Identifying susceptible genes for Parkinson disease employing next-generation sequencer

使用下一代测序仪识别帕金森病的易感基因

基本信息

批准号：
22890041
负责人：
MITSUI Jun
金额：
$ 1.02万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Research Activity Start-up
财政年份：
2010
资助国家：
日本
起止时间：
2010 至无数据
项目状态：
已结题

项目摘要

To identify susceptible genes for Parkinson disease, comprehensive resequencing analysis of candidate genes (responsible genes for lysosomal storage disease) employing a next-generation sequencer was conducted. We arranged 64 DNA samples of patients with Parkinson disease in an 8x8 rectangular array, and formed the pooled DNA sample sets by each row and column (8 rows and 8 columns). Each pooled DNA sample set was subjected to PCR amplifications of candidate genes (approximately 24 kb in total) and every amplicons for each set were mixed. The mixed amplicons for each set was subjected to an analysis of single lane of Illumina GAIIx, single-end, 100bp, according to manufactures' instructions.Reads were aligned to the reference sequences (hg19) of candidate genes by BWA with default parameters, and approximately 1,000-2,000 coverage per allele was obtained. Only 50 bp of reads with more than 20 of QV were used to screen variants. We specifically focused on rare variants not registered in dbSNP, and searching for such variants employing an integer programming algorithm. The results were that 3 variants not registered in dbSNP were obtained; each variant was identified in three different samples in the heterozygous state. All of them were nonsynonymous single nucleotide substitutions. One was reported to be pathogenic for a lysosomal storage disease, and two were unknown variants. All variants were confirmed by an additional direct nucleotide sequence analysis (Sanger method).Based on these results, it was suggested that the matrix pooling approach is an accurate and cost effective testing algorithm for detection of rare variants.

为了鉴定帕金森病的易感基因，采用下一代测序仪对候选基因（溶酶体贮积病的责任基因）进行了全面的重测序分析。我们将 64 个帕金森病患者的 DNA 样本排列成 8x8 的矩形阵列，并按每行和每列（8 行和 8 列）形成混合 DNA 样本集。对每个合并的 DNA 样本组进行候选基因（总共约 24 kb）的 PCR 扩增，并且将每组的每个扩增子混合。根据制造商的说明，对每组的混合扩增子进行 Illumina GAIIx 单泳道、单端、100bp 的分析。通过 BWA 使用默认参数将读数与候选基因的参考序列 (hg19) 进行比对，并且每个等位基因获得大约 1,000-2,000 个覆盖率。仅使用 QV 超过 20 的 50 bp 读取来筛选变体。我们特别关注未在 dbSNP 中注册的罕见变体，并使用整数规划算法搜索此类变体。结果获得了3个未在dbSNP中注册的变异；每个变体均在杂合状态的三个不同样本中被鉴定。它们都是非同义单核苷酸取代。据报道，其中一种对溶酶体贮积病具有致病性，另两种是未知变异。所有变异均通过额外的直接核苷酸序列分析（桑格方法）进行确认。基于这些结果，表明矩阵池方法是一种用于检测罕见变异的准确且具有成本效益的测试算法。