眼咽远端型肌病致病基因NAGK的鉴定与功能分析

Oculopharyngodistal myopathy (OPDM) is a rare inherited muscular disease, of which the causative gene has not been identified. Our previous works have found that in the muscle fibers of OPDM patients there exited rimmed vacuoles, together with ER (endoplasmic reticulum) stress, autophagy dysfunction, and filamentous inclusions. We have also found compound heterozygous NAGK gene mutations in an autosomal recessive inherited OPDM family through whole-exome sequencing, and protein structure prediction suggests it pathogenic. NAGK encodes a key enzyme in the metabolism of sialic acid, and its mutation would probably lead to protein glycosylation dysfunction by affecting sialic acid metabolism of myocyte, and finally result in the aggregation of abnormal proteins as well as the formation of autophagic vacuoles in muscle fibers. The project will carry on thorough analyses on the gene mutation of NAGK and its protein expression in OPDM patients. Besides, a cell model and an animal model of OPDM will be generated: inducing OPDM-iPSCs (induced pluripotent stem cells) and directionally differentiating those into myocytes, as well as constituting a NAGK mutated transgenic mouse model. A series of researches will be carried on these models, including measurement of the content of sialic acid and the expression level of glycosylated proteins, rule of autophagy disruption, epigenetics and proteomics, by the methods of biochemistry, pathology, gene-chip technology, proteomics, etc. Analyses of the molecules interacted with NAGK and their functions will help to study the molecular mechanisms of OPDM caused by NAGK mutation, and finally provide a potential target for the treatment of the disease.

眼咽远端型肌病（OPDM）是一种致病基因未明的罕见肌肉病。我们前期工作发现OPDM的肌纤维内存在自噬性镶边空泡，并出现内质网应激、自噬异常以及管丝样包涵体；通过全外显子组测序在一个常隐遗传的OPDM家系中发现NAGK基因新突变，蛋白结构预测为致病突变。NAGK编码蛋白是唾液酸代谢中的关键酶之一，其基因突变可能通过影响肌细胞的唾液酸代谢而导致蛋白糖基化障碍，从而引发肌纤维内异常蛋白的聚集以及自噬空泡形成。本项目将对OPDM患者深入分析其NAGK基因的突变和蛋白表达；拟诱导产生OPDM-iPSC并定向分化为肌细胞，以及构建NAGK突变转基因鼠模型，运用生化、病理、基因芯片技术和蛋白组学等方法对细胞和动物模型检测唾液酸含量和糖基化蛋白的表达、细胞自噬改变规律以及表观遗传学和蛋白组学研究。通过分析NAGK的相互作用分子及其功能，探讨NAGK突变导致OPDM的分子机制，为该病的治疗提供可能的靶点。

眼咽远端型肌病（OPDM）是一种罕见的遗传性肌肉病，其致病基因未明。前期工作发现OPDM患者肌纤维内存在自噬性镶边空泡，并出现内质网应激、自噬异常以及管丝样包涵体；通过全外显子测序在一个常隐遗传的OPDM家系中发现NAGK基因新突变，蛋白结构预测为致病突变。根据课题研究计划，我们建立OPDM临床随访数据库，完成NAGK基因的突变检测和表达研究，总结OPDM患者的肌肉病理改变，分析内质网应激和自噬通路相关的蛋白表达。并建立Loss-of-function果蝇动物模型和NAGK基因条件性敲除小鼠动物模型，验证NAGK基因功能。但因为NAGK突变未能在更多OPDM患者中得到验证、果蝇动物试验未获阳性结果，我们及时调整研究方案，通过全基因组测序、三代测序、重复引物聚合酶链式反应和荧光扩增产物长度分析聚合酶链式反应相结合的综合策略，首次鉴定出Gene G(文章尚未发表，以Gene G代称致病基因）5’-UTR区(GGC)n异常扩增是OPDM的致病突变。进一步通过甲基化分析和Western-blot，发现OPDM患者Gene G表达水平不受影响，又通过RNA-Seq提示了OPDM机制中涉及的分子和通路。此研究为临床诊断和进一步了解OPDM的发病机制提供理论依据。

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