Development of a comprehensive molecular diagnosis system for neurological diseases based on DNAmicroarrays.

本项目致力于开发基于DNA微阵列的神经系统疾病综合分子诊断系统。为了实现这一目标，我们开发了1.基于DNA微阵列的综合重测序系统，以及2.高密度阵列-CGH系统，以完成缺失/倍增的检测和断裂点的识别。我们开发了基于DNA微阵列的重测序系统，用于肌萎缩侧索硬化症、帕金森病、肾上腺脑白质营养不良和家族性痉挛性截瘫。通过这个系统，我们已经证明了基于DNA微阵列的重测序系统在识别点突变方面是高效的。虽然DNA重测序微阵列在鉴定点突变方面非常有效，但它们在检测缺失或倍增方面效率不高。为了解决这一问题，我们新开发了高密度阵列-CGH系统，以100-200bp的分辨率检测PARK2基因的缺失/倍增。由于分辨率极高，使用位于缺失/倍增两侧的聚合酶链式反应可以很容易地扩增连接片段，从而在核苷酸水平上识别缺失/倍增的断裂点。我们应用该系统研究了常染色体隐性遗传性青少年帕金森综合征(AR，JP)患者PARK2基因缺失/倍增的机制。我们已经确定了299个等位基因的缺失/倍增。断裂点聚集在PARK2与FRA6E(公共脆性位点)中心重合的狭窄区域。事实上，对120个癌细胞株的分析允许31个缺失/倍增，其分布与在ARJP中发现的非常相似。综合这些研究表明，胚系突变(AR-JP)以及体细胞突变(癌细胞株)中的共同机制是缺失/倍增的基础。

This project focused on development of a comprehensive molecular diagnosis system for neurological diseases based on DNA microarrays. lb accomplish this aim, we have developed 1. DNA microarray-based comprehensive resequencing system, and 2. high density array-CGH system to accomplish detection of deletions/multiplications and identification of breakpoints We have developed DNA microarray-based resequencing system for amyotrophic lateral sclerosis, Parkinson disease, adrenoleukodystrophy, and familial spastic paraplegia. With this system, we have shown DNA microarray-based resequencing system is highly efficient to identify point mutations. Although DNA resequencing microarrays are quite effective for identification of point mutations, they are inefficient for detection of deletions or multiplication. To overcome this problem, we have newly developed high density array-CGH system to allow detection of deletions/multiplications of PARK2 gene with the resolution of 100-200bp. Since the resolution is extremely high, the junction segments can be easily amplified by PCR employing PCR primers flanking the deletions/multiplications, allowing identification of breakpoints of deletions/multiplications on nucleotide levels. We applied this system for investigation of the mechanisms of deletions/multiplications of PARK2 in patients with autosomal recessive juvenile Parkinsonism (AR JP). We have determined deletions/multiplications of 299 alleles. The breakpoints clustered in a narrow region of PARK2 that coincides with the center of FRA6E (common fragile site). Indeed analysis of 120 cancer cell lines allowed 31 deletions/multiplications and the distribution is quite similar to that found in ARJP Taken together these studies demonstrate that common mechanisms underlie the of deletions/multiplications in the germline mutations (AR-JP) as well as somatic mutations (cancer cell lines).