鼠中后脑外观、运动和认知能力暂未见明显异常；ndufa1过表达斑马鱼存活率降低、生长发育缺陷、运动神经元发育障碍、运动功能异常。.由于上述部分实验结果不能完全吻合，因此NDUFA1 retrocopy引起RES的作用及分子机制暂未能完全明确，还需后续进一步补充实验获取更多证据支撑NDUFA1 retrocopy获得功能为RES病因和机制，正在计划进行深入研究。

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