电压门控钠通道Nav1.7由SCN9A基因编码,在疼痛敏感性中起关键作用。在患有慢性疼痛的小纤维神经病(SFN)患者中发现了几种SCN9A功能获得(GOF)突变,包括R185H突变.然而,对于大多数这些变体,它们在疼痛表型中的参与仍需要实验阐明.为了描述R185H突变对疼痛敏感性的影响,我们使用CRISPR/Cas9技术建立了Scn9aR185H突变小鼠模型。Scn9aR185H突变小鼠在包含感觉神经元细胞体的背根神经节(DRG)中没有细胞改变,也没有生长或总体健康状态的改变。研究了男女杂合和纯合动物的疼痛敏感性。在甩尾和热板测试中,突变小鼠比野生型小鼠更敏感,丙酮,和vonFrey测试对热的敏感性,冷,和触摸,分别,虽然有性二态效应。新开发的生物信息管道,Gdaphen基于一般线性模型(GLM)和随机森林(RF)分类器以及混合数据的多因素分析,并显示了对疼痛表型贡献最大的定性和定量变量。使用Gdaphen,甩尾,哈格里夫斯,热板,丙酮,冷板,和vonFrey测试,性别和基因型对疼痛表型的贡献最大.重要的是,如在条件位置偏好(CPP)测定中评估的,突变动物显示自发性疼痛。总之,我们的结果表明,Scn9aR185H小鼠表现出疼痛表型,提示在患有慢性疼痛的SFN患者中发现的SCN9AR185H突变有助于他们的症状。因此,我们提供了遗传证据,证明Nav1.7通道的这种突变在具有这种突变的SFN患者的伤害性感受和疼痛中起重要作用.这些发现应有助于探索基于Nav1.7通道的进一步疼痛治疗。
The voltage-gated sodium channel Nav1.7 is encoded by
SCN9A gene and plays a critical role in pain sensitivity. Several
SCN9A gain-of-function (GOF) mutations have been found in patients with small fiber neuropathy (SFN) having chronic pain, including the R185H mutation. However, for most of these variants, their involvement in pain phenotype still needs to be experimentally elucidated. In order to delineate the impact of R185H mutation on pain sensitivity, we have established the
Scn9a R185H mutant mouse model using the CRISPR/Cas9 technology. The
Scn9a R185H mutant mice show no cellular alteration in the dorsal root ganglia (DRG) containing cell bodies of sensory neurons and no alteration of growth or global health state. Heterozygous and homozygous animals of both sexes were investigated for pain sensitivity. The mutant mice were more sensitive than the wild-type mice in the tail flick and hot plate tests, acetone, and von Frey tests for sensitivity to heat, cold, and touch, respectively, although with sexual dimorphic effects. The newly developed bioinformatic pipeline, Gdaphen is based on general linear model (GLM) and random forest (RF) classifiers as well as a multifactor analysis of mixed data and shows the qualitative and quantitative variables contributing the most to the pain phenotype. Using Gdaphen, tail flick, Hargreaves, hot plate, acetone, cold plate, and von Frey tests, sex and genotype were found to be contributing most to the pain phenotype. Importantly, the mutant animals displayed spontaneous pain as assessed in the conditioned place preference (CPP) assay. Altogether, our results indicate that
Scn9a R185H mice show a pain phenotype, suggesting that the
SCN9A R185H mutation identified in patients with SFN having chronic pain contributes to their symptoms. Therefore, we provide genetic evidence for the fact that this mutation in Nav1.7 channel plays an important role in nociception and in the pain experienced by patients with SFN who have this mutation. These findings should aid in exploring further pain treatments based on the Nav1.7 channel.