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

目的：利用从福尔马林固定石蜡包埋(FFPE)尸检组织中提取的DNA，建立基于悬浮阵列的线粒体单倍群分析系统。背景：由于线粒体DNA(MtDNA)的进化速度远高于核DNA，且线粒体DNA具有高度的多态性，线粒体单核苷酸多态(MtSNPs)可能比核DNA中的SNPs对个体间的功能差异有更广泛的贡献。设计/方法：采用乙醇沉淀法从FFPE尸检组织中提取总DNA。根据对mtDNA编码区mtSNPs的综合分析，我们可以估计出7个主要的日本人线粒体单倍群，包括属于2个超单倍群(M和N)的17个亚单群。我们首先从尸检受试者的血液和FFPE组织中检测了大约100个DNA样本，这些受试者的单倍型已经通过冰冻肾脏组织的DNA分析确定。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5161个DNA样本。结果：成功分析4485例(87%)。然而，我们无法分析其余的(13%)，因为由于严重的DNA片段化而导致PCR扩增不充分。我们之前公布了1,500个冷冻肾脏DNA样本的单倍组频率。相比之下，我们开发的系统高估了单倍群D4a，而低估了单倍群M7a、D4b1b和N9b。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人的主要单倍群。我们相信，我们的线粒体单倍群分类系统不仅对病因学研究非常有帮助，而且对流行病学队列研究也很有帮助。

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.