Alloanti-D is still one of the most common causes of severe hemolytic disease of the fetus and newborn in China, as rhesus immunoglobulin (RhIG) prophylaxis is not a routine practice throughout China. HLA plays an important role in the susceptibility to alloimmunisation against red blood cell antigens. This study was designed to identify susceptible and protective HLA alleles for alloanti-D immunisation after pregnancy in the southern Chinese D-negative (D-) pregnant women. In this study, a cohort of 116 true D- pregnant females who had not received prophylactic RhIG prophylaxis, had two or more pregnancies, and did not produce alloanti-D (non-responders group), and 122 true D- pregnant women with alloanti-D immunisation (D responders group), were enrolled. HLA genotyping (HLA-A, -B, -C, -DRB1, -DPA1, -DPB1, -DQA1, and -DQB1) was performed by third generation sequencing with nanopore technology. The phenotypic frequencies of HLA alleles were compared between the D responders group and non-responders group. The results showed that the phenotypic frequencies of HLA-DRB1*08:03 and HLA-DQA1*01:03 alleles in the D responders group were significantly lower than those in the non-responders group: 1.7% versus 13.1% [Odds Ratio (OR): 0.116, 95% CI: 0.026-0.518; pc = 0.029] for HLA-DRB1*08:03 allele, and 5.2% versus 18.8% (OR: 0.235, 95% CI: 0.092-0.600, pc = 0.019) for HLA-DQA1*01:03 allele. Our findings indicated that the presence of HLA-DRB1*08:03 or HLA-DQA1*01:03 alleles can be considered as a protective factor for alloanti-D immunisation in the southern Chinese D- pregnant women.

Neonates in the NICU, particularly those born with very low birth weights, are at heightened risk for healthcare-associated infections (HAIs). Such infections can prolong hospitalization, increase medical costs, and potentially lead to adverse long-term outcomes. Recognizing evolving infection patterns and targeted prevention measures is essential to improving patient care. We retrospectively collected data on neonates admitted to the NICU of our hospital between January 2019 and December 2023. Information regarding HAI incidence, site of infection, device utilization, pathogen distribution, and antibiotic use was extracted and statistically analyzed. Comparisons regarding infection rates and clinical variables were performed using chi-square or Fisher’s exact tests for categorical variables and Wilcoxon rank-sum tests for continuous variables. Among 9786 neonates (70,509 patient-days), 86 HAI cases were identified (0.88

BACKGROUND:Mutation in the FUT1 gene can impact the structure and function of α-(1,2)-fucosyltransferase 1 (α2FucT1). To explain the para-Bombay phenotype of a novel FUT1 allele, three-dimensional (3D) modeling and mutation effect analysis of α2FucT1 were performed by bioinformatic tools.MATERIALS AND METHODS:Blood and saliva samples were collected from a patient who was suspected to be a para-Bombay phenotype. H, A, and B antigens were determined with routine serologic methods for those samples. FUT1 and FUT2 coding regions were determined by Sanger sequencing. The novel heterozygous mutation was confirmed by cloning and sequencing. 3D model of mutant α2FucT1 was built by Phyre 2 and the mutation effect was evaluated by Chimera, PROVEAN, and Polyphen-2.RESULTS:Weak H, A, and B antigens were detected on RBCs of the proband and normal quantities of H, A, and B antigens were observed in his saliva. Cloning sequencing showed that the proband carried a novel FUT1 allele (c.889C>T, p.Leu297Phe) and a null FUT1*01N.06 allele. 3D model showed that the p.Leu297Phe variant in α2FucT1 reduced the number of hydrogen bonds and the mutation effect was predicted to be deleterious and possibly damaging, which suggested that the conformation and activity of the enzyme might be significantly damaged.CONCLUSION:A novel missense mutation led to an amino acid variant p.Leu297Phe in α2FucT1, which was a potential cause of the inactivation of the enzyme. Computational evaluation was a convenient and useful approach for the mutation effect analysis of the enzyme.

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.

Objective To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. Methods ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. Results The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. Conclusion In this case, the development of anti-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

Background: Common alloantibodies leading to severe hemolytic disease of the fetus and newborn (HDFN) could vary among different ethnic groups. The MNS blood group hybrid glycophorin GP.Mur distributes with a high frequency in the regions of Southeast Asia. Alloantibodies against GP.Mur (anti-‘Mia’) often present as mixture of antibodies against several low frequency antigens. In this study, we first described a case of severe HDFN in Guangzhou, China, which was caused by alloantibodies of anti-E in combination with specificities to the GP.Mur including Mia, Mur and Hil. Methods: Blood samples from the newborn boy and parents have been subjected to antibody screening and identification analysis followed by GYP*Mur genotyping. The direct antiglobulin test (DAT) and the eluate technique were also performed for the newborn. Results: The mother was group B, CCDee, Mur−, the father was group B, ccDEE, Mur+, and the newborn was group B, CcDEe, Mur+. Genotyping results showed the mother was absent for GYP*Mur, while the father and the newborn carried heterozygous GYP*Mur allele. DAT test of the newborn was strongly positive with anti-IgG. Anti-E and anti-‘Mia’ were detected in the maternal serum and the newborn’s eluate, whereas anti-E alone was detected in the newborn’s serum. The anti-‘Mia’ specificity was further identified as combination of anti-Mia, anti-Mur and anti-Hil. Conclusions: Because alloantibodies to GP.Mur could cause severe HDFN, it is highly recommended to include GP.Mur red cells in antibody screening cells to avoid miss detection of the alloantibodies in the populations of Southeast Asia.

BACKGROUND:The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary.STUDY DESIGN AND METHODS:Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed.RESULTS:DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women.DISCUSSION:The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.

OBJECTIVES:To screen RhCE variants in the Chinese Southern Han donors for molecular genetic analysis. BACKGROUND:More than hundreds of RhCE variant alleles have been described to resulting in weak and/or partial expression of RhCE antigens, generation of low-prevalence antigens and/or absence of a high-prevalence antigen of Rh system, which mainly reported in the people of African origin. In this study, the serological screening and molecular genetic analysis of RhCE variants were performed in the Chinese Southern Han donors. METHODS:The blood samples of E(+) donors were preliminarily collected. Then, RhCE antigens of the E(+) samples were further typed by using two sets of monoclonal anti-C, anti-c, anti-e and another anti-E. When weak expression of RhCE antigens was found, direct sequencing for 10 exons of RHCE gene, RH genotyping analysis by using multiplex ligation-dependent probe amplification, flow cytometric analysis and even cDNA sequencing were performed. RESULTS:A total of 4487 E(+) samples were collected and four samples with weak expression of antigens were detected. RHCE*Ce375G and RHCE*Ce667T variant alleles were identified in two samples with weak expression of e antigen, respectively. But no variant alleles were found in another two samples with weak expression of C antigen. CONCLUSION:The variant RHCE*Ce375G validated by mRNA sequencing and the deduced RHCE*Ce667T alleles were firstly identified in the Chinese population. The DCE haplotype might account for the weak expression of C antigen in two donors.

BACKGROUND:The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed.STUDY DESIGN AND METHODS:DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast.RESULTS:The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals.CONCLUSION:The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Background The distribution of DI1/DI2 antigens of the Diego blood group system is polymorphic in Mongoloid populations and the corresponding alloantibodies are clinically significant. Here a novelDIvariant was found by donor screening, and the effect of the novel and previously reported mutations on expression of DI1/DI2 antigens and Band 3 protein was explored. Study Design and Methods DNA samples of 1150 Chinese donors were collected.DI*01/DI*02genotyping was determined by Sanger sequencing. For the carrier of novel allele, the expression of Band 3 and DI1/DI2 antigens on red blood cells (RBCs) was detected by Western blot and flow cytometry, respectively. in vitro expression studies were conducted by transfecting the mutant (including the novel and three reportedDI*02(2534T),DI*02(2358_2359insCAC),andDI*02(2572T)alleles) or wild-typeDI*02constructs into HEK 293T cells, the expression of Band 3 and DI1/DI2 antigens was analyzed. Results A novel heterozygous mutation (c.2558C>T, p.Thr853Met), which is located near the DI1/DI2 polymorphism site (c.2561T>C), was identified in a donor with DI:-1,2 phenotype. Reduced expression of DI2 antigen was observed on the RBCs, while weakened expression of Band 3 and absence of DI2 antigen were detected in cells transfected with the mutantDI*02(2558T)construct. In addition, absent or decreased expression of Band 3 and DI2 antigen was also detected in cells transfected with three reported mutant constructs. Conclusion The novelDI*02(2558T)allele and three previously describedDImutations can affect the expression of Band 3 protein and/or DI2 antigen and/or interfere withDI*01/DI*02genotyping result.

目的 建立MN血型高分辨率熔解曲线(HRM)基因分型方法,进行MN血型基因分型,并探讨罕见Mc血型的鉴定方法.方法 收集2013年1月-6月澳大利亚红十字血液服务中心献血者全血标本150例,提取基因组DNA,并利用HRM基因分型方法对MN血型进行基因分型;对HRM分型结果中出现的变异曲线标本,采用直接测序法对GYPA基因外显子2进行测序分析,确证其基因型;同时,利用单克隆抗-M及抗-N通过血清学方法检测待检标本的表型.结果 150例标本中有149例标本的HRM基因分型结果与表型一致;只有1例标本的熔解曲线形态与3个对照标本均不同,提示在扩增片段存在变异位点,血清学结果显示该标本表型为M+N-;但是,该标本的GYPA基因外显子2测序结果显示其在M抗原的1个特异性SNP位点中存在杂合突变:c.71_72GT＞AG,p.Gly5Glu,根据该结果判断其基因型为MMc.结论 HRM基因分型方法能对MN抗原进行准确分型.Mc是介于M与N抗原之间的1种抗原,能与大多数抗-M及少数抗-N反应,因此,仅利用1种抗-M和抗-N试剂容易导致Mc抗原的漏检,基因分型方法更容易发现Mc血型.

Objective To genotype MNS hybrid glycophorin GP. Mur using high resolution melting curve analysis (HRM) and to investigate the reliability of HRM on GP.Mur genotyping. Methods Whole blood samples were collected from 254 blood donors and genomic DNAs were extracted. HRM analysis was conducted to genotype MNS hybrid glycophorin GP.Mur. MLPA analysis and serological tests were performed to confirm the genotype and phenotype of GP. Mur. Direct sequencing was conducted to confirm the variant results. Results A total of 14 donors with heterozygous GYP*Mur allele were identified. Among them, the genotype of 11 donors was consistent with the MLPA genotyping results. And the phenotypes of the other 3 donors were verified by serological tests. Meanwhile, 4 samples with variant melting curves were identified and the sequencing results showed these 4 samples carried point mutations on the GYPB gene. Conclusion HRM is a reliable method for GP.Mur genotyping and presents certain advantages in mutation detection.

BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in- frame insertion, or deletion mutations of the RHD gene and hybrid RHD- CE- D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation- dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next- generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG* 572A or wild- type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c- E- e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c. 572G > A, p. Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG* 572A, the mutant RHAG* 572A and RHD, and the mutant RHAG* 572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG* 572A allele results in weak D expression. It does not affect RhCE expression.

BACKGROUNDMNS hybrid GP(B‐A‐B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B‐A‐B) hybrid glycophorins based on high‐resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population.STUDY DESIGN AND METHODSDNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B‐A‐B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation‐dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles.RESULTSA total of five GYP(B‐A‐B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B‐A‐B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild‐type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5′ inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single‐nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles.CONCLUSIONThe HRM assay could distinguish the GYP(B‐A‐B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B‐A‐B) genotyping kits for the Chinese population.

BACKGROUND: Genotyping platforms for common red blood cell (RBC) antigens have been successfully applied in Caucasian and black populations but not in Chinese populations. In this study, a genotyping assay based on multiplex ligation-dependent probe amplification (MLPA) technology was applied in a Chinese population to validate the MLPA probes. Subsequently, the comprehensive distribution of 17 blood group systems also was obtained.STUDY DESIGN AND METHODS: DNA samples from 200 Chinese donors were extracted and genotyped using the blood-MLPA assay. To confirm the MLPA results, a second independent genotyping assay (ID Core+) was conducted in 40 donors, and serological typing of 14 bloodgroup antigens was performed in 91 donors. In donors who had abnormal copy numbers of an allele (DI and GYPB) determined by MLPA, additional experiments were performed (polymerase chain reaction, sequencing, and flow cytometry analysis).RESULTS: The genotyping results obtained using the blood-MLPA and ID Core+ assays were consistent. Serological data were consistent with the genotyping results except for one donor who had a Lu(a-b-) phenotype. Of the 17 blood group systems, the distribution of the MNS, Duffy, Kidd, Diego, Yt, and Dombrock systems was polymorphic. The Mur and St(a) a antigens of the MNS system were distributed with a frequency of 9% (18 of 200) and 2% (4 of 200), respectively. One donor with chimerism and one who carried a novel DI*02(A845V) allele, which predicts the depression of Di(b) antigen expression, were identified.CONCLUSIONS: The blood-MLPA assay could easily identify the common blood-group alleles and correctly predicted phenotype in the Chinese population. The Mur and St(a) a antigens were distributed with high frequency in a Southern Chinese Han population.

目的 了解广州地区无偿献血者中Lu(a-b-)表型的频率;通过检测Lutheran血型系统编码LU基因和In(Lu)表型相关红细胞转录因子编码KLF1基因,了解Lu(a-b-)表型的分子遗传背景.方法 运用血型血清学方法对5 000名广州地区无偿猷血者的Lub抗原进行筛查,对筛选到的Lub阴性个体,用抗球微柱凝胶卡进行Lu(a-b-)表型确认,扩增Lu(a-b-)表型先证者LU基因15个外显子和KLF1基因3个外显子并进行测序,通过基因序列比对分析测序结果.结果 在广州地区5 000名无偿献血者中筛选到2名Lu(a-b-)表型先证者.测序结果显示,2名先证者LU编码基因编码区及邻近内含子区域未发现任何杂合或纯合突变,进一步分析KLF1基因,发现先证者1携带KLF1基因外显子2的c.895C ＞G (p.His299Asp)杂合突变,先证者2携带KLF1基因外显子2的c.519_526dupCGGCGCC(p.Gly176Argfs*179)杂合突变.结论 Lu(a-b-)表型在广州地区献血者中的频率约为0.04％ (2/5 000).2例均为In(Lu)表型,其表型形成机制与KLF1基因突变相关.

BACKGROUND: The RHCE allele is highly polymorphic; more than 60 variants have been described leading to diminished expression of C, c, E, and e antigens. Not much is known about the prevalence of RHCE variants in the Chinese population. Individuals carrying a variant are at risk to develop alloantibodies in response to mismatched pregnancy or transfusion. In this study, phenotyping and genotyping of the RHCE allele in Chinese donors revealed a new clinically relevant mutation.STUDY DESIGN AND METHODS: Blood samples from 200 D- and 200 D+ Chinese donors were analyzed by the RH multiplex ligation-dependent probe amplification (MLPA) assay and compared to serologically typed RhCE phenotypes, when available. All exons of the RHCE gene were sequenced in samples with aberrant genotyping results. The phenotype of the new variant RHCE allele was tested by transducing cultured human erythroblasts.RESULTS: Aberrant copy numbers for Exon 2 of the RHCE gene were discovered by MLPA in six D-donors (6/200), but not in D+ donors (0/200). Sequencing of the RHCE gene in these six donors identified a new variant RHCE*ce308C>T (p.103Pro>Leu) allele with an allele frequency of 0.015 within the D-individuals in this study. This variant was not detected in D+ individuals showing linkage with the D-haplotype. Serologically weak C expression and loss of c expression was demonstrated on donor red blood cells. In vitro transfection studies of the RHCE*ce308T variant in cDe/ce and CDe/CDe erythroblasts confirmed that the variant is associated with anti-C reactivity while abolishing c expression.CONCLUSION: Genotyping of individuals carrying this variant by standard RHCE genotyping might falsely predict a C-phenotype or a c+ phenotype. This new variant should be taken into account in RHCE genotyping assays designed for the Chinese population.

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.

The Lutheran blood group system consists of 20 antigens encoded by LU gene and includes four pairs of antithetical antigens. The remainders are high-frequency antigens. Lu(a–b–)/Lunull is a rare phenotype first described by Crawford and colleagues in 1961 characterized by the absence of all Lutheran system antigens, which distributed with a frequency of 1:3000 in South East England and 1:5000 in South Wales blood donors. Three molecular backgrounds account for this rare phenotype. First, the rare recessive Lunull phenotype arises from homozygosity for inactivating mutations in the LU gene, which is generally ascertained through the presence of anti-Lu3. Second, a mutation of erythroid transcription factors GATA1 (GATA1-binding protein 1) has been shown to be responsible for X-linked Lu(a–b–) phenotype in one family. Third, the majority of probands with dominant inherited Lu(a–b–)/In(Lu) phenotype result from inactivating heterozygous mutations in KLF1. The red blood cells (RBCs) with this phenotype express traces of Lutheran antigen that could only be detected by absorption/ elution technique; therefore, their blood has been used to satisfy the requirement of patients with anti-Lu3 since they are relatively common. So far, four inactive mutations of LU gene accounting for Lunull phenotype and 11 KLF1 mutations accounting for In(lu) phenotype have been described (see Web Resource). In our study, one male donor with Lu(a–b–) phenotype (20 years old and without transfusion history) was identified accidentally while high-throughput genotyping and corresponding serologic typing were conducted in 90 Chinese donors. Anti-Lu and anti-Lu (Sanquin Reagents, Amsterdam, the Netherlands) were used to type Lu(a–b–) phenotype. Absorption/elution testing was conducted by using the anti-Lu serum. The donor’s RBCs were also tested with anti-AnWj and anti-INFI since the highincidence AnWj (901009) antigen and Indian blood group system antigens including INFI (IN3) are also known to be weakened in the In(Lu) phenotype. HbF level was also tested by using high-performance liquid chromatography (Variant II hemoglobin testing system, Bio-Rad, Hercules, CA). Unfortunately, the samples of the other family members were not available. Furthermore, both LU and KLF1 genes were analyzed by direct sequencing to clarify the molecular background. Genomic DNA was extracted from peripheral blood of Lu(a–b–) proband and 90 random individuals as controls. Exons 1 to 15 of LU gene, the promoter and Exons 1 to 3 of KLF1 gene, and the exon– intron boundaries were amplified by polymerase chain reaction (PCR). Amplification primers, annealing temperature, and PCR condition have been described previously, except that GC Buffer II (Takara, Dalian, China) was used to amplify the Exon_2 of KLF1. PCR products were sequenced using a sequencing kit on a genetic analyzer (ABI BigDye Terminator, Version 3.1, and ABI 3130XL, respectively, Applied Biosystems, Foster City, CA). Heterozygous mutations identified were further examined by TOPO TA cloning (Invitrogen, Carlsbad, CA). The novel mutation identified was tested in 90 random blood donors by direct sequencing. Negative agglutination with anti-Anwj and anti-INFI serum and negative antibody screening results (absence of anti-Lu3) were obtained in this donor. Absorption/ elution testing showed very weak expression of lu antigen compared with normal controls. No mutations were identified in all 15 exons of LU gene. However, a novel heterozygous seven-base duplicated insertion in Exon_2 of KLF1 gene (c.519_525dupCGGCGCC) was detected (Fig. 1A), which resulted in frameshift mutations from Amino Acid 176 (p.Gly176Argfs*179) and created a truncated protein with a premature stop codon (Fig. 1B). This heterozygous mutation was further confirmed by cloning analysis (registered in GenBank as Number JX877554) and was not found in 90 random donors. EKLF encoded by KLF1 gene is an essential transcription factor in the process of erythroid differentiation. The duplicated insertion will disrupt the EKLF transactivation domain in the proline-rich N-terminal region (Amino Acids 1-275) and three zinc finger domain in the C-terminal region (Amino Acids 276-358) that maybe influence the erythroid-specific genes expression resulting in In(lu) phenotype. Based on the weak expression of lu antigen, the absent expression of highfrequency Anwj and INFI antigens, and the novel mutation identified in KLF1 gene, In(lu) phenotype is confirmed in this Chinese donor. Besides, KLF1 mutations are also responsible for persistent hyper– hemoglobin F syndrome. In this donor, mean 0.8% HbF level was obtained compared with 0.7% in controls that indicate the effects of this mutation may be additive for this syndrome. Recently, the heterozygous mutation and compound heterozygous with L51R missense mutation were found in one Korean patient with 1.7% HbF level and one Vietnamese patient with 9.5% HbF level respectively, but the In(lu) phenotype was not tested.