先天性运动性眼球震颤（CMN）是一类单基因遗传病，可能由控制眼球运动的脑部神经网络异常有关。在前一个基金的资助下，我们发现一个新的CMN基因位点NYS7。通过外显子组和全基因组测序、比较基因组杂交，结果排除了NYS7区域全部已知基因，但在一个家系检测到约0.9Mb缺失，缺失区域无已知功能基因，提示有一个尚待克隆的新致病基因。本项目拟以此为基础来克隆该基因。首先以缺失区域内表达序列标签等为线索，应用5'RACE和3'RACE技术从cDNA文库克隆新基因全长cDNA。 然后通过与人类基因组比较确定该基因外显子、内含子的相对位子和序列，结合生物信息分析预测该基因编码的蛋白质序列。继而分析另一个定位于此的家系及其它CMN家系中该基因的突变，初步确定新致病基因。最后初步分析该基因的表达组织及表达定位。这个新功能基因的克隆与鉴定对揭示CMN分子机制和阐明眼球运动控制的机制有重要价值。

Congenital motor nystagmus (CMN) is a group of inherited diseases, in which the primary defect may locate in the central nervous system where the ocular motor is controlled. We identified a novel NYS7 locus responsible for CMN with the support of a previous fund from NSFC. Exome sequencing and whole genome sequencing did not detect any causative mutation in all genes in the linkage interval. However, deletion of a 0.9 Mb fragment in the linkage interval in the patient from one family mapped to NYS7 was identified by whole genome sequencing and array-comparative genomic hybridization (aCGH). An unidentified new gene responsible for CMN was suggested in the deleted region since there is no functional gene in the region except for three pseudogenes, seven UniGene fragments，and 38 expressed sequence tags (ESTs). Based on these important findings, in this project, we propose to clone this novel gene. First, whole length target cDNA will be cloned from cDNA library by 5'RACE and 3'RACE and using ESTs and UniGene as baits. Then the whole length cDNA will be mapped to human genome to define exons and introns. Further bioinformation analysis will be performed in order to clarify the gene structure and to predict the reading frame. Subsequently, primers will be designed to screen for mutations in another CMN family also mapped to this region as well as in other CMN families in order to confirm the role of this gene in causing CMN. Finally, expressed tissue and cell types of the novel gene will be evaluated by using RT-PCR and fluorescence in situ hybridization (FISH). Cloning and identification of a new gene responsible for CMN would be valuable for the understanding of the molecular mechanism of CMN as well as that of ocular motor control in the brain.

先天性运动性眼球震颤（CMN）是以眼球不自主往复摆动为特征的一类单基因遗传病。已知单纯CMN基因位点有6个，其中只有NYS1位点的FRMD7基因被确定；合并眼球震颤的疾病及相关基因则比较多。本项目在我们此前定位的CMN基因位点NYS7的基础上，通过进一步研究发掘该位点致病基因，系统分析CMN的分子遗传学基础。通过外显子组测序分析、全基因组测序分析和传统Sanger测序分析，排除了NYS7定位区域已知基因及可能的功能基因片段与CMN的关系。通过外显子组测序分析73个CMN家系的28个先天性眼球震颤相关基因，结果在40（40/73，54.8%）个家系检测到可能的致病突变，其中35个家系有FRMD7基因突变、3个家系有CACNA1F基因突变、1个家系有CACNA1A基因突变、1个家系有PAX6基因突变。这些结果不仅让我们比较全面了解CMN的致病基因变异频谱、基因表型关系，而且也为CMN基因突变检测的临床应用以及进一步发掘新致病基因奠定了基础。