Application of multiple ligation-dependent probe amplification in allele detection of 2 cases with rare Rh partial D individuals

Abstract

Objective To investigate the application of multiple ligation-dependent probe amplification (MLPA) in allele detection of 2 rare Rh partial D individuals. Methods 2 patients' peripheral blood samples were collected from local hospital in April and September 2012 for RhD phenotyping. Genomic DNA was extracted from peripheral blood after the Rhesus D phenotype had been identified. Multiple ligation dependent probe amplification (MLPA) was used to determinate the RHD and RHCE gene, then the data including gene copy numbers, point mutation or deletion and hybrid fusion were analyzed by GeneMarker software. Results coming from polymerase chain reaction-sequence specific primer (PCR-SSP) and conventional serologic methods were compared with the results of MLPA. Results The MLPA results showed the copy numbers of RHD and RHCE gene of the subject samples as following: RHD was 1.0, RHC and RHc were 1.0, RHE was 0, RHe was 3.0. The copy numbers of D03-380T, D03-455A, D04-602C, D05-667T, D04-514A, D05-787G were 0.Homozygous deletion were found in exon 3-5 of RHD gene. Deletion of exons 3-5 of RHD gene were found in PCR-SSP while alleles of C, c and e gene were found to be positive. Serologic results indicated that RBC of subject samples could be agglutinated with monoclonal anti-D in tubes and the agglutination strength was 2+ , serologic typing results of Rh antigens were C+ c+ E-e+ . The PCR-SSP results and serologic results were basically consistent with MLPA. Conclusions The RhD phenotype of the 2 individuals were D Ⅵ type 4, which was found in Chinese for the first time. As MLPA could detect known point mutation, deletion, hybridization and copy numbers of gene, so it can be used to determine the allele of variant RhD. Key words: Rh Hr blood group system; Genotype; Multiple ligation dependent probe amplification