Structural And Genetic Analysis Of Human RH Gene

Objective: With the cDNA cloning of RHD and RHCE gene, investigation on genetic structures of Rh blood group system has made great progress, and becomes the key of blood transfusion medicine since 1990.However, many disadvantages hamper the development of studies on RH gene. Firstly, domestic research on RH gene is later than overseas, majorities of focus on RH gene confine to Hans nationality, and reagents used, which aiming at Caucasian and mainly detecting some polymorphism, purchage from other countries. Then investigation into structures of RH gene is inadequate, studies on RHD gene exceed on RHCE gene up to now, thus reseach couldn't represent the structures of RH gene. Finally, genetic patterns don't be reported in China yet, and many medical colleges explain genetics of RH gene with outdated and incorreted three-locus theory.The initial investigation shows negative mechanisms of RhD take on national polymorphism. It's rare that individuals With RhD(-) posse different genetic structures for their nationalities. To make genetic structures and inherited characteristics clear has become important duty for Chinese scholars.The investigation expects to reveal genetic structures and inherited characteristics of RHD and RHCE gene, to discover frequent non-expression RHD alleles , and to establish genotyping methods suitable for Chinese by analysizing large quantity of genetic structures of Han, Uigur and other partial minorities' with RhD negative.Methods: Rh antigens were detected with Monoclonal and Multiclonal D,C,c,E,e antibodies routinely. Negative results with routine serology were confirmed with IAT and Del types were detected with Absorption/elution test.The upstream, downstream and hybrid Rhesus boxes were determined with PCP-SSP and mismatched PCR. 10 exons of RHD gene were detected with PCP-SSP utilizing polymorphism points of 10 exons to design SSP. The best annealing temperatures of every PCP-SSP were obtained throughtemperature-laddered SSP. 4 polymorphism in promoter, 37 bp insertion in intron 3, and intron 4 were detected with PCP-SSP and muliti-PCR.RHE/e RHC/c alleles were genotyped and 109 bp insertion in intron 2 of RHCE gene were detected with PCP-SSP. Established technologies were verified and partial special samples were sequenced with four-dyed sequencing technology.Results: The phenotypes out of 228 cases of unrelative Hans with RhD(-) were 130 ccee(57.02%),68 Ccee(29.82%) and 11 CCee (4.82%), without CCEe and CcEE phenotypes. The ccee(88.89%)?Ccee(5.56%) and CCee(5.56%) phenotypes could be detected only out of 72 cases of Uigurs with RhD(-) in Sinkiang, China, and the ratio of Uigurs in ccee and Ccee phenotypes was higher than that of Hans (P<0.005), but the ratio of CCee didn't posses statistical discrepancy (P>0.05), compared to that of Hans. 42 cases of the Del types (18.42%) were detected out of 228 unrelative Hans with RhD(-),however, none of the Del types was obtained out of 72 unrelative Uigurs. 6 cases of weak D belonged to RhD positive with weak agglutination, and 2 cases of Du types were classified to weak D, but the other Du type showed negative with brine agglutination test and weak positive with IAT(belong to low graded weak D).Detected results of Rhesus boxes showed RHD7RHD, RHD/RHD and RHD/RHD genotypes in unrelative Hans with RhD(-) were 61.40%, 34.21% and 4.39% respectively, While the results were 94.44%?2.78% and 2.78% respectively in unrelative Chinese Uigurs, furthermore, all 6 cases of partial minorities were RHD7RHD types. The results in RHD7RHD and RHD/RHD genotypes group from unrelative Chinese Uigurs were higer than in Chinese Hans (P<0.005), while the result showed none of statistical discrepancy (P>0.500).10 exons detected results of RHD gene from 80 cases of RHD/RHD genotypes(78 Hans and 2 Uigurs) representing single RHD gene indicated that usual none-expressed RHD alleles was RHD-CE(2-9)-D2(19.23%) , RHD(1-10)( 17.95%), RHD-CE(2-7)-D2(6.41%), RHD-CE(3-6)-D(2.56%),RHD-CE(6)-D (2.56%) and RHD-CE(5-6)-D (1.28%)respectively. 1 case of RHD-CE(2-...