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

自发性常染色体隐性遗传旋转小鼠相关基因的鉴定和表征

基本信息

批准号：
10302062
负责人：
Jianfeng Xiao
金额：
$ 15.4万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-15 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10302062
关键词：
Affect Applications Grants Backcrossings Behavior Biological Models Brain region Breeding Candidate Disease Gene Cerebellum Cerebral cortex Chromosome Mapping Clinical Communities Corpus striatum structure DNA DNA Sequence Alteration DNA analysis Data Development Dideoxy Chain Termination DNA Sequencing Disease Disease Pathway Dopamine Dystonia Dystonic Disorder Exons Family Family member Functional disorder Gene Expression Profile Genes Genetic Genotype Goals Head Head Movements Hippocampus (Brain)Homologous Gene Homozygote Human Immunohistochemistry Inbreeding Laboratories Lethargies MYO7A gene Midbrain structure Modeling Molecular Movement Movement Disorders Mus Muscle Contraction Mutation Neck Neurosciences Online Mendelian Inheritance In Man Parkinson Disease Pathogenesis Pathway interactions Patients Pattern Phenotype Play Posture RNA Rattus Research Resolution Rivers Role Sampling Signal Pathway Signaling Protein Thalamic structure The Jackson Laboratory Time Tornadoes Tremor United States National Institutes of Health Western Blotting base density genome sequencing hearing impairment human disease improved in silico loss of function mutation melting nervous system disorder neurochemistry novel postnatal proband ranpirnase tool transcriptome sequencing whole genome

项目摘要

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.

我们的实验室发现了一种新的自发性常染色体隐性遗传联合全身性肌张力障碍模型从 Charles River Laboratories 获得的 BALB/cAnNCrl 小鼠品系与来自 Charles River Laboratories 的 C57BL/6J 进行近亲繁殖后杰克逊实验室。与 BALB/cAnNCrl 回交育种和近交后保持表型与 C57BL/6J。纯合子小鼠表现出主动的圆形运动（旋转）和肌张力障碍（头部动作急促，头部扭动，颈部扭曲），但没有听力损失。候选基因包括 Cacng2 (Stargazer 小鼠)、Cacna2d2 (Ducky 小鼠)、Cacnb4 (Lethargic 小鼠)、Cacna1a (Tottering 小鼠) 通过 DNA 和 RNA Sanger 测序以及 QRT-PCR 排除了 Myo7a（Tonado 大鼠）和 Myo7a（小鼠）。核糖核酸对 6 个样本（3 个正常对照和 3 个 Spinning 小鼠）和候选样本进行了测序 (RNA-seq) 生成基因列表，其中包括 Slc16a14、Col6a5、Lrrc66、Marveld3、Armc3、Tc2n、Zfp979 和其他 5 个候选基因。我们的中心假设是，旋转小鼠的盘旋行为很可能是由于可能与多巴胺信号通路有关的新基因的功能丧失突变。这该应用的主要目标是识别旋转小鼠的偶然基因，并表征其功能并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/或全基因组测序以及遗传作图策略的疾病基因。在目标#2中，我们将通过 QRT-PCR、Western blot 和免疫组织化学来表征疾病基因，以鉴定正常对照组和旋转小鼠脑区疾病基因的表达模式；和通过 RNA-seq 和其他计算机分析来识别疾病基因的分子功能途径。我们还将对 3155 名肌张力障碍患者中的至少 2000 名患者进行疾病基因的所有外显子筛查，以确定是否基因也与人类肌张力障碍有关。完成这些目标将改善和扩大我们的了解肌张力障碍和运动障碍。