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

目的：利用福尔马林固定石蜡包埋（FFPE）尸检组织中纯化的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，并且mtDNA具有高度多态性，因此线粒体单核苷酸多态性（mtsnp）在个体之间的功能差异中所起的作用比核DNA中的snp更广泛。设计/方法：我们使用乙醇沉淀法仔细纯化FFPE尸检组织的总DNA。基于mtDNA编码区mtsnp的综合分析，我们可以估计出日本人线粒体7个主要单倍群，其中包含17个亚单倍群，属于2个超级单倍群（M和N）。我们首先测试了大约100个来自尸检受试者血液和FFPE组织的DNA样本，这些受试者的单倍群已通过冷冻肾脏组织的DNA分析确定。为了更充分地检验该系统的效用，我们分析了来自FFPE组织的5161个DNA样本。结果：成功分析4885例（87%）。然而，我们无法分析其余的（13%），因为严重的DNA断裂导致PCR扩增不足。我们之前已经揭示了1500个冷冻肾脏DNA样本的单倍群频率。相比之下，单倍群D4a被高估，而单倍群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原学研究有很大帮助，而且对流行病学队列研究也有很大帮助。

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.