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目的研究中国人群家族性传导系统异常伴心室预激及心肌肥厚患者新错义突变的PRKAG2基因表达和功能变化。方法利用PCR扩增其功能区外显子片段,双脱氧末段终止测序法来构建表达PRKAG2G100S、PRKAG2R302Q及野生型(WT)PRKAG2基因的慢病毒载体,并对其从细胞学水平进行体外实验研究。结果(1)PRKAG2 G100S和PRKAG2 R302Q突变对PRKAG2基因在CCL13细胞中的表达位置没有影响;(2)PRKAG2突变组CCL13/GS和CCL13/RQ与CCL13/WT组相比,PRKAG2蛋白表达量减少,且细胞质及胞核内有较多糖原沉积;(3)PRKAG2基因G100S和R302Q突变降低了CCL13细胞中的AMPK活性,而G100S突变降低AMPK活性的作用弱于R302Q突变(P<0.05或P<0.01)。结论 PRKAG2G100S突变是中国人PRKAG2心脏综合征家系的发病原因,初步证实了突变导致AMPK活性降低及糖原累积是家系的发病机制;G100S突变可能使PRKAG2基因Bateman结构域功能发生变化,但对Bateman结构域功能的影响弱于R302Q突变。
Objective To study the expression of PRKAG2 gene and its functional changes in patients with familial conduction system abnormalities associated with ventricular pre-excitation and cardiac hypertrophy in Chinese population. Methods The lentiviral vector expressing PRKAG2G100S, PRKAG2R302Q and WT PRKAG2 gene was amplified by PCR and sequenced by dideoxy terminator, and its cytotoxicity was studied in vitro . Results (1) PRKAG2 G100S and PRKAG2 R302Q mutations had no effect on the expression of PRKAG2 in CCL13 cells. (2) Compared with CCL13 / WT, the expression of PRKAG2 protein decreased in PRKAG2 mutant group, (3) The G100S and R302Q mutations of PRKAG2 gene decreased the activity of AMPK in CCL13 cells, while the effect of G100S mutation on AMPK activity was weaker than that of R302Q mutation (P <0.05 or P <0.01) ). Conclusions The PRKAG2G100S mutation is the cause of PRKAG2 cardiac syndrome in Chinese families. It is preliminarily confirmed that the mutation causes the decrease of AMPK activity and glycogen accumulation is the pathogenesis of the pedigree. The mutation of G100S may change the function of Bateman domain of PRKAG2 gene. However, The effect of domain function is weaker than the R302Q mutation.