Monitoring Chimerism In The Post Transplantation Of Allogeneic Hematopoietic Stem Cell With Red Cells Blood Group Genes

BackgroundAllogenic hematopoietic stem cell transplantation(allo-HSCT) has been extensively used to treat patients with malignant hematological diseases or other diseases.An accurate quantitative analysis of chimerism kinetics would permit early the absence of engraftment and a delay in engraftment,evaluate the risk of GVHD or those liable to relapse.and help to set up additional therapeutic interventions. At present,STR-PCR is the gold standard in the analysis of chimerism.However, STR-PCR is not a really quantitative technology.The sensitivity of this method is1-10%.This study was aimed to explore a more sensitive detection method of hematopoietic chimerism by real-time fluorescent quantitative PCR with red cell blood group genes.Methods1. EDTA-anticoagulated blood samples from random donors were collected and identified by serological technology.Genomic DNA was extracted. The5’-untranslated region,extron5-7and intron5-6in ABO gene,extron4-11of SLC14A1gene in Kidd system were amplified by PCR.The sequencing of purified PCR product was performed bidirectionally.2、The mainly selective criterion of polymorphism sites by which TaqMan probes were designed in RQ-PCR was high heterozygosity in Chinese population and high specificity of sequence.3、EDTA-anticoagulated blood samples from random donors were collected and identified by serological technology.Genomic DNA was extracted from samples of JK(a+b+) type,B type and O type.The screening of B101and non-B101alleles as well as O02and non-002alleles were performed by sequencing.The heterozygous samples of Jk(a+b+),B101/non-B101and001/002were reserved. Genomic DNA from specific sites of these sample was amplified by PCR.The fragment of purified PCR product was ligated into PCR-4-TOPO vector using TOPO TA cloning kit(Invitrogen). The recombined plasmids were identified by PCR and analyzed by sequencing. Artificial chimeric DNA samples on the model of distinct chimerism and standard substance were prepared by plasmid DNA. With special TaqMan MGB probes and primers designed on the polymorphism of red cells blood group gene, artificial chimeric DNA samples were detected by RQ-PCR and analyzed by Standarded curves.Results1、ABO blood group system:There were4ABO alleles which were highly heterozygous in Chinese population according to polymorphism analysis of extron5-7 and intron5-6.They were001(33.693%)、O02(22.302%)、B101(20.624%) and A102(19.784%),respectively.The frequencies of the others were all <2%.796C>A+803G>C could be used to discriminate the B allele and non-B allele,which was two vicinal SNPs. The heterozygosity is0.33. IVS5+103insCCC could be used to discriminate the O allele and non-0allele which were three consecutive SNPs and whose heterozygosity is0.35..According to polymorphism analysis of ABO5’-untranslated region,we had found more than20polymorphism sites.9of them had been published.11were new,which were-4682A>G、-4597C>T、-4581C>T、-4238A>G、-4105A>C、-2848G>A、-2818C>T、-2247--2232del CCCTTCCT (8bp-reps)、-2081G>A、-840G>T、-593C>T,respectively.16SNPs were distant from each other.2、Kidd blood group system:There were5SNPs found in extron4-11of kidd gene which were-99A>G.130G>A、499A>G、588G>A and838G>A.The frequencies of the alleles were0.479(-99A),0.521(-99G),0.583(130G),0.417(130A),0.788(499A),0.212(499G),0.925(588G),0.075(588A),0.479(838G),0.521(838A). The heterozygosity are0.50(-99A>G)、0.49(130G>A),0.33(499A>G),0.14(588G>A)和0.50(838G>A).respectively.3、The theoretic and practical measured value of artifical chimeric DNA had no significant deviation(P>0.05).4、The sensitivity of838G>A,796C>A+803G>C, and IVS5+103insCCC was1.56%,0.39%and0.195%by RQ-PCR, respectively. Conclusions1、796C>A+803G>C in extron7and IVS5+103insCCC in intron5of ABO gene as well as-99A>G、130G>A and838G>A in extron4-11and intron of Kidd gene all meet the requirement of molecular markers in monitoring chimerism in the posttransplantation of stem cell.2、The result of artificial chimeric DNA samples was reliable by RQ-PCR with red cell blood group genes.3、The specificity and sensitivity of the method could be improved by the increasing the number of nucleotide polymorphisms in RQ-PCR with red cell blood group genes.4、We have successfully established the detection method of artificial chimeric DNA by RQ-PCR with red cell blood group genes.