@misc{oai:repo.qst.go.jp:00069927,
 author = {Michikawa, Yuichi and Suga, Tomo and Iwakawa, Mayumi and Imai, Takashi and 道川 祐市 and 菅 智 and 岩川 眞由美 and 今井 高志},
 month = {Nov},
 note = {Purpose: A haplotype GGTT of four SNPs (rs187116, rs3794116, rs3794107, rs8193) that spans 80 kb distance in CD44 gene has been reported by statistical association analysis to show significant greater early adverse skin reaction risk (odds ratio = 2.17; 95% confidence interval, 1.07–4.43) after radiotherapy of Japanese breast cancer patients (Suga et al. Int J Radiat Oncol Biol Phys. 2007, 69, 685-693). For future individualized clinical applications, we developed the igMDA (in-gel multiple displacement amplification) method (Michikawa et al. Anal Biochem. 2008, 383, 151-158) and improved to be capable to use frozen human blood samples, instead of the frozen EB virus-transformed human B lymphoblastoid cells of the original method. In this study, we applied the improved protocol for direct determination of the individuals diplotype in CD44 gene.  
\nMaterials and Methods: Frozen blood samples from breast cancer patients who registered from 2001 to 2005 for RadGenomics Project were used in this study. All the patients provided written informed consent to participate in the study, which was approved by the Ethical Committee at NIRS and by each collaborating institution. The frozen blood samples were thawed at room temperature and centrifuged at 3,000 x g for 5 min to remove liquid component of bloods. Blood cells were then washed by PBS once and number of cells with nuclear DNA was microscopically counted by staining their nuclear DNA with PicoGreen. The cellular DNA-embedded agarose gel solution was first solidified at room temperature then small pieces (approximately 1 micro liter) were picked to aliquot, instead of directly aliquoting the embedded agarose gel solution before solidification in the original conditions. The multiple displacement amplification reaction that uses Phi29 DNA polymerase to comprehensively amplify limited amount of DNA embedded in the aliquoted agarose gel pieces was used with the original conditions. Post-amplification screening of CD44 gene containing-amplicons was carried out using a PCR primer set designed to avoid the copy number variation region shown in the database of this gene. Genotyping of the four tag SNPs were carried out by the allele-specific real-time quantitative PCR. 
\nResults: Number of nuclear DNA-positive cells and volume of the cell suspension to be embedded into agarose gel solution were carefully titrated to make an appropriately diluted condition to isolate sufficient number of single homologous chromosomes. Resulting conditions provided average 23.2 (SD+-9.2) positive amplicons in a set of 94 amplicons (n=40). Full length coverage ratio of the positive amplicons was average 15.8 % (SD+-8.7). Overall successful haplotype determination rate was 80%. 
\nConclusions: Frozen human blood samples can be used for direct determination of radiosensitivity-associated haplotype of CD44 gene by the improved igMDA method., Biotecnologia Habana 2009, Medical Applications of Biotechnology},
 title = {Direct determination of the radiosensitivity-associated haplotype in CD44 gene by igMDA method using frozen human blood samples},
 year = {2009}
}