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目的:房室传导阻滞是心脏电激动传导过程中,发生在心房和心室之间的电激动传导异常,可导致心律失常。前期,我们发现一个遗传性房室传导阻滞伴肥厚型心肌病家系,本次工作主要对其致病基因及机制进行研究。方法:采用候选基因测序的方法对已经确诊为房室传导阻滞的心脏病患者进行致病基因分析,对报道与心脏病相关的TRP4、SCN5A、SCN1B、NKFX2.5、CLCA2和LMNA共6个基因测序并确定致病基因;构建致病基因的野生型和突变型质粒;克隆野生型和突变型,激光共聚焦显微镜检测野生和突变型蛋白在细胞定位,利用Western blot检测目的蛋白的表达情况对该突变功能进行分析。结果:遗传学分析显示在家系中患者LMNA基因第10号外显子缺失,并与疾病共分离。Western blot结果显示突变型lamin蛋白表达水平正常,定位结果显示,野生型蛋白位于细胞核核膜,而突变型蛋白在核膜上和核内均有表达。LMNA异常可引起房室结细胞死亡与纤维化,导致房室传导速率降低,并发生房室传导阻滞。自噬和凋亡是细胞死亡的重要形式,LC3-Ⅱ是检测自噬的标志蛋白。应用Western blot检测LC3-Ⅱ,结果显示LMNA突变不影响该蛋白表达。结论:本次研究中,我们发现了一个新的导致遗传性房室传导阻滞伴肥厚型心肌病的LMNA基因突变,该突变可使其编码的蛋白不能正确定位于细胞核膜,导致细胞核核纤层结构异常。核纤层在细胞分裂中呈现周期性的变化,因此核纤层的异常会对细胞正常周期产生重要影响从而导致细胞的异常死亡。这可能是导致该家族成员发生房室传导阻滞的原因。
PURPOSE: Atrioventricular block is an abnormal electrical conduction that occurs between the atria and ventricles during electrical conduction of the heart. It can lead to cardiac arrhythmias. Early, we found a hereditary atrioventricular block with hypertrophic cardiomyopathy pedigree, this work mainly on its pathogenic genes and mechanisms for research. Methods: Candidate gene sequencing method was used to analyze the causative genes of heart disease patients who had been diagnosed as atrioventricular block. A total of 6 cases of TRP4, SCN5A, SCN1B, NKFX2.5, CLCA2 and LMNA were reported Gene sequencing and identification of the causative genes; Construction of wild-type and mutant plasmids of the causative genes; Cloning of wild-type and mutant, confocal laser scanning microscopy of wild and mutant proteins in the cell localization, the use of Western blot detection target protein expression The mutation function was analyzed. RESULTS: Genetic analysis showed deletion of the LMNA gene exon 10 in the pedigree and co-segregation with the disease. The result of Western blot showed that the expression level of mutant lamin protein was normal. The localization results showed that the wild type protein was located in the nucleus nuclear membrane, while the mutant protein was expressed on the nuclear membrane and nucleus. Abnormal LMNA can cause cell death and fibrosis of atrioventricular node, leading to atrioventricular conduction rate decreased, and the occurrence of atrioventricular block. Autophagy and apoptosis is an important form of cell death, LC3-Ⅱ is a marker protein to detect autophagy. LC3-Ⅱ was detected by Western blot, and the results showed that LMNA mutation did not affect the expression of this protein. CONCLUSIONS: In this study, we found a new LMNA gene mutation that leads to atrioventricular block and hypertrophic cardiomyopathy that leads to the inability of the encoded protein to localize on the nuclear membrane, leading to a nuclear fiber Layer structure is abnormal. The nuclear lamina changes periodically in cell division, so abnormalities in the lamina can have an important effect on the normal cell cycle and lead to abnormal cell death. This may be the cause of atrioventricular block in this family member.