SLC25A32基因新突变引起代谢性肌病的致病作用及机制研究

Metabolic myopathies refer to a group of hereditary muscle disorders caused by energy generation defect of cells due to deficiencies of enzymes involved in glycogen, fatty acids or mitochondrial metabolism. Patients always present with muscle weakness and exercise intolerance, and have a high risk of morbidity and mortality. Recently, we found a patient with metabolic myopathy had novel heterozygous mutations in SLC25A32 gene by the second-generation sequencing, and the mutations were highly pathogenic according to our preliminary research. SLC25A32 gene was found pathogenic in 2016, but its pathogenic mechanism is unclear. the gene encodes an inner mitochondrial membrane carrier, however, whether the protein transfers flavin adenine dinucleotide (FAD) or folate is unclear right now. The results of metabolites analysis in plasma and urine of our patient indicated that both fatty acids and mitochondrial metabolism had disorder due to reduced activities of several mitochondria FAD-dependent flavoprotein enzymes. We hypothesis that SLC25A32 novel mutations may cause low activities of FAD-dependent flavoprotein by mitochondrial FAD transport defect and lead to fatty acids and energy metabolism disorder, then induced metabolic myopathy. We have generated slc25a32 heterozygous mutations mouse model successfully, and preliminary experiment showed that the disease mouse had similar metabolic spectrum to that of our patient. In this study, we are going to use the mouse model to verify our hypothesis by investigating mouse activity, histopathology, levels of ATP in liver and muscle, the amount of FAD in mitochondria, the abilities of mutated slc25a32 protein binding and transport FAD, and the activities of several flavin protein enzymes. This research will clarify the function and pathogenic mechanism of SLC25A32 novel mutations in metabolic myopathy, which will benefit accurate diagnosis and treatment of this kind of disease.

代谢性肌病是一组由于糖、脂肪或线粒体代谢异常引起细胞产能障碍的疾病，以肌无力、运动不耐受为主要表现，严重致残致死。项目组前期二代测序发现一例该病患儿携带SLC25A32基因复合杂合新突变，疑似致病。该基因是2016年才报道的新致病基因，机制不明，其编码线粒体内膜转运蛋白，但底物为黄素腺嘌呤二核苷酸（FAD）还是叶酸尚有争议。患儿生化检测提示参与脂肪和线粒体产能的多种以FAD为辅酶的黄素蛋白酶活性下降，推测SLC25A32突变引起FAD转运障碍、黄素蛋白酶活性下降、脂肪和线粒体代谢障碍致病。本项目已成功构建slc25a32复合杂合新突变小鼠模型，其代谢物谱与患儿相似，拟进一步分析小鼠运动功能、肌肉病理、肝脏和肌肉ATP水平、线粒体内FAD含量、slc25a32突变蛋白结合和转运FAD的能力及黄素蛋白酶活性，以验证推测。本项目将明确SLC25A32新突变致病作用和机制，为精准诊疗提供科学依据。

代谢性肌病是一组由于糖、脂肪或线粒体代谢异常引起细胞产能障碍的疾病，以肌无力、运动不耐受为主要表现，具有严重致残性。项目组在临床发现一例代谢性肌病患儿携带有SLC25A32基因复合杂合新突变，疑似致病。SLC25A32基因编码线粒体内膜转运蛋白，但底物为黄素腺嘌呤二核苷酸（FAD）还是叶酸尚有争议。. 本项目采用CRISPR-Cas9 基因编辑技术,构建了三个小鼠模型（Slc25a32 Y174C/Y174C, Slc25a32 K235R/K235R, 和Slc25a32Y174C/K235R），通过分析小鼠运动能力、线粒体内FAD含量、线粒体摄取FAD的能力及黄素蛋白酶活性测定的等实验，研究SLC25A32变异的致病机制。结果表明Slc25a32 Y174C/K235R和Slc25a32 K235R/K235R小鼠的肌力、耐力和平衡能力均比同窝野生型小鼠弱，其代谢物谱与患者代谢物谱改变一致。模型成功构建，证实了SLC25A32基因新突变具有致病性。另外，研究显示Slc25a32变异主要累及脑和骨骼肌，而肝脏受累不明显。与野生型小鼠比，Slc25a32K235R/K235R和Slc25a32Y174C/K235R骨骼肌线粒体中FAD，FMN和核黄素含量明显下降，其线粒体摄取FAD含量也显著下降，但摄入核黄素和叶酸的能力未见异常，从而明确了Slc25a32是线粒体FAD转运蛋白，而非叶酸转运蛋白。对线粒体内多种黄素蛋白酶的酶活性和含量结果显示，Slc25a32K235R/K235R小鼠骨骼中参与氨基酸和脂肪酸代谢的多个黄素蛋白酶活性均明显下降，反应体系中加入FAD后，各种酶活性有不同程度的恢复。进一步Western-Blot试验显示Slc25a32K235R/K235R小鼠酶活性不能通过外源性FAD恢复的黄素蛋白酶，其含量均显著下降。该研究证实了Slc25a32变异不仅剥夺了线粒体内多个黄素蛋白酶的辅酶因子，还使其载体蛋白含量下降，从而导致多种酰基CoA脱氢酶缺乏。. 本项目建立了筛查和诊断SLC25A32基因突变所致神经性肌病的一系列方法，均可直接实现临床应用。对致病机制的研究为治疗方案的改进奠定了基础。

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