CLN3 AND SUBUNIT 9 GENES AND NEURONAL SURVIVAL

巴顿病包括婴儿期晚期和幼年型 神经元性蜡样脂褐素沉着症(LINCL和JNCL)。潜在的 这类疾病的病理生理学仍知之甚少。这个 在分子水平上确定负有责任的遗传缺陷 将1)解释临床亚型之间和内部的异质性；2) 通过提供特定于亚型的载体来革命性地改变诊断方法 和产前检测；3)提供有价值的见解 神经细胞的快速死亡和缺陷基因产物在神经细胞死亡中的作用 发育中的神经系统。详细的临床和关联研究 调查人员已将LINCL和JNCL确定为两个不同的实体。 我们已经证明JNCL与结合珠蛋白基因连锁。LINCL一直被 并将仍然是连锁研究的糟糕底物，因为 多个受影响的家庭由于这些疾病的早期死亡而受到限制 病人。幸运的是，最近关于体内蓄积的生化证据 三磷酸腺苷合成酶-9亚基和焦磷酸二乙酯的溶酶体 已经经过加工的低聚糖牵涉到 巴顿病发病机制中四个基因中的至少一个：这些 这两个基因是线粒体亚单位-9前体的编码基因吗 三磷酸腺苷合成酶与微粒体葡萄糖苷酶和葡萄糖苷酶基因II。 本申请建议1)克隆亚基-9的正常cDNA，并 “锚定聚合酶链式反应”对人类亚基-9表达调控的研究 Northern印迹分析；2)确定基因的表达类型和水平 LINCL中的亚单位-9用正常的cDNA作为探针，检测在 通过克隆和测序获得LINCL-9亚基的cDNAs，并将其克隆到 确认LINCL基因组DNA中存在这些突变 等位基因特异性杂交单剂或双剂患者；3) 确定 LINCL和JNCL中微粒体葡萄糖苷酶I和II的活性水平 人成纤维细胞和淋巴母细胞检测方法的建立 比色法和放射性衬底；4)测定 微粒体葡萄糖苷酶I和II对亚基-9积累的影响 利用亲和力进行Western Blot和ELISA法检测正常细胞和巴顿细胞 纯化的亚基-9特异性抗体。希望这些研究 将提供重要和新颖的病因学和 巴顿病的发病机制。

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.