| Budget Amount *help |
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The aim of this project is to establish a novel method of chimerism analysis, which is to determine the ratios of donor-derived hematopoietic cells after allogeneic hematopoietic stem cell transplantation(CST) , with insertion-deletion polymorphisms(indels) as genetic markers. We first intended to establish a real-time PCR-based method, however, because the running costs for this method as a routine clinical examination turned out to be too high, we tried to establish fluorescence-labeled PCR-based method(the indels-PCR method).
We first chose 8 indels markers(insertion sized 21-40 base pairs) , which were reported to be highly polymorphic in the Japanese population from a database, and assessed the utility for chimerism analysis DNA of peripheral blood cells from 71 CST donor/recipient pairs was analyzed, and the selected 8 markers could discriminate genetic differences in 67 pairs. With the addition of the X- and Y-chromosome-linked amelogenin marker, chimerism analysis could be performed for all the 71 pairs. Thus, chiraerism analysisis possible with a lelatively small number of indels markers.
We next compared the indels-PCR method to conventional short tandem repeat-based method(the STR-PCR method) . The two recognized drawbacks of the STR-PCR method are 1) stuttered peak formation that could obscure the distinction of alleles specific for the donor and the recipient, and 2) the inconsistency of amplification efficiency between alleles of different sizes that makes the calculation of donor-derived cell ratios difficult. The indels-PCR method was free from the former problem, and for the latter problem it showed higher consistency compared to the STR-PCR method. The indels-PCR method is more accurate to assess the ratios of donor-derived cells after CST compared to the conventional STR-PCR method, and it could be a practically useful method for chimerism analysis.
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