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

该项目的长期目标是确定导致尼曼-皮克C型（NP-C）的基因，研究其在疾病发病机制中的作用，并利用这些信息帮助治疗这种疾病。NP-C是一种常染色体隐性的神经内脏脂质储存疾病，表现为可变肝脾肿大、垂直核上眼麻痹、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的杂交，我们已经确定了导致这种疾病的基因。首先，我们在含有m-npc的区域使用了0.1cM的小鼠遗传连锁图谱，使用了一系列亚种间小鼠回交。接下来，我们利用从含有NP-C基因的人类DNA序列中克隆的DNA片段，将小鼠遗传图谱与人类遗传图谱和物理图谱整合起来。最后，我们从cDNA克隆库和从人类生理结构中分离的捕获的外显子片段中评估了作为NP-C候选基因。通过Northern blot， Southern blot， SSCP和测序分析，将从小鼠自发突变体中分离的样本与等基因野生型对照进行比较，我们发现一个基因NPC1具有反转录转座子插入，导致突变小鼠正常基因产物功能丧失。我们也在NPC患者中发现了突变。我们还开展了一项自然历史研究，以确定疾病预防的时间进程，并作为治疗干预的基础。我们正在使用反义方法来模拟这种疾病的动物模型来评估治疗干预措施。我们正在评估基因表达的变化，以确定潜在的生物标志物，并探索筛选，以确定潜在的干预措施。

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 models, 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 mutant 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 using antisense approaches to mimic animal models of this disease to assess therapeutic interventions. We are assessing changes in gene expression to identify potential biomarkers and exploring screens to identify potential interventions.