遗传因素与脑内铁缺乏被认为在不安腿综合征（RLS）的发病与耐药机制中发挥重要作用。申请者在前期的临床研究中新发现了一个与RLS高度关联的风险SNP，位于MEIS1基因启动子区，其携带者血清中铁平衡关键调节激素-Hepcidin的含量显著增高。推测该SNP可调控MEIS1基因转录，MEIS1作为转录结合因子进一步参与Hepcidin编码基因（HAMP）的转录调控，从而影响脑内铁平衡。本项目将在人神经母细胞系中验证该SNP对MEIS1基因的转录调控，上调和下调MEIS1基因表达，检验其对HAMP基因表达的影响；采用染色质免疫共沉淀高通量测序、体外凝胶迁移实验确认MEIS1与HAMP的结合部位；通过染色体构象捕获技术探索该SNP直接调控HAMP基因或其上游基因的可能性。采用CRISPR构建该SNP对应突变小鼠模型，验证其临床表型及MEIS1突变对铁平衡和多巴胺能药物反应的影响。

Restless Legs Syndrome (RLS) is a common sensorimotor disorder of unknown cause and lacking of long-term effective treatment.Genetic factors and brain iron deficiency are recognized as the cardinal components of the disease aetiology.Hepcidin, the master regulator of iron homeostasis, has currently been indicated to play an important role in dysregulation of brain iron equilibrium in RLS patients.Our previous clinical study found a new risk SNP highly associated with RLS, which is located in the promoter region of MEIS1 gene, and the proportion of higher serum hepcidin level increased significantly in this risk SNP carriers of RLS patients. We presume that, this risk SNP can regulate the transcription of MEIS1 gene, and MEIS1, as a transcription binding factor, may be involved in the transcription regulation of HAMP gene (hepcidin encoded by HAMP gene),consequently change the iron balance in brain. In this project,we will verify the transcriptional regulation effect of this SNP on MEIS1 gene in human neuroblastoma cell line, and test whether up-regulation and down-regulation of MEIS1 gene affect the expression of HAMP. Using Chromatin Immunoprecipitation sequencing (ChIP-seq) and electrophoretic mobility shift assay (EMSA) to identify the binding sites between MEIS1 and HAMP. We will use Chromosome Conformation Capture and related method (4C) to explore the possibility of the SNP directly regulating the HAMP gene or its upstream genes.CRISPR will be used to construct the SNP corresponding mutation mouse model to verify the clinical phenotype and the effect of MEIS1 mutation on iron hemostasis and dopaminergic drug response.