Preliminary Study Of Digital PCR Monitoring Of Hematopoietic Stem Cell Transplantation Chimera And Analysis Of The Linkage Relationship Between HLA-DPB1 And Rs9277534

Objective:According to the single nucleotide base polymorphism in Chinese population,the digital PCR based quantitative detection system was developed which aim to detecting chimerism.Also,we conducted the preliminary study on the clinical application value of the method in early chimerism monitoring after allogeneic hematopoietic stem cell transplantation.Methods:1.Establishment of SNP-dPCR quantitative detection systemIn terms of the distribution characteristics of SNP in Chinese population,we selected appropriate SNP loci from dbSNP database.200 unrelated volunteers from Sichuan Han nationality were selected from Sichuan CMDP,followed by further screening through TaqMan probe,consequently developing SNP loci complex typing system suitable for this study.Based on the data of Han population in the Beijing and the South from 1000 Genomes Browser database,the effective recognition of SNP loci complex typing system among unrelated individuals was analyzed.To detect different sites between donors and recipients and analyze effective recognition of SNP loci complex typing system among sibling donors and recipients,200 clinical samples for transplantation consisting of 100 pairs of 8/8 HLA-identical sibling donors and recipients were selected from Sichuan umbilical cord blood bank.In vitro,the feasibility of this method was evaluated by simulating chimeric specimen to determine the specificity and sensitivity of SNP-dPCR quantitative system.2.Detection of early chimerism after hematopoietic stem cell transplantationA total of 21 clinical samples were collected from West China Hospital of Sichuan and General Hospital of Chengdu Military Region.Prior to hematopoietic stem cell transplantation,the specimens of donors and recipients were collected and then screen the difference sites among the donors and recipients.And the peripheral blood anticoagulant specimens were collected from patients with hematopoietic stem cell transplantation on day 7,day 14,day 21,day 30,day 60,day 90 and day 120,respectively.CD3+T cells were separated by immunomagnetic beads and the chimerism of whole blood cells and CD3+T cells was quantitatively detected by SNP-dPCR.Results:1.Establishment of SNP loci complex typing system13 SNP locus were selected from dbSNP database and a typing method for theTaqMan probe was established.The 13 SNP locus of allele frequency were between 0.4 and 0.6 among the unrelated healthy individuals of the Han population in Sichuan.2.Effective recognition of SNP loci complex typing systemIn the 100 pairs of 8/8 HLA-identical sibling donors and recipients,98 pairsshowed one or more than one difference site(98%),94 pairs performed at least two difference sites(94%)and 94 pairs displayed no less than three difference sites(90%).In the 1000 Genomes Browser database,there were at least four effective difference sites between any two unrelated individuals in CHB and at least six effective difference sites between any two unrelated individuals in CHS.3.The detection of Chimeric specimen by digital PCR quantitative systemAll SNP-TaqMan probes could be used for effective and specific typing on digital PCR,and the sensitivity of simulated chimeric specimens against digital PCR could reach 0.01%.4.Detection and analysis of clinical transplantation specimens21 clinical specimens were collected and detected and analyzed by the method developed in this study.During the early stage after transplantation,the whole blood cells of donors in the peripheral blood of patients reached complete chimera earlier than CD3+T cells of donors(+14d vs +21d).Moreover,the chimeric rate of CD3+T cells was different from different donor sources.In the early +21d after transplantation,the chimeric rate of CD3+T cells was significantly higher in the unrelated matched donor group than that in the sibling matched donor group(P<0.05)and the complete chimerism of T cell was achieved earlier than that in the sibling matched donor group.Cord blood microchimerism for adjuvant therapy could also be detected by the method during the early period after transplantation(+21d～+42d).Conclusions:This study established a SNP-dPCR quantitative detection method.It can be used to detect chimerism of donor in recipient body during the early chimerism monitoring after hematopoietic stem cell transplantation,which has high sensitivity and good stability.This method can make a good distinction between the donor and recipient of 21 hematopoietic stem cell transplantation patients,so as to obtain accurate and reliable test results.This study preliminarily revealed the significance of dynamic monitoring of T cell chimerism in early chimerism monitoring after hematopoietic stem cell transplantation.Objective:To investigate the distribution feature of HLA-DPB1 and rs9277534 alleles and analyze linkage disequilibrium in China Marrow Donor Program of Sichuan Han population.Methods:Screened samples from 200 unrelated healthy blood donors from CMDP of Sichuan.The DNA of blood donors were extracted by blood DNA extraction kit.The method of PCR-SBT was applied to HLA-DPB1 genotyping,and TaqMan was applied to rs9277534 genotyping.Arlequin 3.1 software was used for analysis of HLA-DPB1 and rs9277534 alleles linkage disequilibrium.Results:The genotype of DPB1 *05:01:01G allele was the most common which the frequency is 0.4125(165/400)in CMDP of Sichuan Han Population.The allele frequencies of A and G were 0.4(160/400)and 0.6(240/400).AA,GG and AG genotype frequencies were 0.165(33/200),0.375(75/200)and 0.46(92/200).10HLA-DPB1 alleles were found to be in significant LD with the rs9277534 SNR The DPB1 *04:02:01G,DPB1*02:01:02G,DPB1*02:02:01G and DPB1*17:01:01G were in complete linkage with the rs9277534 A allele,while DPB1*05:01:01G,DPB1*03:01:01G? DPB1*13:01:01G,DPB1*14:01:01G and DPB1*21:01,were in complete LD with the rs9277534 G allele.While not completely,DPB1*04:01:01G(D5=0.95;p<0.01)were found to be in significant LD with the rs9277534 A allele.Conclusion:This study expounds the LD between specific HLA-DPB1 and rs9277534A/G alleles in CMDP of Sichuan Han Population.There is a reference value for the clinical choice of donor in hematopoietic stem cell transplantation.