应用患者来源iPS细胞模型解析线粒体遗传病MERRF综合征心肌损伤的新机制

Myoclonus epilepsy with ragged-red fiber (MERRF) syndrome is an inherited mitochondrial disease which is associated with severe defects and limited treatment. The mechanism underlying the cardiac phenotypes associated in MERRF syndrome remains unknown. In our previous study, 6 MERRF patient-derived iPS cell lines with different m.A8344G heteroplasmy were generated using a non-integrating reprogramming technique. MERRF-iPSCs showed lowered cardiac differentiation ability. Using transcriptome analysis, we identified a nuclear gene ZFPM2, which was downregulated and involved in the defective cardiac differentiation procedure in MERRF-iPS cells. Using ChIP assay, histone methylation and epigenetic regulations were indicated to be the key mechanism for m.A8344G mutation to affect ZFPM2 gene expression. In this study, we plan to further clarify the role of ZFPM2/GATA4 in defective differentiation procedure of MERRF-iPS. We will then focus on the regulatory mechanism by which m.A8344G mutation affects the expression of ZFPM2. Using our existing MERRF-iPS lines with various mutation heteroplasmy, we will identify the pathogenic heteroplasmic threshold for the cardiac differentiation phenotype in MERRF cells. We will finally evaluate the application potent of ZFPM2 during clinical diagnosis and prevention of MERRF syndrome. As very few studies have been reported on researching MERRF syndrome using iPS models, this work will uncover the molecular pathogenic mechanism of MERRF syndrome in cardiac defect, and may provide candidate bio-target for the diagnosis and treatment in the future.

MERRF综合征是一种危害严重且无有效治疗方法的线粒体遗传病，所引起的心肌损伤分子机制仍不清晰。前期研究中，我们采用非整合重编程技术成功构建了患者来源的6个MERRF-iPS细胞系；心肌细胞分化过程中MERRF-iPS显示出明显的分化缺陷；基因表达谱分析和ChIP实验表明线粒体m.A8344G突变可通过表观遗传机制调控核基因ZFPM2表达导致心肌细胞异常。本研究拟：确认ZFPM2/GATA4在MERRF-iPS细胞心肌分化缺陷中的作用；明确m.A8344G突变对ZFPM2基因表达的调控机制；进一步寻找MERRF-iPS出现心肌分化缺陷的突变比率阈值；并评估ZFPM2在MERRF诊断和防治中的作用。目前通过iPS细胞模型对MERRF综合征的机制研究鲜有报道，本研究将揭示MERRF综合征在心肌损伤方面的发病分子机制，为该疾病的诊断和防治提供潜在靶标。

肌阵挛性癫痫伴破红纤维(myoclonus epilepsy associated with ragged-red fibers, MERRF) 综合征是一组严重的临床异质性线粒体疾病(mitochondrial disease, MD)，直接或间接影响线粒体呼吸链和氧化磷酸化功能系统的功能。目前MERRF综合征等线粒体疾病的主要治疗方法是对症治疗，以延缓疾病的进展，尚无可治愈的治疗方法。本项目主要研究内容是通过患者来源iPS细胞模型阐述MERRF综合征致心肌发育异常的分子机制和疗法开发。我们在检测了多种不同m. 8344A>G突变比例的iPS细胞心肌分化的表型后，我们发现突变率在62%以上为出现心肌分化异常的阈值。通过在高突变率iPS细胞同低突变率iPS细胞上进行代谢组学分析，进一步探索m.8344A>G突变导致心肌分化异常的内在机制，我们发现高突变率iPS细胞在多能干细胞水平及分化所得心肌水平存在L-谷氨酰胺等多种代谢物及精氨酸生物合成途径等多种代谢途径异常，揭示了相关代谢物及代谢途径在MERRF发病机制中的作用。另一方面，我们尝试了mitoTALENS及DdCBE两种不同的策略进行线粒体DNA的突变修复。通过使用两对不同组合的A8344G-mitoTALENS,并在转染前后进行焦磷酸测序分析，我们发现其中一对mitoTALENS可以实现m.8344A>G mtDNA突变异质度短期内少量降低，另一对无明显变化。关于DdCBE，我们在废弃的3PN胚胎中评估该技术的应用潜能。在3PN胚胎上注射不同DdCBE组合后，我们成功针对G3733和G8363位点实现了较高的编辑效率，但仍存在一定的脱靶风险。这些结果表明，DdCBE技术是应用于线粒体遗传病疗法开发的优良工具，但在临床应用前仍有待进一步优化。