Background There is increasing evidence that de novo CACNA1D missense mutations inducing increased Cav1.3 L-type Ca2+-channel-function confer a high risk for neurodevelopmental disorders (autism spectrum disorder with and without neurological and endocrine symptoms). Electrophysiological studies demonstrating the presence or absence of typical gain-of-function gating changes could therefore serve as a tool to distinguish likely disease-causing from non-pathogenic de novo CACNA1D variants in affected individuals. We tested this hypothesis for mutation S652L, which has previously been reported in twins with a severe neurodevelopmental disorder in the Deciphering Developmental Disorder Study, but has not been classified as a novel disease mutation. Methods For functional characterization, wild-type and mutant Cav1.3 channel complexes were expressed in tsA-201 cells and tested for typical gain-of-function gating changes using the whole-cell patch-clamp technique. Results Mutation S652L significantly shifted the voltage-dependence of activation and steady-state inactivation to more negative potentials (~ 13-17 mV) and increased window currents at subthreshold voltages. Moreover, it slowed tail currents and increased Ca2+-levels during action potential-like stimulations, characteristic for gain-of-function changes. To provide evidence that only gain-of-function variants confer high disease risk, we also studied missense variant S652W reported in apparently healthy individuals. S652W shifted activation and inactivation to more positive voltages, compatible with a loss-of-function phenotype. Mutation S652L increased the sensitivity of Cav1.3 for inhibition by the dihydropyridine L-type Ca2+-channel blocker isradipine by 3-4-fold.Conclusions and limitationsOur data provide evidence that gain-of-function CACNA1D mutations, such as S652L, but not loss-of-function mutations, such as S652W, cause high risk for neurodevelopmental disorders including autism. This adds CACNA1D to the list of novel disease genes identified in the Deciphering Developmental Disorder Study. Although our study does not provide insight into the cellular mechanisms of pathological Cav1.3 signaling in neurons, we provide a unifying mechanism of gain-of-function CACNA1D mutations as a predictor for disease risk, which may allow the establishment of a more reliable diagnosis of affected individuals. Moreover, the increased sensitivity of S652L to isradipine encourages a therapeutic trial in the two affected individuals. This can address the important question to which extent symptoms are responsive to therapy with Ca2+-channel blockers.

中文翻译：

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CACNA1D 从头突变的生物物理学分类为严重神经发育障碍的高风险突变。


背景 越来越多的证据表明，CACNA1D 错义突变可诱导 Cav1.3 L 型 Ca2+ 通道功能增强，从而导致神经发育障碍（有或没有神经和内分泌症状的自闭症谱系障碍）的高风险。因此，电生理学研究表明，典型的功能获得性门控变化是否存在，可以作为区分受影响个体中可能致病的 CACNA1D 变异和非致病性新发 CACNA1D 变异的工具。我们测试了这一突变 S652L 的假设，该突变此前曾在破译发育障碍研究中在患有严重神经发育障碍的双胞胎中报道过，但尚未被归类为新的疾病突变。方法 为了进行功能表征，野生型和突变型 Cav1.3 通道复合物在 tsA-201 细胞中表达，并使用全细胞膜片钳技术测试典型的功能获得门控变化。结果突变 S652L 显着地将激活和稳态失活的电压依赖性转移到更负的电位（约 13-17 mV），并增加了亚阈值电压下的窗口电流。此外，它在类似动作电位的刺激过程中减慢了尾电流并增加了 Ca2+ 水平，这是功能获得变化的特征。为了提供证据证明只有功能获得性变异才会带来高疾病风险，我们还研究了在表面健康个体中报告的错义变异 S652W。 S652W 将激活和失活转移到更正的电压，与功能丧失表型兼容。突变 S652L 使 Cav1.3 对二氢吡啶 L 型 Ca2+ 通道阻滞剂伊拉地平抑制的敏感性增加了 3-4 倍。结论和局限性我们的数据提供的证据表明，功能获得性 CACNA1D 突变（例如 S652L）而非功能丧失突变（例如 S652W）会导致包括自闭症在内的神经发育障碍的高风险。这将 CACNA1D 添加到破译发育障碍研究中确定的新疾病基因列表中。尽管我们的研究没有深入了解神经元中病理性 Cav1.3 信号传导的细胞机制，但我们提供了功能获得性 CACNA1D 突变的统一机制作为疾病风险的预测因子，这可能有助于建立更可靠的诊断受影响的个人。此外，S652L 对伊拉地平的敏感性增加，鼓励在两个受影响的个体中进行治疗试验。这可以解决一个重要问题：症状在多大程度上对 Ca2+ 通道阻滞剂治疗有反应。