Homozygous mutation in TXNRD1 is associated with genetic generalized epilepsy
Homozygous mutation in TXNRD1 is associated with genetic generalized epilepsy
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DOI：
10.1016/j.freeradbiomed.2017.02.040
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发表时间：
2017-05-01
影响因子：
7.4
通讯作者：
Kunz, Wolfram S.
中科院分区：
文献类型：
--
作者：
Kudin, Alexei P.;Baron, Gregor;Kunz, Wolfram S.
氧化应激的增加与各种形式的人类癫痫的发病机制有广泛的关系。在这里，我们报告了遗传性全面性癫痫家族TXNRD1（硫氧还蛋白还原酶1）的纯合突变。TXNRD1是一种必需的含硒酶，参与活性氧（ROS）解毒和氧化还原信号传导。通过对该患者血液DNA的全外显子组测序，鉴定了影响高度保守氨基酸残基的TXNRD1突变p.p o190leu。检测到的突变及其在家族中的分离-指标患者的所有兄弟姐妹都是纯合的，父母是杂合的-通过Sanger测序得到证实。在指标患者的骨骼肌活检和皮肤成纤维细胞的亚细胞组分中测定TXNRD1活性，并通过Se-75标记和Western blot分析评估突变蛋白的表达水平。由于突变，TXNRD1的活性在患者的成纤维细胞和骨骼肌中降低（分别为对照组的34 +/- 3%和16 +/- 8%）。在成纤维细胞中，我们检测到酶的se -75标记降低（对照组的41 +/- 3%）。重组突变TXNRD1的深入体外动力学分析表明，k(cat)/Se值降低了30-40%。因此，经Western blotting和Se-75标记证实，患者组织样品中酶活性降低可以解释为：(i)酶周转率降低，（ii）突变酶丰度降低。突变的成纤维细胞也被发现对过氧化氢的抗性较低。我们的数据与遗传性全身性癫痫中ROS解毒不足的潜在作用一致。
Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family- all siblings of the index patient were homozygous and the parents heterozygous-were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by Se-75 labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34 +/- 3% and 16 +/- 8% of controls, respectively). In fibroblasts, we detected reduced Se-75-labeling of the enzyme (41 +/- 3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered k(cat)/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and Se-75 labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy.