In vivo, in vitro and in silico correlations of four de novo SCN1A missense mutations

Mutations in the SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.SCN1A基因（SCN1A gene）编码电压门控钠通道NaV1.1（voltage-gated sodium channel NaV1.1），其突变可引发Dravet综合征（Dravet syndrome）——一种重型发育性癫痫性脑病。临床指南推荐在患者幼年早期开展该基因的遗传学检测。然而，由于轻度癫痫综合征也可能与SCN1A新生错义突变（de novo missense mutation）相关，因此预测此类突变的临床结局颇具挑战。本研究将临床病情严重程度与通道活性的体外电生理功能检测、以及突变损伤效应的生物信息学预测结果进行了关联分析。3例携带p.Gly177Ala、p.Ser259Arg及p.Glu1923Arg突变的患者，自幼便出现频繁难治性癫痫发作，并伴随认知与行为功能退化，符合典型Dravet综合征表型。上述突变在哺乳动物表达系统中均无法检测到钠电流，提示通道功能完全丧失。第4例患者携带p.Met1267Ile突变，虽自幼出现癫痫发作，但癫痫负荷更低、认知功能保留更好，符合边缘型Dravet综合征表型。与之相符的是，功能分析显示该突变可产生钠电流，但存在部分功能丧失。与之形成对比的是，6种预测突变致病性的生物信息学工具均显示所有突变的影响程度相近。同样，对蛋白二级与三级结构进行同源建模后，未发现蛋白质折叠异常。综上，本研究提示采用膜片钳（patch clamp）技术开展的功能检测可作为预后评估工具：可检测到钠电流提示表型相对轻微，而无钠电流则提示预后不良。未来自动化膜片钳系统的研发，将推动这类功能检测纳入个体化患者诊断方案中。