OBJECTIVETo explore the phenotype types and genetic mutation mechanism of Rhesus D variant individuals.

METHODSFouty-eight peripheral blood samples of pregnancies and blood donors who had been identified as Rhesus D variant by using routine serologic methods were collected from January 2013 to October 2015 in our center. The multiple ligation-dependent probe amplification(MLPA) was used to determine the RHD after genomic DNA had been extracted from the blood sample, then the data including gene copy number variations, point mutations, deletions and hybrid fusions were analyzed by GeneMarker software. All exons of blood sample RHD were amplified via PCR and analyzed by sequencing when its MLPA results were not in accordance with serologic results. Cloning and haplotype sequencing were performed if novel allele had been found.

RESULTSRh phenotypes of the 48 samples were typed as following: 20 cases out of 48 were CcDee(41.7%, 20/48),12 cases were ccDEe (25%,12/48), 11 cases were CCDee(22.9%, 11/48), 5 cases were CcDEe (10.4%, 5/48), respectively. The MLPA analysis showed that 38 cases possessed only 1 variant allele(RHD zygosity was Dd), while 10 cases possessed 2 variant alleles(RHD zygosity was DD). In Dd type individuals, point mutations were found in 18 cases and RHD/CE hybrid fusions were found in 20 cases. In DDindividuals, point mutations combined with RHD/CE hybrid fusions were found in 9 cases, deletion combined with RHD/CE hybrid fusions were found in 1 case. Variant alleles analysis basing on MLPA showed that 14 cases were weak D 15 and 22 cases were RhD VI type 3, however, the variant alleles were not identified in 7 cases due to lack of detecting probes and were identified via sequencing analysis. Two novel mutations, 79-81delCTC and 689G>A were also certificated by sequencing in 2 cases.

CONCLUSIONCcDee is the major Rh phenotype in RhD variants, weak D 15 and RhD VI type 3 are the main serologic type of RhD variants, point mutation and RHD/CE hybrid fusions are main molecular mechanism for RhD variant phenotype. Besides, 79-81delCTC and 689G>A are two novel alleles.