RhD Epitopes Research Articles

Molecular Mechanism of a Rhesus D Variant Individual with RHD*845A/1227A

To explore the genetic mutation mechanism of a rare Rhesus D variant individual. Regular serological assay was used for determination of Rh type for the sample. Indirect anti-human globulin test (IAT) was used to confirm the RhD antigen and screen the antibodies. D-screen reagent was used to analyze the RhD epitopes of the sample. RHD genotype and RHD zygosity testing of the sample were detected by palymerase chain reaction with sequence-specific primers (PCR-SSP). The full length coding region of RHD gene was sequenced. RHD mRNA was detected using reverse transcription polymerase chain reaction (RT-PCR). The PCR products were cloned and sequenced. The RhD blood group of the sample was determined as weak D, and the Rh phenotype was CcDEe. The antibody screening was negative. The sample tested with all monoclonal anti-Ds in D-screen showed the D epitope profiles as partial D types. The analysis of RHD gene sequence indicated that the individual with RHD c.845G/A and RHD c.1227G/A base heterozygosis. Three kinds of alternative splicing isoforms were obtained by TA cloning and sequencing. The object has RHD c.845G/A and RHD c.1227G/A mutation. This heterozygous mutation is responsible for the low expression of RhD antigen on the red blood cells of the sample.

The Significance of RHD Genotyping and Characteristic Analysis in Chinese RhD Variant Individuals.

BackgroundRhD is the most important and complex blood group system because of its highly polymorphic and immunogenic nature. RhD variants can induce immune response by allogeneic transfusion, organ transplantation, and fetal immunity. The transfusion strategies are different for RhD variants formed by various alleles. Therefore, extensive investigation of the molecular mechanism underlying RhD variants is critical for preventing immune-related blood transfusion reactions and fetal immunity.MethodsRhD variants were collected from donors and patients in Zhejiang Province, China. The phenotypes were classified using the serologic method. The full coding regions of RHD gene were analyzed using the PCR-SBT method. The multiplex ligation-dependent probe amplification (MLPA) assay was used to analyze the genotype and gene copy number. SWISS-MODLE and PyMOL software were used to analyze 3D structures of RhD caused by the variant alleles. The effect of non-synonymous substitutions was predicted using Polymorphism Phenotyping algorithm (PolyPhen-2), Sorting Intolerant From Tolerant (SIFT), and Protein Variation Effect Analyzer (PROVEAN) software.ResultsIn the collected RhD variants, 28 distinct RHD variant alleles were identified, including three novel variant alleles. RH-MLPA assay is advantageous for determining the copy number of RHD gene. 3D homology modeling predicted that protein conformation was disrupted and may explain RhD epitope differential expression. A total of 14 non-synonymous mutations were determined to be detrimental to the protein structure.DiscussionWe revealed the diversity of RHD alleles present in eastern Chinese RhD variants. The bioinformatics of these variant alleles extended our knowledge of RhD variants, which was crucial for evaluating their impact to guide transfusion support and avoid immune-related blood transfusion reactions.

IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.

Detergent-free isolation of native red blood cell membrane complexes

Over the past few decades, studies on the red blood cell (RBC) membrane gave rise to increasingly sophisticated although divergent models of its structural organization, since investigations were often performed in denaturing conditions using detergents. To access soluble isolated RBC membrane complexes with the preservation of their interactions and conformations, we decided to apply the recent SMALP (Styrene Maleic Acid Lipid Particles) technology to RBC ghosts. Depending on the ionic strength of buffers in which ghost membranes were resuspended, the isolated proteins within SMALPs could differ on Coomassie–stained gels, but with few changes when compared to ghost membrane SDS lysates. We subsequently produced SMALPs derived from ghosts from two different blood group phenotypes, RhD-positive and RhD-negative, both types of RBC expressing the RhCE proteins but only RhD-positive cells being able to express the RhD proteins. This allowed the isolation, by size exclusion chromatography (SEC), of soluble fractions containing the Rh complex, including the RhD protein or not, within SMALPs. The use a conformation-dependent anti-RhD antibody in immunoprecipitation studies performed on SEC fractions of SMALPs containing Rh proteins clearly demonstrated that the RhD protein, which was only present in SMALPs prepared from RhD-positive RBC ghosts, has preserved at least one important conformational RhD epitope. This approach opens new perspectives in the field of the erythroid membrane study, such as visualization of RBC membrane complexes in native conditions by cryo-electron microscopy (CryoEM) or immuno-tests with conformation-dependent antibodies against blood group antigens on separated and characterized SMALPs containing RBC membrane proteins.

A case with a novel weak D type

To report on a novel weak D type identified in a Chinese individual. Peripheral blood sample was collected for a voluntary blood donor with weakened expression of D antigen. Routine serological testing was carried out to determine the D, C, c, E and e antigens of the Rh blood group. A D-screening kit was used to analyze the RhD epitopes. The 10 exons and flanking intronic regions of the RHD gene were sequenced. The zygosity of RHD was determined with a sequence-specific primer PCR method. A novel RHD allele, RHD (1022T>A), was found in the subject with a weak D phenotype. Serological testing of the RhD epitopes has coined with the weak D phenotype. A novel weak D allele has been identified in Chinese population.

A D+ blood donor with a novel RHD*D-CE(5-6)-D gene variant exhibits the low-frequency antigen RH23 (D(W) ) characteristic of the partial DVa phenotype.

Blood donors whose red blood cells (RBCs) exhibit a partial RhD phenotype, lacking some D epitopes, present as D+ in routine screening. Such phenotypes can exhibit low-frequency antigens (LFAs) of clinical significance. The aim of this study was to describe the serologic and genetic profile for a blood donor with an apparent D+ phenotype carrying a variant RHD gene where D Exons 5 and 6 are replaced by RHCE Exon (5-6). Anti-D monoclonal antibodies were used to characterize the presentation of RhD epitopes on the RBCs. RHD exon scanning and DNA sequencing of short- and long-range polymerase chain reaction amplicons were used to determine the RHD structure and sequence. Extended phenotyping for LFAs RH23 (D(W) ) and Rh32 was performed. The donor serology profile was consistent with partial RhD epitope presentation. The donor was hemizygous for an RHD variant allele described as RHD*D-CE(5-6)-D hybrid. The RHCE gene insert is at least 3.868 kb with 5' and 3' breakpoints between IVS4 + 132-c.667 and IVS6 + 1960-IVS6 + 2099, respectively. The sequence for this hybrid was assigned GenBank Accession Number KT099190.2. The RBCs were RH23 (D(W) )+ and Rh32-. A novel RHD*D-CE(5-6)-D hybrid allele encodes a partial RhD epitope and carries the LFA RH23 (D(W) ). This and the epitope profile resemble the partial DVa phenotype. Given that RBCs from this individual lack some RhD epitopes, there is an alloimmunization risk if the donor is exposed to D+ RBCs. Conversely, transfusions of RH23 (D(W) )+ cells to RH23 (D(W) )- recipients also pose an alloimmunization risk.

Serologic and molecular investigations of DAR1 (weak D Type 4.2), DAR1.2, DAR1.3, DAR2 (DARE), and DARA

The partial D variant DAR1 (weak D Type 4.2) is caused by three single-point mutations, 602C>G, 667T>G, and 1025T>C. Here we report a molecular study on different D variants belonging to the DAR category (DAR1, DAR1.2, DAR1.3, and DAR2) and their serologic data. A total of 42 samples belonging to the DAR category were screened for the presence of the silent mutations 744C>T and 957G>A. The samples were phenotyped for RhD and RhCE, characterized for RhD epitope expression, and sequenced for RHD exons. Flow cytometry was performed to determine RhD antigen density. The silent mutation 744C>T was found in all six samples previously typed as RHD*DAR2 (602C>G, 667T>G, 957G>A, 1025T>C). In addition to the three nucleotide changes originally reported for the RHD*DAR1 allele, the silent mutations 744C>T and 957G>A were found in 14 of 16 samples previously typed as RHD*DAR1. In the remaining two samples one additional silent mutation, 744C>T, was found. Serologically the DAR1.2 and DAR1.3 samples analyzed in this study showed no distinct difference in their anti-D reaction pattern compared to each other. The anti-D reaction pattern of DARA/DAR2 showed some distinct differences compared to those of DAR1.2 and DAR1.3. RHD*DARA and RHD*DAR2 are the same allele. Furthermore, the alleles RHD*DAR1.2 and RHD*DAR1.3 both exist; however, the silent mutation 957G>A (V319) showed no influence on the RhD phenotype.

ANTI-hrs associated with the RHD-RHCE HAPLOTYPE DAR-ceAR in a patient with Sickle Cell Trait

Introduction: The RH blood group system is the most polymorphic and immunogenic among blood groups and some rare Rh phenotypes are found exclusively in the black population. The RH 18 phenotype (Hr-negative) that is one of these phenotypes is characterized by a high incidence antigen. The production of anti-RH 18 imposes to use equivalent rare antigen-negative red blood cells (RBCs), or deleted-Rh RBCs (Rhnull or D- -) for transfusion.Case Report: A 24-year-old female patient with two previous abortions and one ectopic pregnancy and miscarriage had to be transfused with a RBC unit. She was typed as blood group A, Dccee phenotype with weak D, positive RBC antibody screening, auto-control negative and TAD negative. The antibody was anti-e (title 1/64) determined by testing serum against a commercial RBC panel (Fresenius-Kabi) by IgG indirect antiglobulin test tube (PEG-IAT), and nontreated (LissCoombs) and papain-treated RBCs on a gel matrix (DiaMed, Latino America). After adsorption of serum on Rhe-negative RBC (DccEE), the retrieving antibody was an anti-e-like antibody reacting with all normal Rhe-positive RBCs. Molecular and serologic Rh typing was also performed on blood samples obtained from five relatives of the patient and her husband.Results: Patient and relatives cDNA sequence of gene RHD and RHCE transcripts were analyzed for RHCE exons 4 to 5 and exon 6; for RHD exons 4 to 5, 6 and 7. Sequencing of cDNA from the patient, her father and her sister showed in RHD the presence of the DAR allele point mutation 602 C>G in exon 4; 667 T>G and 744 C>T in exon 5; 957 G>A and 1025 T>C in exon 7; while on gene RHCE we found the ceAR allele carrying 712 A>G, 733 C>G e 787 A>G in exon 5. Hemoglobin tests revealed that the patient, her father and sister had a sickle cell trait (patient HbA1=55%; HbA2=2,8%; HbS=41%; HbF=1,2%; father HbA1=58,5%; HbA2=1,4%; HbS=37,7%; HbF=2,4%; sister HbA1=57,4%; HbA2=2,5%; HbS=38,6%; HbF=1,5%).Discussion: The family study showed that the haplotype DAR-ceAR was inherited from her father. Individuals carrying the ceAR allele may produce anti-hrs antibody that is defined as a Rhe-like which does not react with rare Rhe positive variant RBCs. The antibody may be clinically significant and represents a great risk for those patients who are not transfused with a similar rare phenotype variant. Serologically, DAR has shown weaker reactions with anti-D, it is characterized by complete loss of at least 9 of 37 RhD epitopes, and may produce anti-D. Both mutated alleles show a replacement of RHD exons by RHCE counterparts.

P35 Two Cases of Anti‐D in DAR Individuals

Introduction Current monoclonal anti‐D reagents will react with most D‐variant RBCs. A small number of individuals with D variant phenotypes (e.g. IIIa, IV, V, VI, VII, DAU) appear to be stimulated to produce allo anti‐D more readily than others. DAR is characterised by the complete loss of at least nine of 37 RhD epitopes. We are reporting two cases of allo anti‐D in patients who have the DAR phenotype.Case history Case 1: An African woman aged 20 was admitted for termination of pregnancy with no clinical history available. She was grouped as B, Ro. Her D phenotype using BioVue was weakly positive and also with anti‐D reagents RUM1 and MS201 by tube. The antibody screen was positive, and suggestive of anti‐D. Our laboratory found her RBCs strongly positive with RUM1 and MS201 anti‐Ds by tube and with DiaMed grouping cards. The Diagast D‐screen suggested a weak D antigen. No plasma was supplied for antibody identification. A weak anti‐D by tube LISS IAT was detected in a subsequent sample. The D status was identified by IBGRL as DAR. Case 2: A pregnant African female, aged 35 was grouped by the referring hospital as O, D+; the antibody screen was suggestive of anti‐D by Capture R. Our laboratory had tested her D status in Feb. 2003 after a miscarriage. We reported her as weak D with a negative antibody screen. No anti‐D Ig prophylaxis was given. In our laboratory the most recent sample grouped as O, Ro. Her RBCs gave strongly positive reactions with RUM1 and MS201 by tube and with DiaMed grouping cards. The Diagast D‐screen suggested weak D. Anti‐D was detected with a level of <0.1 IU mL−1. The D status was identified by IBGRL as DAR.Conclusion Immunisation risk for DAR is estimated to be >1% per D+ transfusion, which is less than for anti‐K and anti‐c but similar to anti‐Fya and anti‐Jka. The incidence of DAR has been reported as one in 4233 from a population with ‘mixed ethnicity’ in Canada. There is no routine serological method to identify all partial D types. In these two cases the D‐screen indicated both were weak D, and the current monoclonal anti‐D typing reagents used gave positive reactions. Although both of the patients were immunised, the anti‐D in both cases was weak which concurs with our previous experience of anti‐D in partial D individuals. Molecular studies may help to provide further information about the likelihood of the risk of immunisation.

Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells

AbstractIn the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)

Surface expression of Rh-associated glycoprotein (RhAG) in nonerythroid COS-1 cells

In the Rh blood system, RhAG (Rh-associated glycoprotein, or Rh50) is thought to be involved in Rh30 (D, CE) expression by forming a protein complex on the red cell surface. To obtain further insight into the Rh complex, we chose nonerythroid COS-1 cells instead of proerythroblast-like K562 cells, which produce endogenous Rh proteins as cell host, for the expression of both RhAG and RhD. The RhAG cDNA was subcloned into a retroviral vector, and a stable COS-1 cell line was then established via retroviral transduction. Surface expression of RhAG on the COS-1 cells was monitored by flow cytometry using mouse monoclonal anti-RhAG(2D10). Under these conditions, we detected significant expression of RhAG on the cell surface, compared to stable COS-1 cells transduced with the vector alone. To confirm the results, we isolated RhAG by immunoprecipitation from the lysate of the COS-1 cells, which were metabolically labeled with [35S]-methionine. A strong band of the 32 kd on SDS-PAGE was obtained, corresponding to the results obtained from other cultured cells (K562 cell and others), which always produce partially glycosylated RhAG with a molecular weight of 32 kd. Thus, RhAG was expressed without Rh30 and other Rh-related glycoproteins (LW, glycophorin B) in nonerythroid cells. Using the same strategy, however, we could not express RhD epitopes on COS-1 cells even in the presence of RhAG cDNA, suggesting that other factors might be required for the surface expression of RhD antigen. (Blood. 2000;95:336-341)

Three Molecular Structures Cause Rhesus D Category VI Phenotypes With Distinct Immunohematologic Features

Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype III occurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.

Three Molecular Structures Cause Rhesus D Category VI Phenotypes With Distinct Immunohematologic Features

AbstractRhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III,was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype IIIoccurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.

RHD epitope density profiles of RHD variant red cells analyzed by flow cytometry.

For DII and DIVa, RHD antigen sites per cell were tested previously only with a limited number of radio labelled anti-D. We determined RHD antigen densities (sites/cell) by flow cytometry with 64 anti-D in four partial RHD red cells. Epitope densities detected (mean +/- SD) were: DVII 8,000 +/- 1,665 (number of anti-D used for calculation: n = 55; percentage of reference cells: 47%); DNU (DIIlike) 6,869 +/- 1,381 (n = 47; 29%); and DHMii 20,626 +/- 4,320 (n = 55; 87%). One DIV cell revealed two peaks with a low fluorescence range of 18,158 +/- 2,504 (n = 11; 76%) and a high range of 35,477 +/- 2,485 (n = 6; 149%). We established epitope density profiles with a large panel of anti-D for four RHD variant cells and determined the number of RHD antigens per cell.