[Objective] To further identify the RhD phenotype and RHD genotype in the individual who have RhD negative phenotype in the primary screening, and to analyze the effect of c. 801+2T>G mutation on RhD phenotype by minigene splicing assay. [Methods] The serologic test was performed for RhD phenotype identification and absorption-elution test was performed by using monoclonal anti-D. Sanger sequencing was used to analyze the sequence of RHD genes and the newly identified splicing site mutations of RHD genes were used to construct pSplicePOLR2G micro gene expression plasmids. By using an in vitro micro gene splicing system, the mRNA splicing results were detected and analyzed using agarose and capillary electrophoresis to predict their impact on RhD phenotype. [Results] The serological test results showed that the patient's blood type was RhD-negative, but the anti-D absorption-elution test was positive, indicating a Del phenotype. The rare genotype RHD*(1227A/801+2G) was identified in this individual. The c. 801+2T>G was a novel mutation at 5'-splice site of intron 5. The minigene splicing assay showed that c. 801+2T>G resulted in a complete skipping of RHD exon 5 in the mature transcript, forming a transcript without exon 5. [Conclusion] An individual carrying a novel mutation c. 801+2T>G in the RHD gene was found to exhibit a Del phenotype, but also carry the Asian Del allele c. 1227G>A. It was speculated that the c. 801+2T>G mutation caused RhD negative or Del phenotype based on the results of minigene splicing assay in vitro.

目的 制备能用于Mur抗原血清学检测的抗-Mur试剂,并将其应用于献血者标本的筛查中.方法 采用偶联了载体蛋白——钥孔血蓝蛋白(KLH)的Mur抗原特异性多肽免疫BALB/c小鼠,通过淋巴细胞杂交瘤技术获得分泌抗体的细胞株,用Mur抗原阳性细胞筛选阳性克隆,利用谱细胞鉴定单克隆抗体的特异性,利用制备的单克隆抗体对1 062例献血者标本的Mur抗原进行检测.结果 经筛选及鉴定后共获得4个能稳定分泌抗-Mur的单克隆,均为IgM型Kappa轻链,效价分别为64、128、256和256.制备的抗-Mur单克隆抗体与日本惠赠的人源单克隆抗-Mur分别检测了 1 062例献血者标本,其检测结果符合率为100％.结论 成功建立了能稳定分泌抗-Mur单克隆抗体的杂交瘤细胞株,可用于临床上Mur抗原的血清学快速检测.

Background GP.Mur belongs to the GP(B-A-B) hybrid glycophorin family, which is the most common hybrid glycophorin in Southeast Asia. Antibodies against GP.Mur may cause a clinically significant haemolytic disease of the foetus and newborn (HDFN) although, so far, not many cases have been reported in mainland China. Materials and methods Two Chinese women with a history of severe hydrops foetalis were seen in our centre. Alloantibody identification and GYP.Mur genotyping analysis were used for prenatal evaluation. Intrauterine transfusion was performed in two pregnancies in case 1. The features of these two women are described and literature-reported cases of HDFN related to antibodies against GP.Mur are summarised. Results The phenotype of both mothers was Mia- Mur-, while the fathers' was Mia+ Mur+ with a heterozygous GYP.Mur hybrid gene as determined by a high-resolution melting method of genotyping. In case 1, the antibodies against GP.Mur were detected in the mother's serum and the cord blood of two foetuses. Fortunately, the latest foetus was successfully saved after intrauterine transfusion. In case 2, hydrops foetalis occurred in the first two pregnancies, but the risk of HDFN was excluded for the third foetus because of the GP.Mur negative phenotype. The literature review showed that 68.8% (11/16) of the reported cases of HDFN related to antibodies against GP.Mur occurred in the Chinese population, and that 37.5% (6/16) of them were cases of severe HDFN. Discussion More cases of severe HDFN caused by antibodies against GP.Mur are presumably undetected as GP.Mur cells are not included in the panel of obligatory screening tests in most Southeast Asian countries including mainland China. The high-resolution melting method for GYP.Mur genotyping and zygosity detection is helpful in prenatal management.

OBJECTIVE:To explore the correlation between special A/O genotype and the O phenotype.METHODS:Group O samples with partially reduced or lack of isoagglutinins were collected to determinate the ABO genotype with a PCR-sequence specific primer (PCR-SSP) assay. Seven samples with A/O genotype were selected for further study. Serological tests including forward and reverse typing, H antigen determination and adsorption/elution were carried out with a tube method. Genomic DNA was genotyped by amplifying and sequencing of the coding regions of exons 1 to 7 of the ABO gene.RESULTS:Seven samples were serotyped as group O by the forward typing test. However, reduced anti-A activity was found in 5 samples by the reverse typing test, reduced anti-A and anti-B activities were found in 1 sample, and no anti-A isoagglutinin activity was found with 1 sample. H antigen was determined in all samples by routine serologic method. Neither anti-A nor anti-B was eluted from red cells derived from all samples. Three samples were genotyped as Ael02/O02, whilst the remainders were Ael02/O13, Ael02/O65, Am04/O75, Ael06/O02, respectively.CONCLUSION:Special A/O genotype may not express the A antigen, leading to the generation of group O red cells. Reduced or missed anti-A activity is the typical serological feature of this special group of O phenotype, for which ABO*Ael02 and ABO*O02 are the major alleles. Group O individuals with isoagglutinin detection problem should be grouped by serological tests and genomic DNA analysis.

The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3′ breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

Characterisation of the genetic changes characteristic of hybrid glycophorins by SNV mapping, provides a reliable basis for prediction of the complex phenotypes associated with these clinically significant variants. In particular Gp.Mur is found at prevalences up to 10% of East Asian ethnic groups. Use of genotyping to screen for and characterise these glycophorins is, however, complicated by 'silent' genetic change. In one case of silent genetic change the 5' end of the BAB allele is in intron 2 and 3' end is in the beginning of pseudoexon 3. The insertion of GYPA sequence does not extend to the splice site at the 5' end of intron A3 and splice site of GYPB pseudoexon 3 is homozygous in the individual with this new BAB allele. As the pseodoexon remains silent, no is antigenic change is expected. In a second case a BAB gene conversion typical of Gp.Mur was detected but a further genetic change resulted in inactivation of splice site at the 5' end of intron A3, preventing phenotypic expression of the hybrid exon. To predict phenotype the use of sequencing as well as SNV mapping is required to specifically confirm the predicted phenotype.

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.

Objective To study the molecular genetic mechanism of para-bombay phenotype in two individuals.Methods The proband was a female.When the proband donated blood,because the forward blood group wasn't coincident with her reverse blood group,the blood and saliva specimen from proband and her family members were sent to Guangzhou Blood Center for further identification.Routine serological techniques were used to determine proband's and her family members' blood group and ABH antigen in saliva.The coding regions of FUT1 and FUT2 gene,exon 6 and exon 7 of ABO gene were amplified by polymerase chain reaction using proband's and her family members' genomic DNA.All amplified products were analyzed after being directly sequenced.The two-base deletion regions of FUT1 gene were certified by cloning and haplotype sequencing.Results Proband's and her little brother's blood group were identified as para-bombay while other family members' blood group were normal.Two-base deletion heterozygous mutations of FUT1 gene were found in proband and her brother,AG deletion at position 547-552 and TT deletion at position 880-882,which caused a reading frame shift and a premature stop eodon.Meanwhile,880-882del TT heterozygous mutation was found in proband's grandfather and her father and 547-552del AG heterozygous mutation was found in proband's mother and her little sister.Results Of cloning and haplotype sequencing certified that these two-base deletion mutations occurred at 547-548 and 881-882 position respectively.Three new mutations were found in FUT2 gene,390C ＞ T,418A ＞ T and 749G ＞ A,which could cause the change of amino acid at position 140Ile ＞ Phe and 250Arg ＞ Gln.Conclusions Two-base deletion heterozygous mutations in different positions in FUT1 gene were found in 2 individuals,which maybe the molecular genetic mechanism of para-bombay phenotype.Heterozygous deletion mutation in one-strand DNA wouldn't change the ABO blood group.Three new mutations were also found in FUT2 gene.( Chin J Lab Med,2012,35:815-819)