Abstract：
BACKGROUND: Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families.
OBJECTIVE: The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M.
METHODS: We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases.
RESULTS: Our results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R.
CONCLUSIONS: Our results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.
OBJECTIVE: The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M.
METHODS: We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases.
RESULTS: Our results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R.
CONCLUSIONS: Our results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.
摘要：
背景：Brugada综合征（BrS）是一种遗传性致心律失常疾病，易导致患者心源性猝死。它与SCN5A的突变有关，编码心脏钠通道α亚基（NaV1.5）。BrS相关突变在家族内具有不完全的外显率和可变的表达率。
目的：确定患者特异性遗传背景对NaV1.5_p携带者细胞和临床表型的作用。V1525M。
方法：我们使用全细胞膜片钳技术研究了来自患者特异性人诱导多能干细胞衍生心肌细胞（hiPSC-CM）和异源转染HEKtsA201细胞的钠电流。我们通过qPCR测定基因和蛋白质的表达，RNA-seq,和蛋白质印迹，并进行了心律失常性疾病的遗传小组。
结果：我们的结果表明，与来自非载体的hiPSC-CM相比，来自两个V1525M单核苷酸变体（SNV）载体的hiPSC-CM的INa密度大大降低，表明NaV1.5_p的显性负(DN)效应。V1525M通道。INa在源自也携带NaV1.5_p的V1525MSNV载体的hiPSC-CM中不受影响。H558R多态性。V1525M在HEK-293T细胞中的杂合表达产生了INa功能的丧失,当该变体与H558R一起表达时没有观察到。此外,抗心律失常药物美西律在hiPSC-CM中拯救了INa功能。SCN5A表达在也表达NaV1.5_p的V1525M载体中增加。H558R.
结论：我们在患者特异性hiPSC-CM中的结果指出NaV1.5_p的显性负效应。V1525M,可以通过NaV1.5_p的存在来恢复。H558R.总的来说,我们的数据表明,患者特异性遗传背景是BrS不完全外显率的决定因素.
目的：确定患者特异性遗传背景对NaV1.5_p携带者细胞和临床表型的作用。V1525M。
方法：我们使用全细胞膜片钳技术研究了来自患者特异性人诱导多能干细胞衍生心肌细胞（hiPSC-CM）和异源转染HEKtsA201细胞的钠电流。我们通过qPCR测定基因和蛋白质的表达，RNA-seq,和蛋白质印迹，并进行了心律失常性疾病的遗传小组。
结果：我们的结果表明，与来自非载体的hiPSC-CM相比，来自两个V1525M单核苷酸变体（SNV）载体的hiPSC-CM的INa密度大大降低，表明NaV1.5_p的显性负(DN)效应。V1525M通道。INa在源自也携带NaV1.5_p的V1525MSNV载体的hiPSC-CM中不受影响。H558R多态性。V1525M在HEK-293T细胞中的杂合表达产生了INa功能的丧失,当该变体与H558R一起表达时没有观察到。此外,抗心律失常药物美西律在hiPSC-CM中拯救了INa功能。SCN5A表达在也表达NaV1.5_p的V1525M载体中增加。H558R.
结论：我们在患者特异性hiPSC-CM中的结果指出NaV1.5_p的显性负效应。V1525M,可以通过NaV1.5_p的存在来恢复。H558R.总的来说,我们的数据表明,患者特异性遗传背景是BrS不完全外显率的决定因素.
