AGL基因剪切突变和糖原累积症Ⅲ型肌肉受累的相关性研究

Glycogen storage disease type Ⅲ is autosomal recessive metabolic disorders of glycogen metabolism.result from deficiency glycogen debrancher enzyme activity.The enzyme gene was isolated on 1p21,called AGL. Hepatomegaly, hypoglycemia,short stature and slowly progressive muscle weakness arethe predominant feature in children with type Ⅲ disease. During infancy serum AST,ALT,LDH,ALP levels are markedly elevated. The applicant has confirmed the relationship between the splicing mutations of AGL in the Chinese population of GSD III. This study will further explore the function of the AGL gene in the GSD III survey of their mutation types and sites, as well as different types of mutations in children with myopathy (eg, muscle weakness, cardiomyopathy) and development relations, understanding of the genotype and clinical phenotype correlation. On this basis, to study the molecular mechanism of splicing mutation in the gene function, a clear lack of expression in vitro enzyme activity in the complete gene function in cells, further reveal the relationship of the AGL gene inactivation patterns and disease progression and prognosis of the disease gene differences qualitative more in-depth discussion. For the first time, further revealing the molecular mechanisms of the development and prognosis of the course. For great significance, improving our understanding and treatment for the disease of the world.

糖原累积症Ⅲ型为常染色体隐性遗传的代谢性疾病，患儿主要表现为低血糖，肝酶升高，以及进行性发展的肌无力和心脏受累。此病是由于AGL基因突变引起糖原脱支酶合成障碍，糖原不能分解所致。申请人曾首次证实AGL剪切突变和中国人群中GSDⅢ的关系。本研究将进一步探索AGL在GSDⅢ中的基因功能，调查GSDⅢ患者AGL基因突变的类型及位点，以及不同种类的突变与患儿进行性肌病（如肌无力，心肌病）的发生和发展的关系，了解此病的基因型和临床表现的联系。同时，在此基础上研究其基因功能中剪接突变的分子机制，明确体外缺失表达完整基因功能细胞中的酶活性，进一步揭示AGL基因失活类型和疾病进展及预后的关系，对疾病的基因异质性作出更深入的探讨。该研究在国内外尚属首次，对进一步揭示此病的分子机制，病程的发展及预后，提高中国乃至世界的认识和治疗水平具有重要意义。

本研究按计划建立了肝糖原累积症三型AGL基因外周血RNA RT-PCR的分析方法。结合DNA的PCR检测，我们在50例患者中发现了AGL基因53种突变，其中26种为新突变，丰富了中国人AGL基因突变谱，明确了剪切突变c.1735+1G>A为最常见突变，占24.21%。另外第一次报道了剪切突变是中国人AGL基因突变发生率最高的突变类型,占49.5%。.本研究第一次针对AGL基因的15种剪切突变进行了RNA的RT-PCR分析，发现了13种剪切效应，并且第一次报道了c.665-1G>A剪切突变可以在不同的患者中造成2种不同剪切效应, 即外显子4跳跃或外显子5跳跃；发现了2种不同剪切突变（c.664+1G>A和c.665-1G>A；和c.1423+1G>C和c.1424-1G>C）可以造成一种剪切效应（外显子5跳跃；和外显子11跳跃）；第一次证明了错义突变c.958G>A，p.E320K是有剪切效应的致病突变。.通过分析患者剪切突变携带状况与肌肉（CK值）和心脏受累（心脏彩超异常）关系，发现携带2个剪切突变的患者CK升高的可能性较没有剪切突变的患者有增高趋势；但是，携带2个剪切突变的患者心脏彩超异常的可能性较其他基因型明显减少, 同时携带2个剪切突变是否对心脏有保护作用有待进一步更大样本的观察分析。同时也提示有必要采用更精确的检测手段评估心脏受累的程度。