| Objective1.To establish the pyrosequencing method to determine Rhesus box of RHD gene.2.To establish the pyrosequencing method to detect RHC/c allele.3.Assessing and applying the pyrosequencing methods for Rhesus box of RHD gene and RHC/c allele detection.Methods1.Rh phenotypes were tested using agglutination with monoclonal anti-D,anti-C and anti-c.For RhD-negative blood samples,we further confirmed the results with modified indirect anti-human globulin test(IAT).2.On the basis of our previous study,we selected study samples with known Rhesus box zygosity of RHD gene,which had been analyzed by mismatch PCR-SSP assay.Primers were designed based on polymorphic sites located at the upstream,downstream and hybrid Rhesus box of the RHD gene to amply the DNA fragment including the polymorphic sites(amplicon).We then pyrosequenced the amplicon with a pyrosequencer.RHD+/RHD+ homozygote,RHD+/RHD-heterozygote and RHD-/RHD-homozygote could be distinguished by analyzing the results of pyrosequencing.3.To validate the accuracy of pyrosequencing results for Rhesus box of RHD gene,all the amplified products were sequenced with Sanger sequencing method.The results of Sanger sequencing were compared with BLAST in the Genebank,as well as compared with that of pyrosequencing method.We also compared the detected results of Rhesus box zygosity of RHD gene from previous PCR-SSP method with that of the pyrosequencing method.4.Samples for RHC/c allele study were selected according to the RhC and Rhc phenotypes.Sequence specific primers were designed on the basis of nt455 polymorphic sites on exon 3 of RHCE gene to specifically amplify the whole exon 2 of RHCE gene.The amplified products were used as the templates to amplify the nt178 polymorphic site(amplicon)of RHC/c allele.Then we used pyrosequencing technology to sequence amplicons which including the nt178 polymorphic site of RHC/c allele.RHCC,RHCc and RHcc genotypes were then distinguished by analyzing the results of pyrosequencing.5.To validate the accuracy of pyrosequencing results,all the amplicons of the whole exon 2 of RHCE gene with PCR-SSP method and ampl icons containing the nt178 polymorphic site of RHC/c allele were sequenced with Sanger sequencing method.The results from Sanger sequencing method were performed BLAST research in the Genebank,as well as compared with that of pyrosequencing results.Results1.A total of 96 samples were used to detect and validate Rhesusbox zygosity of the RHD gene with pyrosequencing method,of which 36 samples(37.50%)were RhD-positive phenotypes and 60 samples(62.50%)were of RhD-negative phenotypes.2.Among all the 96 samples,pyrosequencing results showed that 32(33,33%)samples were RHD+/RHD+ homozygotes,with the C and T proportions of(50.84± 2.08)%(mean ± S.D.,95%CI 50.09%to 51.59%)and(49.22 ± 2.12)%(mean± S.D.,95%CI 48.45%to 49.98%);36(37.50%)samples were RHD+/RHD-heterozygotes,with the C and T proportions of(66.78 ± 1.82)%(mean ± S.D.,95%CI 66.16%to 67.39%)and(33.22 ± 1.82)%(mean ± S.D.,95%CI 32.61%to 33.84%);28(29.17%)samples were RHD-/RHD-homozygotes,with the C and T proportions of(97.86±1.21)%(mean± S.D.,95%CI 97.39%to 98.33%)and(2.14±1.21)%(mean ±S.D.,95%CI 1.67%to 2.61%).The cytosine proportion distribution between above three groups had statistically significant(F=5424.99,P=0.00).The cytosine proportion distribution between RHD+/RHD+homozygotes and RHD+/RHD-heterozygotes group(t=137.41,P=0,00)and between RHD+/RHD+ homozygotes and RHD-/RHD-homozygotes group(t=222.60,P=0.00),as well as between RHD+/RHD-heterozygotes and RHD-/RHD-homozygotes group(t=425.48,P=0.00)had statistically significant.The thymine proportion distribution between above three groups had statistically significant(P=5340.90,P=0.00).The thymine proportion distribution between RHD+/RHD+homozygotes and RHD+/RHD-heterozygotes group(t=228.57,P=0.00)and between RHD+/RHD+homozygotes and RHD-/RHD-homozygotes group(t=336.29,P=0.00),as well as between RHD+/RHD-heterozygotes and RHD-/RHD-homozygotes group(t=444.00,P=0.00)had statistically significant.3.In the previous study,the mismatch PCR-SSP method detected 32(33.33%)samples,39(40.63%)samples and 25(26.04%)samples as the RHD+/RHD+homozygotes,the RHD+/RHD-heterozygotes,and the RHD-/RHD-homozygotes,respectively.From this data,there were only 3 cases of the results were not consistent with that of the pyrosequencing data.In fact,there were five cases different between them,namely the mismatch PCR-SSP method mistook 4 cases of RHD-/RHD-homozygosity(by DNA pyrosequencing)as RHD+/RHD-heterozygosity,and 1 case of RHD+/RHD-heterozygosity(by DNA pyrosequencing)as RHD-/RHD-homozygosity.There were no significant difference between DNA pyrosequencing method and mismatch PCR-SSP assay(X2=0.03,P=0.06).4.A total of 28(29.17%)samples were detected as RHD-/RHD-homozygotes with Sanger sequence method,and the results were the same as that of the pyrosequencing method.Since Sanger sequencing is not a quantitative method,it can't discriminate the RHD+/RHD+ homozygote from RHD+/RHD-heterozygote according to the sequencing peak figures.Consequently altogether 68(70.83%)samples of RHD+/RHD+ homozygotes and RHD/RHD-heterozygotes were identified with the Sanger sequencing method.5.A total of 68 samples were chosen to to detect and validate for RHC/c allele study with pyrosequencing method.Of which 30(44.12%)samples,30(44.12%)samples,and 8(11.76%)samples were phenotyped as RhCC,RhCc,and Rhcc phenotypes,respectively.6.The results of RHC/c allele detected by DNA pyrosequencing method showed that 30(44.12%)samples were genotyped as RHCC homozygotes,with the A and C proportions of(96.17±4.14)%(mean ± S.D.,95%CI 94.62%to 97.71%)and 3.83±4.14%(mean ± S.D.,95%CI 2.29%to 5.38%);30(44.12%)samples were RHCc heterozygotes,with the A and C proportions of(45.97 ± 3.95)%(mean ±S.D.,95%Cl 44.49%to 47.44%)and(54.07 ±3.96)%(mean ±S.D.,95%CI 52.59%to 55.54%);8(11.76%)samples were the RHcc homozygotes,with the A and C proportions of(3.38±3.34)%(mean ± S.D.,95%CI 0.59%to 6.16%)and(96.63±3.34)%(mean ± S.D.,95%CI 93.84%to 94.41%).The adenine proportion distribution between above three groups has statistically significant(F=2219.77,P=0.00).The adenine proportion distribution between RHCC homozygotes and RHCc heterozygotes group(t=126.54,P=0.00)and between RHCC homozygotes and RHcc homozygotes group(t=63.85,P=0 00),as well as between RHCc heterozygotes and RHcc homozygotes group(t=3.25,P=0.00)had statistically significant.The cytosine proportion distribution between above three groups had statistically significant(F=2218.91,P=0.00).The thymine proportion distribution between RHCC homozygotes and RHCc heterozygotes group(p=5.04,P=0.00)and between RHCC homozygotes and RHcc homozygotes group(t=75,10,P=0.00),as well as between RHCc heterozygotes and RHcc homozygotes group(t=81.89,=0.00)had statistically significant.7.The specific amplicon from exon 2 of RHCEgene with PCR-SSP were verified including the whole exon 2 of RHCE gene with the Sanger sequencing method.Detected results of ntl78 polymorphic site from RHC/c allele with Sanger sequencing method showed that 30 samples(44.12%),30 samples(44.12%),and 8 samples(11.76%)were PRHC/C homozygotes,RHC/c heterozygotes,and RHc/c homozygotes,respectively,and the results were the same as that of the pyrosequencing method.Conclusion1.We successfully established the pyrosequencing method to analyze Rheses box zygosity of RHD gene,and then identified RHD genotypes.The method is suitable to detect massive clinical samples with a fast,accurate,and electrophoresis-free quality.2.We set up the pyrosequencing method to detect RHC/callele firstly.The pyrosequencing method for detecting RHC/c allele could avoid false-positive or false-negative results caused by gene mutation or sequence homology,which made the best of quantitive characteristics of pyrosequencing technology. |
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