The biophysical characterization of the first SCN5A mutation R1512W identified in Chinese sudden unexplained nocturnal death syndrome.
The biophysical characterization of the first SCN5A mutation R1512W identified in Chinese sudden unexplained nocturnal death syndrome.
复制标题
中国不明原因夜间死亡综合征中首次发现的 SCN5A 突变 R1512W 的生物物理特征
DOI：
10.1097/md.0000000000003836
复制
发表时间：
2016-06
期刊：
影响因子：
1.6
通讯作者：
Cheng J
中科院分区：
文献类型：
--
作者：
Zheng J;Zhou F;Su T;Huang L;Wu Y;Yin K;Wu Q;Tang S;Makielski JC;Cheng J
摘要：越来越多的临床表型证据表明，睡眠期间突然出现的呼吸障碍可能在不明原因夜间死亡综合征（SUNDS）的发病机制中发挥重要作用。报道的 Brugada 综合征导致心脏钠通道 R1512W 突变 &agr;亚基编码基因SCN5A在既往体外研究中未发现明显的心脏钠通道功能丧失，被我们确定为中国人SUNDS的第一个遗传原因。 R1512W携带者是一名38岁的男性SUNDS受害者，他在夜间睡眠时呼吸急促而突然死亡，没有任何结构性心脏病。为了检验我们的假设，即轻微酸中毒条件可能导致 SUNDS 基础突变心脏钠通道功能的显着丧失，在 pH 7.0 的细胞外和细胞内轻微酸中毒下对 SCN5A 突变 R1512W 进行了生物物理表征。 R1512W 的 cDNA 是使用 pcDNA3 质粒载体中的定点诱变方法创建的。将野生型 (WT) 或突变型心脏钠通道 R1512W 瞬时转染至 HEK293 细胞中。室温下用全细胞膜片钳法在 HEK293 细胞中转染 24 小时后测量宏观电压门控钠电流 (INa)。在 pH 7.4 的基线条件下，与 WT (-254±±23pA/pF，P< 0.05) 相比，R1512W (−175 ± 15 pA/pF) 的 INa 峰值降低了约 30%。在pH 7.0的酸中毒条件下，R1512W（−130 ± 17 pA/pF）与WT（−243 ± 23 pA/pF，P < 0.005）相比，INa峰值显着降低近50%。与pH 7.4的基线条件相比，pH 7.0的酸中毒并不影响WT中的INa峰值（P > 0.05），但降低了R1512W中的INa峰值（P < 0.05）。这项针对中国 SUNDS 受害者 SCN5A 突变的初步功能研究表明，酸中毒加剧了突变通道 R1512W 的功能丧失，并表明夜间睡眠障碍相关的轻微酸中毒可能引发遗传缺陷背景下 SUNDS 病例猝死的致命性心律失常。
AbstractIncreasing evidence observed in clinical phenotypes show that abrupt breathing disorders during sleep may play an important role in the pathogenesis of sudden unexplained nocturnal death syndrome (SUNDS). The reported Brugada syndrome causing mutation R1512W in cardiac sodium channel &agr; subunit encoded gene SCN5A, without obvious loss of function of cardiac sodium channel in previous in vitro study, was identified as the first genetic cause of Chinese SUNDS by us. The R1512W carrier was a 38-year-old male SUNDS victim who died suddenly after tachypnea in nocturnal sleep without any structural heart disease. To test our hypothesis that slight acidosis conditions may contribute to the significant loss of function of mutant cardiac sodium channels underlying SUNDS, the biophysical characterization of SCN5A mutation R1512W was performed under both extracellular and intracellular slight acidosis at pH 7.0. The cDNA of R1512W was created using site-directed mutagenesis methods in the pcDNA3 plasmid vector. The wild type (WT) or mutant cardiac sodium channel R1512W was transiently transfected into HEK293 cells. Macroscopic voltage-gated sodium current (INa) was measured 24 hours after transfection with the whole-cell patch clamp method at room temperature in the HEK293 cells. Under the baseline conditions at pH 7.4, R1512W (−175 ± 15 pA/pF) showed about 30% of reduction in peak INa compared to WT (−254 ± 23 pA/pF, P < 0.05). Under the acidosis condition at pH 7.0, R1512W (−130 ± 17 pA/pF) significantly decreased the peak INa by nearly 50% compared to WT (−243 ± 23 pA/pF, P < 0.005). Compared to baseline condition at pH 7.4, the acidosis at pH 7.0 did not affect the peak INa in WT (P > 0.05) but decreased peak INa in R1512W (P < 0.05). This initial functional study for SCN5A mutation in the Chinese SUNDS victim revealed that the acidosis aggravated the loss of function of mutant channel R1512W and suggested that nocturnal sleep disorders-associated slight acidosis may trigger the lethal arrhythmia underlying the sudden death of SUNDS cases in the setting of genetic defect.