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

点击翻译按钮获取中文摘要

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.