| Objective1. To comparatively study on distributionin characteristics of Rh phenotypes in Chinese Han and Tibet Tibetan population.2. To investigate SNPs of SMP1gene in Chinese Han and Tibetan population.3. To explore the relationship of SMP1gene SNPs with RhC/c phenotypes and with RHC/c alleles in Chinese Han and Tibetan population.4. To research on the correlation of polymorphism of RHD gene promoter with RhD-negative mechanism in Chinese Han, Tibetan and Uighur population.5. To study on intron4sequnence and structure of RHD and RHCE gene in Chinese Han and Tibetan population.6. To discover the polymorphism in noncoding region of RHD and RHCE gene, and investigate the genetical mechanism of the Rh blood group system in China, as well as compare diversities of the genetical backgroup among different nations.Methods1. Rh phenotypes were detected wih monoclonal Anti-D, Anti-C Anti-c, Anti-E, and Anti-e antibodies. RhD-negative samples screened with igM-monoclonal Anti-D antibody were confirmed with modified indirect antiglobulin test (IAT).2. Based on previously sequencing, SNPs of SMP1gene were detected by PCR-SSP method using specific primers, and results were verifyed with sequencing.3. Upstream box, downstream box and hybrid box of RHD gene, as well as polymorphic loci of RHD gene promoter were detected by PCR-SSP method, and partial results were verified with sequencing. 4. Intron4sequence and structure of RHD and RHCE gene were investigated with bi-directional sequencing.5. Studty data were analyzed with SPSS13.0statistical software.Results1. Six kinds of Rh phenotypes were detected in200samples from random unrelated Chinese Hans, which were DCCee (55.00%), DCcEe (29.00%), DCcee (7.00%), DccEE (5.50%), DccEe (3.00%) and dccee (0.50%), respectively. The frequencies of main haplotypes were DCe (0.730)> DcE (0.215)> dce (0.055), and the frequencies of RH alleles were D (0.993)> e (0.785)> C (0.730)> c (0.270)> E (0.215)> d (0.007).2. Five kinds of Rh phenotypes were detected in50samples from random unrelated Tibetans in Tibet, which were DCcEe (54.00%), DCCee (28.00%), DccEE (8.00%), DccEe (8.00%) and DCcee (2.00%), respectively. The frequencies of main haplotypes were DCe (0.560)> DcE (0.390)> Dce (0.050), and the frequencies of RH alleles were D (1.000)> e (0.610)> C (0.560)> c (0.440)> E (0.390)> d (0).3. The gene frequencies of SMP1gene exon7were0.739for nt1351C and nt1726A,0.261for nt1351T and nt1726G in Chinese Hans. The gene frequencies of SMP1gene exon7were0.860for nt1351C and nt1726A,0.140for nt1351T and nt1726G in Tibetans. The gene frequency of nt1351C in Tibetans was higher than that in Chinese Hans (x2=6.36, P<0.05), while the gene frequency of nt1351T in Tibetans was lower than that in Chinese Hans (x2=6.36, P<0.05). The gene frequency of ntl726A in Tibetans was higher than that in Chinese Hans (x2=6.36, P<0.05), while the gene frequency of nt1726G in Tibetans was lower than that in Chinese Hans (x2=6.36, P<0.05).4. The genotype frequencies of1351CC,1351TT and351CT in exon7of SMP1gene in Chinese Hans were0.550,0.071and0.379, respectively. The genotype frequencies of1726AA,1726GG and1726AG in exon7of SMP1gene in Chinese Hans were also0.550,0.071and0.379, respectively. The genotype frequencies of1351CC,1351TT and351CT in exon7of SMP1gene in Tibetans were0.263,0.140and0.600, respectively. The genotype frequencies of1726AA,1726GG and1726AG in exon7of SMP1gene in Tibetans were also0.263,0.140and0.600, respectively. The genotype frequencies both nt1351CC and1726AA in Chinese Hans were higher than that in Tibetans (both were x2=9.77, P<0.01), while the genotype frequencies both nt1351CT and1726AG were lower than that in Tibetans (both were x2=9.28, P<0.01). The gene frequencies both1351TT and1726GG in Chinese Hans had no difference compared with that in Tibetans (both were x2=0.01, P>0.05).5.182samples from random unrelated Chinese Hans were phenotyped as101RhCC phenotypes,16Rhcc phenotypes and65RhCc phenotypes, respectively. Among101samples with RhCC phenotypes, of which nt1351C and nt1726A of SMP1gene could be detected in100samples (99.01%), and nt1351T and nt1726G of SMP1gene could be detected only in1sample (0.99%). Nt1351T and nt1726G of SMP1gene could be detected in all16samples with Rhcc phenotypes, furthermore, of which nt1351C and nt1726A of SMP1gene could be detected in3samples (18.75%). Among65samples with RhCc phenotypes, of which nt1351C, nt1351T, nt1726A and nt1726G of SMP1gene could be detected in64samples (98.46%), and only nt1351C and nt1726A of SMP1gene could be detected.in1sample (1.54%).6.182samples from random unrelated Chinese Hans were genotyped as100RHCC genotypes,16RHcc genotypes and66RHCc genotypes, respectively. Only nt1351C and nt1726A of SMP1gene could be detected in all100samples with RHCC genotypes, and nt1351T and nt1726G of SMP1gene could not be detected. Nt1351T and nt1726G of SMP1gene could be detected in all16samples with RHcc genotypes, furthermore, of which nt1351C and nt1726A of SMP1gene could be detected in3samples (18.75%). Among66samples with RHCc genotypes, of which nt1351C, nt1351T, nt1726A and nt1726G of SMP1gene could be detected in65samples (98.48%), and only nt1351C and nt1726A of SMP1gene could be detected in1sample (1.52%).7.50samples from random unrelated Tibetans in Tibet were phenotyped as13RhCC phenotypes,8Rhcc phenotypes and29RhCc phenotypes, respectively. Only nt1351C and nt1726A of SMP1gene could be detected in all13samples with RhCC phenotypes, and nt1351T and nt1726G of SMP1gene could not be detected. Nt1351T and nt1726G of SMP1gene could be detected in all8samples with Rhcc phenotypes, furthermore, of which nt1351C and nt1726A of SMPl gene could be detected in4samples (50%). Among29samples with RhCc phenotypes, of which nt1351C, nt1351T, nt1726A and nt1726G of SMPl gene could be detected in27samples (93.10%), and only nt1351C and nt1726A of SMPl gene could be detected in2samples (6.90%).8.50samples from random unrelated Tibetans in Tibet were genotyped as 13RHCC genotypes,8RHcc genotypes and29RHCc genotypes,respectively. Only nt1351C and nt1726A of SMP1gene could be detected in all13samples with RHCC genotypes, and nt1351T and nt1726G of SMPl gene could not be detected. Nt351T and nt1726G of SMP1gene could be detected in all8samples with RHcc genotypes, furthermore, of which nt1351C and nt1726A of SMPl gene could be detected in4samples (50%). Among29samples with RHCc genotypes, of which nt1351C, nt1351T, nt1726A and nt1726G of SMP1gene could be detected in27samples (93.10%), and of which only nt1351C and nt1726A of SMPl gene could be detected in2samples (6.90%).9. Among7RhD-negative samples of Tibetan in Tibet, of which3samples with RhCc phenotype and RHCc genotype nt1351C, nt1351T, nt1726A and nt1726G of SMP1gene could be detected simultaneously, and4smaples with Rhcc phenotype and RHcc genotype only nt1351T and nt1726G could be detected and nt1351C and nt1726A of SMPl gene could be detected.10. Out of200RhD-positive samples of Chinese Hans,189samples (94.50%) could detect upstream box and downstream box of RHD gene, which meaned these samples had RHD+/RHD+genotypes, and11samples (5.50%) could detect upstream box, downstream box and hybrid box. Out of50RhD-positive samples of Tibetans in Tibet,47samples (94%) could detect upstream box and downstream box of RHD gene, and3samples (6%) could detect upstream, downstream and hybrid box. Out of50RhD-positive samples of Uighurs in Xinjiang,45samples (90%) could detect upstream and downstream box of RHD gene, and5samples (10%) could detect upstream, downstream and hybrid box. The frequencies of RHD+/RHD+genotype with RhD-positive phenotype samples showed no significant difference between Chinese Han and Tibetan polulation, between Han and Tibetan polulation, as well as between Uighur and Tibetan polulation (x2=0.04, P>0.05; x2=0.71, P>0.05; x2=0.14, P>0.05, respectively). The frequencies of RHD+/RHD-genotype with RhD-positive phenotype samples showed no significant difference between Chinese Han and Tibetan polulation, between Han and Tibetan polulation, as well as between Uighur and Tibetan polulation (x2=0.04, P>0.05; x2=0.71, P>0.05; x2=0.14, P>0.05, respectively).11.Out of228RhD-negative samples of Chinese Hans,164samples (71.93%) could only detect hybrid box of RHD gene wich meaned these samples had RIID-/RIID genotypes,63samples (27.63%) could detect upstream box, downstream box and hybrid box, and only1sample (0.44%) could detect upstream box and downstream box. Out of70RhD-negative samples of Uighurs in Xinjiang,59samples (84.29%) could only detect hybrid box of RHDgene, while11samples (15.71%) could detect upstream box, downstream box and hybrid box. Out of7RhD-negative samples of Tibetans in Tibet,6samples (85.71%) could only detect hybrid box of RHD gene, while1sample (14.29%) could detect upstream box, downstream box and hybrid box of RHD gene. Frequencies of RHD+/RHD-genotype with RhD-negative phenotype showed significant difference between Chinese Han and Uighur polulation (x2=4.08,0.01<P<0.05), but showed no significant difference between Chinese Han and Tibetan, as well as between Tibetan and Uighur polulation (x2=0.12, P>0.05; x2=0.2, P>0.05, respectively). Frequencies of RHD-/RHD-genotype with RhD-negative phenotype showed significant difference between Chinese Han and Uighur polulation (x2=4.34,0.01<P<0.05), but showed no significant difference between Chinese Han and Tibetan polulation, as well as between Tibetan and Uighur polulation,(x2=0.14, P>0.05; x2=0.2, P>0.05, respectively).12. Upstream box, downstream box and hybrid box of RHD gene could be detected in all6weak D samples of Chinese Hans. Out of42samples with Del phenotype,41could detect upstream box, downstream box and hybrid box of RHD gene, while1sample could only detect upstream box and downstream box of RHD gene.13. Four polymorphic loci in RHD gene promoter could be detected in all424samples with RHD+/RHD-or RHD+/RHD+genotype, including200RhD-positive Chinese han samples,50RhD-positive Tibetan samples,50RhD-positive Uighur samples, as well as including6weak D samples,42Del samples,64RhD-negativeChinese Han samples,11RhD-negative Uighur samples, and1RhD-negative Tibetan sample. The four polymorphic loci in RHD gene promoter could not be detected in all229samples with RHD-/RHD-genotype, including164RhD-negative Chinese Han samples,59RhD-negative Uighur samples, and6RhD-negative Tibetan samples.14. Intron4length of RHCE gene in Chinese Han and Tibetan population was the same, which was1078bp long including a Alu sequence. Intron4length of RHD gene in Chinese Han and Tibetan population was also the same, but intron4length of RHD gene was652bp shorter than that in RHCE gene. Intron4of RHD and RHCE gene in Chinese Han and Tibetan population had the same sequence with that of Caucasian. Compared with Janpanese intron4of RHCE gene, there were three bases insertion and two bases substitution in the652bp deletion sequence in Chinese Hans and Tibetans.Conelusions1. Distribution characteristics of Rh phenotypes in Tibetan population were similar with Chinese population, but it also had its own distribution characteristics in Tibetan population.2. There existed1351C>T and1726A>G polymorphisms in SMP1gene exon7of Tibetan and Han population. The gene frequencies of nt1351C and nt1726A in Chinese Hans were lower than that in Tibetans, while the gene frequencies of nt1351T and nt1726G in Chiese Hans were higher than that in Tibetan. The genotype frequencies of1726AA and1351CC in Chiese Hands were higher than that in Tibetans, while the gene frequencies of1351TT、1726GG、1351CT and1726AG in Chinese Hans were lower that in Tibetans.3. In exon7of SMP1gene, nt1351C was linkage with nt1726A, and nt1351T was linkage with nt1726G.4. Nt1351C and nt1726A in exon7of SMP1gene existed relationship with RhC antigen and RHC allele in Chinese Hans and Tibetans, while nt1351T and nt1726G had relationship with Rhc antigen and RHc allele in Chinese Hans and Tibetans.5. In Chinese Hans, Tibetans and Uighurs, RHD genotypes with RhD-positive phenotypes were mainly RED+/RED+homozygotes, while RHD genotypes with with RhD-negative phenotypes were mainly RHD-/RHD homozygotes.6. As long as existence of the whole or partial sequence of RHD gene, four polymorphic loci of RHD gene promoter could be detected whether in samples with RhD-positive, RhD-negative, weak D phenotypes, or in Del type samples. Four polymorphic loci of RHD gene promoter could not be detected in samples with D-negative and RHD-/RHD-genotype.7. The sequence and structure of intron4of RHD and RECE gene in Chinese Han and Tibetan population were the same with Caucasian population. |
PDF Full Text Request