CLN3 AND SUBUNIT 9 GENES AND NEURONAL SURVIVAL

巴滕氏病包括婴儿晚期和青少年类型 神经元蜡样脂褐质沉积症（LINCL和JNCL）。 底层 这组疾病的病理生理学仍然知之甚少。 的 在分子水平上确定相关的遗传缺陷 将1）解释临床亚型之间和内部的异质性; 2） 通过提供亚型特异性载体， 和产前检测; 3）提供有价值的洞察机制， 快速神经元细胞死亡和缺陷基因产物在神经细胞死亡中的作用 神经系统发育。 详细的临床和连锁研究， 调查人员将LINCL和JNCL确定为两个不同实体。 我们已经证明JNCL与触珠蛋白位点相连。 LINCL已 并将仍然是一个贫穷的基板联系研究的数量， 多受影响的家庭是有限的，由于这些早期死亡， 患者 幸运的是，最近的生化证据表明， ATP合酶9亚基溶酶体与长痛酰焦磷酸 已经经历过加工的低聚糖意味着 Batten病发病机制中的四个基因中的至少一个：这些 是编码线粒体膜蛋白亚基-9前体的两个基因， ATP合酶和微粒体葡萄糖苷酶和葡萄糖苷酶II的基因。 本申请提出1）克隆亚基-9的正常cDNA， 通过"锚定PCR"研究人类亚基-9表达调控， 北方印迹分析; 2）确定的表达类型和水平 使用正常cDNA作为探针，检测LINCL中亚基-9的突变， 通过克隆和测序，并 证实了LINCL基因组DNA中这些突变的发生 通过等位基因特异性杂交进行单剂量或双剂量的患者; 3） 确定 LINCL和JNCL中微粒体葡萄糖苷酶I和II的活性水平， 开发人成纤维细胞和成淋巴细胞的测定方法， 比色和放射性底物; 4）确定 微粒体葡萄糖苷酶I和II活性对亚基-9蓄积的影响 正常和Batten细胞系通过Western印迹和ELISA使用亲和 纯化的亚基-9特异性抗体。 希望这些研究 将提供重要的和新的信息的病因， Batten病的发病机制。

Batten's disease comprises the late infantile and juvenile types of neuronal ceroid-lipofuscinosis(LINCL and JNCL). The underlying pathophysiology of this group of diseases remains poorly understood. The determination of the responsible genetic defects at a molecular level will 1) explain heterogeneity between and within clinical subtypes; 2) revolutionize diagnostic methods by providing subtype-specific carrier and prenatal detection; 3) provide valuable insight into mechanisms of rapid neuronal cell death and the role of defective gene products in the developing nervous system. Detailed clinical and linkage studies by the investigator have established LINCL and JNCL as two distinct entities. We have shown JNCL to be linked to the haptoglobin locus. LINCL has been and will remain a poor substrate for linkage studies as the number of multiaffected families are limited due to the early demise of these patients. Fortunately, recent biochemical evidence on accumulation in the lysosome of subunit-9 of ATP synthase and dolichyl-pyrophosphate oligosaccharides that have already undergone processing implicates at least one of four genes in the pathogenesis of Batten's disease: These are the two genes coding for the precursors of subunit-9 of mitochondrial ATP synthase and the genes for microsomal glucosidase and glucosidase II. This application proposes to 1) clone the normal cDNAs for subunit-9 and study regulation of subunit-9 expression in humans by "anchored PCR" and Northern blot analysis; 2) determine the type and level of expression of subunit-9 in LINCL using the normal cDNAs as probes, detect mutations in the cDNAs for subunit-9 in LINCL by cloning and sequencing., and to confirm the occurrence of these mutations in genomic DNA from LINCL patients in single or double dose by allele-specific hybridization; 3) determine activity levels of microsomal glucosidase I and II in LINCL and JNCL by developing assay methods in human fibroblasts and lymphoblasts using colorimetric and radioactive substrates; 4) determine the effects of microsomal glucosidase I and II activity on subunit-9 accumulation in normal and Batten cell lines by Western Blot and ELISA using an affinity purified subunit-9-specific antibody. It is hoped that these studies will provide important and novel information on the etiology and pathogenesis of Batten's disease.