Identification and characterization of the gene associated with the spontaneous autosomal recessive Spinning mice

本实验室发现了一种新的自发性常染色体隐性遗传综合性肌张力障碍模型 在将从Charles River Laboratories获得的BALB/cAnNCrl小鼠系与来自 杰克逊实验室。与BALB/cAnNCrl回交育种和近交后保持的表型 C57BL/6J纯合子小鼠表现出在圆周方向上的主动运动（旋转）和肌张力障碍 （头部运动不稳定，头部摆动并扭曲颈部），但无听力损失。候选基因 包括Cacng 2（Stargazer小鼠）、Cacna 2d 2（Ducky小鼠）、Cacnb 4（Lethargic小鼠）、Cacna 1a（Tottering小鼠 小鼠）和Myo 7a（Tonado大鼠）通过DNA和RNA桑格测序和QRT-PCR排除。RNA 在6个样品（3个正常对照和3个旋转小鼠）和一个候选小鼠中进行测序（RNA-seq 建立了Slc 16 a14、Col 6a 5、Lrrc 66、Marveld 3、Armc 3、Tc 2n、Zfp 979等5个基因的基因表 候选基因我们的中心假设是，旋转小鼠的盘旋行为很可能是由于 一种可能与多巴胺信号通路相关的新基因的功能缺失突变。的 本申请的主要目的是鉴定纺纱小鼠的因果基因，并表征其功能 揭示其人类同源基因是否与肌张力障碍患者相关。在目标1中，我们将确定 使用RNA-seq和/或全基因组测序的疾病基因和遗传作图策略。在目标2中， 我们将通过QRT-PCR、Western blot和免疫组化来表征疾病基因，以确定 在正常对照和Spinning小鼠中，疾病基因在脑区域中的表达模式;以及 通过RNA-seq等计算机模拟分析确定致病基因的分子功能通路。我们 我们还将对3155名肌张力障碍患者中至少2000名患者的疾病基因的所有外显子进行筛查，以确定 基因也与人类肌张力障碍有关。这些目标的实现将改善和扩大我们的 了解肌张力障碍和运动障碍。

A new spontaneously autosomal recessive model of combined generalized dystonia was identified in our lab after outbreeding of BALB/cAnNCrl mouse line obtained from Charles River Laboratories with C57BL/6J from The Jackson Laboratory. Phenotypes maintained after backcross breeding to BALB/cAnNCrl and inbreeding with C57BL/6J. Homozygote mice display active movement in a circular direction (Spinning) and dystonia (jerky head movements, head tossing with the neck twisted), but without hearing loss. Candidate genes including Cacng2 (Stargazer mice), Cacna2d2 (Ducky mice), Cacnb4 (Lethargic mice), Cacna1a (Tottering mice), and Myo7a (Tonado rat) were excluded by DNA and RNA Sanger sequencing and QRT-PCR. RNA sequencing (RNA-seq) was performed in 6 samples (3 normal controls and 3 Spinning mice) and a candidate gene list was generated which includes Slc16a14, Col6a5, Lrrc66, Marveld3, Armc3, Tc2n, Zfp979, and other 5 candidate genes. Our central hypothesis is that the circling behavior in the Spinning mice is most likely due to a loss of function mutation of a novel gene which may be related to the dopamine signaling pathway. The major goal of this application is to identify the casual gene of the Spinning mice, and characterize its function and reveal if its human homologue gene are related with dystonia patients. In Aim #1, we will identify the disease gene using RNA-seq and or whole genome sequencing, and genetic mapping strategies. In Aim #2, we will characterize the disease gene by QRT-PCR, Western blot, and immunohistochemistry to identify the expression pattern of the disease gene in brain regions, among normal controls and the Spinning mice; and identify the molecular functional pathway of the disease gene with RNA-seq and other in-silico analysis. We will also screen all exons of the disease gene in at least 2000 of our 3155 dystonia patients to identify if the gene is also associated with human dystonia. Completion of these objectives will improve and expand our understanding of dystonia and movement disorders.