THE USE OF THE MOUSE NPC MODEL TO CLONE THE HUMAN DISEASE GENE

该项目的长期目标是确定尼曼-匹克C型（NP-C）的基因，研究其在疾病发病机制中的作用，并利用这些信息来帮助治疗这种疾病。NP-C是一种常染色体隐性遗传的神经内脏脂质储存障碍，表现为可变的肝脾肿大、垂直核上性眼肌麻痹、进行性共济失调、肌张力障碍和痴呆。利用人类定位克隆和与自发小鼠模型杂交，我们已经确定了导致这种疾病的基因。首先，我们利用一系列亚种间小鼠回交，在含有m-npc的区域中使用0.1cM小鼠遗传连锁图。我们接下来使用从含有NP-C基因的人DNA的重叠群产生的克隆DNA片段将鼠遗传图谱与人遗传图谱和物理图谱整合。最后，我们评估了从cDNA克隆池中作为NP-C候选基因的基因，并捕获了从人类物理重叠群中分离的外显子片段。利用北方印迹、南方印迹、SSCP和测序分析，将从自发突变小鼠中分离的样品与它们的同基因野生型对照进行比较，我们发现一个基因NPC 1具有反转录转座子插入，导致突变小鼠中正常基因产物的功能丧失。我们还在NPC患者中发现了突变。 我们还建立了自然史研究，以确定疾病预防的时间进程，并作为治疗干预的基础。 我们正在利用反义方法来模拟这种疾病的动物模型，以评估治疗干预措施。

The long term goal of this project is to identify the gene responsible for Niemann-Pick Type C (NP-C), to study its role in the pathogenesis of the disorder and to use this information to aid in the treatment of this disease. NP-C is an autosomal- recessive, neurovisceral lipid storage disorder and presents as variable hepatosplenomegaly, vertical supranuclear ophthalmoplegia, progressive ataxia, dystonia, and dementia. Using human positional cloning and crosses with spontaneous mouse modles, we have identified the gene responsible for this disorder. First, we used a 0.1cM mouse genetic linkage map in the region containing m-npc using a series of intersubspecific mouse backcrosses. We next integrated the murine genetic map with the human genetic and physical maps using cloned DNA fragments generated from the contig of human DNA containing the NP-C gene. Finally we evaluated genes as candidates for NP-C from a pool of cDNA clones and trapped exon fragments isolated from the human physical contig. Using Northern blot, Southern blot, SSCP and sequencing analyses using samples isolated from spontaneous mouse mutants in comparison to their isogenic wild type controls we found that one gene, NPC1 has a retrotransposon insertion resulting in a loss-of-function of the normal gene product in mutatn mice. We have also found mutations in human individuals with NPC. We have also established a natural history study in order to determine the timecourse of disease prevention and as a basis for therapeutic interventions. We are utilization antisense approaches to mimic animal models of this disease to assess therapeutic interventions.