Experimental Study Of RHCE Genotyping

BackgroundRh blood group system is one of the most polymorphic and immunogenic systems known in human. As a good gene marker, Rh blood group system is widely applied in forensic practice. In China, traditional serological typing methods are still used for typing of above Rh antigens, which have many disadvantages. Moreover, as to some special questions of today's forensic practice, such as prenatal parentage testing and parentage testing of person who has ever received transfusion, traditional methods are rarely helpful. Research of Rh blood group system has already been penetrated to molecular level since Rh cD-NA was cloned in 1990. The technique of RH genotyping appeared at this time and became investigative hot spot. Now, RH genotyping assays are mainly based on research in whites, research of RHCE genotyping is rare in China. The sequences of RH genes may vary with the ethnic background of a person. It is important to be aware of the differences in genetic sequences in order to develop genotyping methods that are reliable in a multiracial society. In my study,the previously described RHCE genotyping assays were tested on large panels of se-rologically typed donors from different Chinese ethnic backgrounds, so that suitable RHCE genotyping methods can be found for Chinese population and used in the future practice.Materials and Methods1. Samples1.1 Samples for population census: A total of 400 EDTA blood samples were obtained from 200 Han population ( China Medical University, ForensicMedicine School) and 200 Ii population (Hainan Medical College).1. 2 Samples for parentage diagnosis: 5 cases of parentage diagnosis were obtained from Forensic Medicine School in China Medical University.2. Methods2.1 Serologic typing: RBCs of all donors were phenotyped for D, C, c, E and e by using standard monoclonal antisera.2.2 Genotyping for RHCE: Genomic DNA was isolated from peripheral blood WBCs. There are 4 PCR applied for genotyping of RHCE, several exons and introns of RHCE gene are studied, mainly including RHCE exon2, inton2 and exon 5. The PCR production are detected by the PAGE and AgNO₃ stain.Results1. Genotyping results1.1 Chinese Han There are 9 kinds of genotypes in Chinese Han, they are RHCCEE 1(0.5%), RHCCEe 3(1.5% ), RHCCee 88(44% ),RHCcEE 4 (2% ) , RHCcEe 20(10% ) , RHCcee 54(27% ) , RHccEE 1 (0. 5% ), RHc-cEe 22(11%), RHccee 7(3.5%).1.2 Chinese Ii There are 8 kinds of genotypes in Chinese Ii, they are RHCCEE 2(1%), RHCCEe 2(1%), RHCCee 106(53% ) , RHCcEE 7(3. 5%), RHCcEe 62(31%), RHCcee 10(5% ), RHccEE 3(1. 5% ), RHccEe 8(4%).The genotyping results are in accordance with the serologic results2. Parentage diagnosis resultsParentage diagnosis results show that they are in accordance with the results of 13 STRs which recommended by international forensic genetics communication.ConclusionPCR - SSP method can detect the RHCE genotyping of Chinese Han and Ii without mistakes.