A Novel Kv7.3 Variant in the Voltage-Sensing S4 Segment in a Family With Benign Neonatal Epilepsy: Functional Characterization and in vitro Rescue by β-Hydroxybutyrate
A Novel Kv7.3 Variant in the Voltage-Sensing S4 Segment in a Family With Benign Neonatal Epilepsy: Functional Characterization and in vitro Rescue by β-Hydroxybutyrate
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DOI：
10.3389/fphys.2020.01040
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发表时间：
2020-09-04
影响因子：
4
通讯作者：
Sands, Tristan T.
中科院分区：
文献类型：
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作者：
Miceli, Francesco;Carotenuto, Lidia;Sands, Tristan T.
KCNQ 2和KCNQ 3中的致病性变体，编码Kv7.2和Kv7.3电压门控K（+）通道亚基的旁系同源基因，导致早发性发育/癫痫疾病，其特征为从良性家族性新生儿癫痫（BFNE）到早发性发育和癫痫性脑病（DEE）的异质性临床表型.KCNQ2变异体占BFNE家系的大多数，KCNQ 3变异体占小得多的亚组，但这种不平衡的原因尚不清楚。需要对其他家系进行分析，以进一步阐明这种遗传异质性的性质，并改善对新变异致病性的预测。我们确定了一个BFNE家庭，有两个兄弟姐妹和一个受影响的父母。对父母和兄弟姐妹样本的外显子组测序显示了一种新的KCNQ 3变体（c.719T>G; p.M240R），在三个受影响的个体中分离。M240残基在人Kv7.2-5中是保守的，并且位于最靠近电压敏感S（4）跨膜区段的细胞内侧的两个丝氨酸（R5和R6）之间。全细胞膜片钳记录在中国仓鼠卵巢（CHO）细胞显示，同源Kv7.3 M240 R通道是没有功能的，而异聚体通道纳入Kv7.3 M240 R突变亚基与Kv7.2和Kv7.3显示去极化位移约10 mV的激活门控。分子模拟结果表明，M240 R取代优先稳定的静息状态，并可能不稳定的激活状态的Kv7.3亚基，结果与功能数据一致。暴露于β-羟基丁酸酯（BHB）（生酮饮食（KD）期间产生的酮体）可逆转M240 R变体诱导的通道功能障碍。总之，我们描述了在BFNE患者中鉴定的Kv7.3的S（4）段内的第一个错义功能丧失（LoF）致病变体。在模拟杂合性的条件下研究，M240 R变体主要影响电压灵敏度，与先前分析的降低电流密度的BFNE Kv7.3变体相反。我们的药理学结果为在Kv7.2或Kv7.3亚基中携带LoF变体的患者中使用KD提供了理论基础。
Pathogenic variants inKCNQ2andKCNQ3, paralogous genes encoding Kv7.2 and Kv7.3 voltage-gated K(+)channel subunits, are responsible for early-onset developmental/epileptic disorders characterized by heterogeneous clinical phenotypes ranging from benign familial neonatal epilepsy (BFNE) to early-onset developmental and epileptic encephalopathy (DEE).KCNQ2variants account for the majority of pedigrees with BFNE andKCNQ3variants are responsible for a much smaller subgroup, but the reasons for this imbalance remain unclear. Analysis of additional pedigrees is needed to further clarify the nature of this genetic heterogeneity and to improve prediction of pathogenicity for novel variants. We identified a BFNE family with two siblings and a parent affected. Exome sequencing on samples from both parents and siblings revealed a novelKCNQ3variant (c.719T>G; p.M240R), segregating in the three affected individuals. The M240 residue is conserved among human Kv7.2-5 and lies between the two arginines (R5 and R6) closest to the intracellular side of the voltage-sensing S(4)transmembrane segment. Whole cell patch-clamp recordings in Chinese hamster ovary (CHO) cells revealed that homomeric Kv7.3 M240R channels were not functional, whereas heteromeric channels incorporating Kv7.3 M240R mutant subunits with Kv7.2 and Kv7.3 displayed a depolarizing shift of about 10 mV in activation gating. Molecular modeling results suggested that the M240R substitution preferentially stabilized the resting state and possibly destabilized the activated state of the Kv7.3 subunits, a result consistent with functional data. Exposure to beta-hydroxybutyrate (BHB), a ketone body generated during the ketogenic diet (KD), reversed channel dysfunction induced by the M240R variant. In conclusion, we describe the first missense loss-of-function (LoF) pathogenic variant within the S(4)segment of Kv7.3 identified in patients with BFNE. Studied under conditions mimicking heterozygosity, the M240R variant mainly affects the voltage sensitivity, in contrast to previously analyzed BFNE Kv7.3 variants that reduce current density. Our pharmacological results provide a rationale for the use of KD in patients carrying LoF variants in Kv7.2 or Kv7.3 subunits.