Study On Polymorphism Of RHCE Gene In Southern Chinese Hans And Tibetans

BackgroudSince the Rh blood group system was discovered in1939. it has been well known that the Rh blood group could induce Rh-incompatible hemolytic transfusion reactions, hemolytic disease of newborn (HDN), and autoimmune hemolytic anemia. It is one of the most complex and polymorphic blood group systems, whose importance is secondary to the ABO blood group system. RH gene presents rich ethnic diversity and polymorphism which existed not only in coding region but also in non-conding region.RH gene is consist of RHD and RHCE gene both gens having more than96%homology. RH gene located on1p34.3-1p36.1, of which RHD gene encode RhD polypeptide, and RHCE gene encode RhC/c and RhE/e polypeptide. Compared with RHc allele, RHC allele has six different nucleotides locating on48、150、178、201、203, and307sites, which are C、T、A, G、G, and T in RHC allele, otherwise are G、C、C、A、A and C in RHc allele. Among these sites, only the103proline are encoded by nt307, which determines the antigenicity and specificity of Rhc antigen.It was reported that all of the Caucasian population carrying Rhc antigen had C nucleotide on the307positon of exon5. It could gain correct results using this polymorphism to genotype RHc allele. However, other findings indicated that it existed high false positive and false negative errors as for RHC genotyping. Some study indicated that all the Caucasian population carrying RHC had109insert in intron2of RHCE gene. However, other study showed that using this insert to detect RHC allele would make false negative error. It was reported that all the Caucasian population carrying RHC had C on nt48of exonl in RHCE gene, nevertheless,5.2%of the Caucasian population carrying RHc had C, as well as74%of Africa blacks carrying Rhcc also had C in this location, which would make false positive error using this polymorphism to genotype RHC allele. No related reports in China had been seened. It was reported that there had733C＞G polymorphism in exon5of RHCE gene, and the polymorphism was2%in Caucasian population and50%in Africa blacks respectively, No related data about Asia had been reported.Comparative study on RHCE gene between Chinese Han and Tibetan population is helpful to provide insight into RH gene structure and ethnic genetic differences, to explore the genetic mechanism of RH gene in evolution, recombination and mutation, as well as to reveal genetic background of RHD and RHCE polymorphism, and it also contributed to genoty RHCE gene correctly.ObjectiveTo investigate the48G>C polymorphism in exon1,109bp insert in intron2, and733C＞G polymorphism in exon5of RHCE gene in Chinese Han and Tibetan population. To compare the genetic diversities of above polymorphism between Chinese Hans and Tibetans population, and to investigate the correct methods to genotype RHCE gene.Methords1. Samples collection.187random unrelated samples from Southern Chinese Hans were provided by the Guangdong Provincial Hospital of TCM, and51random unrelated samples from Tibetans were kindly provided by the Blood Center of the Tibet Autonomous Region.2. All the Rh phenotypes from Southern Chines Hans and from Tibetans were detected with microgel method. RhD-negative samples were confirmed with indirect anti-human globulin test.3. RHCE gene were genotyed by polymerase chain reaction with specific primers.4.48G>C polymorphism in exon1and109bp insertion in intrion of RHCE gene were detected by PCR-SSP.5. In order to investigate the correct genotyping methods for RHCc allele, the samples whose RHCc genotypes were inconsistent with corresponding phenotypes were sequencing.6.733C＞G polymorphism in exon5of RHCE gene were detected by PCR-SSP, and the method were proved by sequencing.Results1. There existed48G>C polymorphism in exon1of RHCE gene in Southern Chinese Han and Tibetan population, which was3.23%in Chises Hans and12%in Tibetan respectively (X2=6.2, P<0.01). It would results in3.23%false positive rate in Southern Chinese Han population and12%false positive rate in Tibetan population using48G>C polymorphism in exon1of RHCE gene to genotype RHC allele.2. The deletion rate of109bp insertion in intron2of RHCE gene in individuals carrying RhC were1.08%in Southern Chinese Han population and0in Tibetan population, which showed no difference between Chinese Han and Tibetan population (X2=1.2, P>0.05). It would results in1.08%false negative rate in Southern Chinese Han population using109bp insertion in intron2of RHCE gene to genotype RHC allele, wich need to further validate if109bp insertion was suitable to genotype RHC allele in Tibetan population.3.733C＞G polymorphism in exon5of RHCE gene was not detected in Southern Chinese Han and Tibetan population, and all the samples had the G at the nt733in exon5of RHCE gene.Conelusion1. There existed deletion of109bp in intron1,48G>C polymorphism in exon1, however,733＞G polymorphism in exon5was not detected in Southern Chinese Han population2. There existed48G>C polymorphism in exon1, however, deletion of109bp in intron1and733C＞G polymorphism in exon5was not detected in Tibetan population3. There exited difference between the Southern Chinese Han population and Tibetan population on48G>C polymorphism. However, the difference on deletion of109bp in intron1and733C＞G polymorphism in exon5was not detected.4. It would results in false positive and false negative errors using48G>C polymorphism in exon1and109bp insertion in intron of RHCE gene to genotype RHC allele in Southern Chinese Han population and Tibetan population.