Development of mutation analysis system by multiplex PCR and suspension array for paraffin-embedded autopsy samples

石蜡包埋尸检样本多重PCR和悬浮阵列突变分析系统的开发

• 批准号: 21590411
• 负责人: HOSOYA Hiroko
• 金额: $ 3.08万
• 依托单位: Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21590411/
• 关键词: 病理解剖; パラフィン包埋切片; 生活習慣病; 脳梗塞; 動脈硬化; 健康長寿; ミトコンドリアゲノム多型; 遺伝子増幅; ミトコンドリアDNA; 変異; パラフィン; 剖検例; Luminex; 網羅的解析

Objective : To establish a suspension array-based analysis system for determining mitochondrial haplogroups, using DNA purified from formalin-fixed paraffin-embedded(FFPE) autopsy tissues. Background : Because the evolutionary rate of mitochondrial DNA(mtDNA) is much higher than that of nuclear DNA, and because mtDNA is highly polymorphic, mitochondrial single nucleotide polymorphisms(mtSNPs) are expected to contribute more extensively to the functional differences among individuals than those SNPs in the nuclear DNA. Design/Methods : We carefully purified total DNA from FFPE autopsy tissues by using ethanol precipitation. By using our system, we could estimate 7 major mitochondrial haplogroups for Japanese containing 17 subhaplogroups belonging to 2 super haplogroups(M and N), based on information obtained from the comprehensive analysis of mtSNPs in the coding region of mtDNA. We first tested approximately 100 DNA samples from blood and FFPE tissues of autopsy subjects whose haplogroups had been identified by DNA analysis of frozen-kidney tissues. To examine more fully the utility of the system, we analyzed 5, 161 DNA samples from FFPE tissues. Results : We could analyze 4, 485(87%) of them successfully. However, we could not analyze the remainder(13%) because of an insufficient PCR amplification due to severe DNA fragmentation. We had previously revealed the haplogroup frequencies of 1, 500 frozen-kidney DNA samples. In comparison, the haplogroup D4a was overestimated, but the haplogroups M7a, D4b1b and N9b were underestimated, by the present system we developed. Conclusions : We have established a method to purify DNA from FFPE tissues and developed a suspension array-based analytic system to estimate major haplogroups of Japanese individuals. We believe that our system for the classification of mitochondrial haplogroups will be very helpful not only for pathogenetical studies, but also for epidemiological cohort studies.

目的：建立基于悬浮芯片的分析系统，利用从福尔马林固定石蜡包埋（FFPE）尸检组织中纯化的 DNA 来确定线粒体单倍群。背景：由于线粒体DNA（mtDNA）的进化速度远高于核DNA，并且mtDNA具有高度多态性，因此线粒体单核苷酸多态性（mtSNP）预计比核DNA中的SNP对个体之间的功能差异有更广泛的贡献。设计/方法：我们使用乙醇沉淀从 FFPE 尸检组织中仔细纯化总 DNA。通过使用我们的系统，根据对mtDNA编码区mtSNP的综合分析获得的信息，我们可以估计日本人的7个主要线粒体单倍群，其中包含属于2个超级单倍群（M和N）的17个亚单倍群。我们首先测试了来自尸检受试者血液和 FFPE 组织的约 100 个 DNA 样本，通过对冷冻肾脏组织的 DNA 分析鉴定了其单倍群。为了更全面地检验该系统的实用性，我们分析了来自 FFPE 组织的 5, 161 个 DNA 样本。结果：我们可以成功分析其中的 4, 485 个（87%）。然而，由于严重的 DNA 碎片导致 PCR 扩增不足，我们无法分析其余部分（13%）。我们之前已经揭示了 1, 500 个冷冻肾脏 DNA 样本的单倍群频率。相比之下，通过我们开发的现有系统，单倍群 D4a 被高估，但单倍群 M7a、D4b1b 和 N9b 被低估。结论：我们建立了一种从 FFPE 组织中纯化 DNA 的方法，并开发了一种基于悬浮阵列的分析系统来估计日本个体的主要单倍群。我们相信，我们的线粒体单倍群分类系统不仅对病理学研究非常有帮助，而且对流行病学队列研究也非常有帮助。

项目成果

ピルビン酸療法

丙酮酸盐疗法

• 发表时间: 2010
• 作者: Masayo Minami;Tomomi Kato;Yoshiki Miyata;Toshio Nakamura and Quan Hua;田中雅嗣
• 通讯作者: 田中雅嗣

Association of a Polymorphism of BTN2A1 With Hypertension in Japanese Individuals

• DOI: 10.1038/ajh.2011.74
• 发表时间: 2011-08-01
• 期刊: AMERICAN JOURNAL OF HYPERTENSION
• 影响因子: 3.2
• 作者: Horibe, Hideki;Kato, Kimihiko;Yamada, Yoshiji
• 通讯作者: Yamada, Yoshiji

The establishment of an expanded simultaneous swift diagnosing system for mitochondrial DNA pathological mutations.

建立了线粒体DNA病理突变的扩展同步快速诊断系统。

• 发表时间: 2009
• 作者: Xie ZH;et. al.;Nishigaki Y
• 通讯作者: Nishigaki Y

Polymorphisms in the Control Region of Mitochondrial DNA Associate with Elite Japanese Athlete Status

线粒体 DNA 控制区的多态性与日本优秀运动员的地位相关

• 发表时间: 2011
• 作者: Eri Mikami;Noriyuki Fuku;Hideyuki Takahashi;Nao Ohiwa;Yannis P Pitsiladis;Mitsuru Higuchi;Takashi Kawahara;Masashi Tanaka
• 通讯作者: Masashi Tanaka

Comprehensive Mitochondrial Haplogroup Analysis System Based On Suspension-Array Technology Using DNA From Formalin-Fixed Paraffin-Embedded Autopsy Tissues

基于悬浮阵列技术的综合线粒体单倍群分析系统，使用福尔马林固定石蜡包埋尸检组织中的 DNA

• 发表时间: 2012
• 作者: Yutaka Nishigaki;Hiroko Hosoya;Makiko Naka-Mieno;Tomio Arai;Motoji Sawabe;Masashi Tanaka
• 通讯作者: Masashi Tanaka