RhD Proteins Research Articles

Opposing actions of JIP4 and RILPL1 provide antagonistic motor force to dynamically regulate membrane reformation during lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson's disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

Molecular dynamics of the human RhD and RhAG blood group proteins.

Introduction: Blood group antigens of the RH system (formerly known as "Rhesus") play an important role in transfusion medicine because of the severe haemolytic consequences of antibodies to these antigens. No crystal structure is available for RhD proteins with its partner RhAG, and the precise stoichiometry of the trimer complex remains unknown. Methods: To analyse their structural properties, the trimers formed by RhD and/or RhAG subunits were generated by protein modelling and molecular dynamics simulations were performed. Results: No major differences in structural behaviour were found between trimers of different compositions. The conformation of the subunits is relatively constant during molecular dynamics simulations, except for three large disordered loops. Discussion: This work makes it possible to propose a reasonable stoichiometry and demonstrates the potential of studying the structural behaviour of these proteins to investigate the hundreds of genetic variants relevant to transfusion medicine.

Search of a genomic sequence database for potential novel blood group antigens: Investigation into why some amino acid substitutions are not immunogenic.

Polypeptide blood group antigens are typically identified through investigation of the antibodies they induce. Human genome sequence databases are a new tool to identify AA substitutions that potentially create blood group antigens. The Erythrogene genomic sequence database was searched for missense mutations not known to be blood group antigens in the extracellular domains of selected RBC proteins in European populations. Any mutations found with prevalence of 1%-90% and not known to have induced antibodies in transfusion practice were analyzed using protein structural analysis and epitope prediction programs to determine why they apparently lack immunogenicity. Thirteen missense mutations not known to create blood group antigens were identified in the extracellular domains of Kell, BCAM, and RhD proteins, but not in RhCE, Urea Transporter 1 (Kidd), Atypical Chemokine Receptor 1 (Duffy), glycophorin A or glycophorin B. While 11 of the 13 mutations had low prevalence (<1%), a Kell Ser726Pro substitution and a BCAM Val196Ile substitution had predicted phenotype prevalences of 43.2% and 5.7%, respectively. Ser726Pro had multiple properties of a linear B-cell epitope, but possible suboptimal protein location for B-cell receptor binding and limited T-cell epitope possibilities. Val196Ile was not predicted to be in a linear B-cell epitope. Multiple potential new blood group antigens of low prevalence were identified. Whether they are antigenic remains to be determined. Two higher prevalence variants in Kell and BCAM are unlikely antigens, otherwise their antibodies presumably would already have been identified. Possible reasons for their poor immunogenicity were identified.

Patients with Asian-type DEL can safely be transfused with RhD-positive blood

AbstractRed blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D–) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; none of the recipients (0/42; 95% confidence interval, 0-8.40) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4045 serologic D– pregnant women throughout China; alloanti-D was found only in individuals with true D– (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). We further retrospectively examined 127 serologic D– pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

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.

Weak D Type 4.2.2 (DAR1.2) in an African child: Serology and molecular characterization

The weak D phenotype is represented by a group of RHD genotypes that code for alterated RhD proteins associated with a reduced RhD expression on red blood cell. By routine serology, some partial D variants are likely to be missed.In this report we describe the case of a three-year-old Black African child with a “unclear” reaction with monoclonal anti-D. We analyzed the blood sample of the child with different methods to conclude that it is a case of DAR 1.2 (weak D 4.2.2) and that it must be transfused with D negative erithrocytes.

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The Velvet Underground Emerges

Comparison of RH Genotypes In Rh Alloimmunized and Non-Alloimmunized Patients with Sickle Cell Disease.

AbstractAbstract 1122Red cell alloimmunization remains a frequent and potentially serious complication of transfusion for patients with Sickle Cell Disease (SCD). RBC antigen disparities between African American patients and European American blood donors, particularly in the Rh system, are thought to contribute to the elevated frequency of alloimmunization in SCD. Extended RBC antigen-matching has been shown in single institutional and prospective multi-center experiences to reduce the incidence of alloantibody production in SCD. Some transfusion services also supply RBCs from African American donors when possible, but its effect on alloimmunization is not well-studied. Despite a program of providing D, C, E, and K-matched RBCs from primarily African-American donors, we have continued to observe Rh alloimmunization. Many patients produced anti-D, -C and –e, even though their RBCs type D+, C+, and e+, respectively. To examine whether Rh alloimmunization was associated with inheritance of diverse RH alleles that encode altered Rh proteins, we characterized RHD and RHCE in 50 patients with SCD: 27 who were alloimmunized to at least one Rh antigen, and 23 patients who had received transfusions but were not Rh alloimmunized. RH testing consisted of PCR-RFLP, AS-PCR and/or amplification and sequencing of exons and Rh-cDNA. In the Rh alloimmunized group, the average number of unit exposures was 275, and the median was 189. Twelve patients had anti-e despite typing e+ serologically, and all 12 had genotypes associated with weak or altered e antigen suggesting the antibody formed in response to exposure to conventional e antigen. Three of these patients subsequently formed anti-E when placed on e-negative transfusion protocols. Of 12 patients with anti-C, 7 typed serologically as C+. Of these, 3 had a hybrid RHD-CE-D that expresses an altered C antigen. However, the remaining 5 had at least one RHCE encoding conventional C antigen. Interestingly, anti-D was reported in 14 patients who typed serologically as D+. Genotyping revealed weak and/or partial D variants in 3, but 11 patients had at least one conventional RHD. These Rh antibodies were almost all transient, particularly unusual for anti-D. In the non-Rh-alloimmunized group, the average number of unit exposures was 189, and the median was 120. RH genotyping of the non-alloimmunized group revealed significant genetic diversity as well, with most patients carrying altered RH alleles. Of 23 e+ patients, 15 had only variant RHce, while 8 had at least one conventional RHce (only 3 were homozygous for conventional RHce). All 4 C+ patients had conventional RHCe genotypes. All 23 non-Rh-alloimmunized patients typed serologically D+, of which 15 had at least one conventional RHD and 8 had altered RHD. In conclusion, we demonstrate that some but not all Rh alloimmunization in patients with SCD transfused with D, C, E, and K phenotype-matched RBCs from primarily African American donors is due to recipients with variant RH genes who are exposed to conventional Rh proteins. In this small study of 50 patients, all 12 allo anti-e occurred in patients with only variant RHce, likely due to mismatched e antigen. Three cases of anti-C in C+ patients and 3 anti-D in D+ patients were also explained by variant RH. However, we found a number of cases of anti-D and -C in patients who had at least one conventional allele and would not be expected to make alloantibodies. More studies are needed, but one possible explanation is that variations in Rhce or RhD proteins may alter the entire Rh antigenic complex in the RBC membrane, as most of these patients had changes in at least one RH allele. Alternatively, these transient Rh antibodies could be stimulated by exposure to units from African American donors expressing variant Rh antigens, and these antibodies could represent “mimicking” or “cross-reactive” specificities. Since the RH genotypes of most of the non-alloimmunized patients also demonstrated variant RH alleles, future studies should evaluate which Rh variants are immunogenic. These data suggest 1) RH genotyping is a valuable complement to serologic phenotyping, 2) it may become important to use molecularly characterized reagent cells for antibody identification, and 3) knowledge of donor genotypes will be important for understanding Rh alloimmunization and possibly for selecting blood for patients with SCD. Disclosures:Westhoff:Immucor scientific advisory board: Membership on an entity's Board of Directors or advisory committees.

Specific amino acid substitutions cause distinct expression of JAL (RH48) and JAHK (RH53) antigens in RhCE and not in RhD

Westhoff and colleagues1 and Hustinx and coworkers2 have recently reported a total of three molecular causes for the low-prevalence Rh antigen JAL (RH48). Quantitative weakening and qualitative effects are documented for these variant RhCE proteins. Similar serological effects have been observed in the JAHK+ (RH53),3 ceSL,4 and RhCe(S122P)5 variants of the RhCE protein. We have observed 4 RhD variants harboring similar amino acid substitutions at positions 114 or 122 (Table 1). Table 1 RHD alleles encoding amino acid substitutions at positions 114 and 122 Most Rh antigens are known to be associated with molecular variants of either the RHD or RHCE gene. However, distinct Rh antigens, like c (RH4), G (RH12), Rh32 (RH32), Evans (RH37) and FPTT (RH50), can be expressed by RhD and RhCE variants (Table S1, available online). We collated all low-prevalence Rh antigens caused by single nucleotide substitutions and identified pairs of RHD and RHCE alleles that harbor identical substitutions (Table S2, available online).1–4,6 Only amino acid substitutions at positions 114 and 122 were currently found to fulfill both criteria. In RhCE these substitutions cause JAL or JAHK antigen expression. Hence, we assessed the possible expression of JAL and JAHK by RhD proteins harboring similar amino acids at positions 114 or 122. A DVII sample harboring a substitution at 110 was used as negative control. All RhD samples with amino acid substitutions at position 114, weak D type 17, type 25 and type 47, were negative for the JAL antigen (Table 2). The CeMA and Rhce(R114Q) control samples were JAL positive, as expected. Because the antigen density of weak D type 17 is very low (Table 1), we corroborated its lack of JAL expression by a negative adsorption-elution test (not shown). Table 2 Serologic results of RhD and RhCE variants with amino acid substitutions at positions 114 and 122 The weak D type 54 sample with an amino acid substitution at position 122 was negative for the JAHK antigen (Table 2). The RhCe(S122L) control sample was positive with the 3 anti-JAHK sera, as expected. ceSL has been reported to be JAHK negative.4 We confirmed this result with the original red blood cell (RBC) samples and the anti-JAHK P4.53A serum used previously. However, the two anti-JAHK sera of greater titer revealed that ceSL samples were in fact JAHK positive (Table 2). Both RhCe(S122P) samples were JAHK negative with all 3 sera. The two sera P4.53B and P4.53C were identified from a series of 138 samples collected from pregnant women with a positive antibody screen.7 Both women had neither been transfused nor were their newborns or the putative fathers positive for the JAHK antigen.7 Hence, anti-JAHK is possibly a rather frequent, naturally occurring antibody associated with pregnancy. The titers of anti-JAL (Table 2) determined with CeMA and Rhce(R114Q) differed in congruence with the known weaker expression of JAL in the Rhce variants Rhce(R114Q) and ces(340) compared to the RhCe variant CeMA.2 In contrast to Rhce(R114Q) harboring Gln114, ces(340) and CeMA are caused by mutations encoding for Trp114.1,2 The model presented by Westhoff et al.1 suggests conformational changes due to Trp114 that contribute to the expression pattern of the JAL antigen. The weak JAL expression in Rhce(R114Q) may be due to the different biochemical properties of Trp (W) and Gln (Q). However, the JAL expression in ces(340), which also harbors Trp114, is distinctly weaker than in CeMA and, hence, requires a different molecular explanation (Table S3, available online):1,2,8 The additional Leu245Val substitution, known to cause a weak expression of the e antigen,8 could also induce the weak expression of the JAL antigen. Currently observed alleles cannot exclude the possibility that the weak JAL expression in both Rhce variants is caused by the 4 amino acid residues at positions 48, 60, 68, and 103, which typically differ between the RhCe and Rhce proteins. We conclude that JAL and JAHK antigens are expressed by Ce and ce variants of the RhCE protein. We found that neither antigen was expressed by any of the 4 RhD protein variants possessing similar amino acid substitutions at positions 114 and 122.

Two Alleles of NF-κB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities

BackgroundNF-κB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-κB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain.Methods/Principal FindingsWe characterized two NF-κB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-κB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-κB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-κB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-κB proteins affect DNA binding as in human NF-κB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant.Conclusions/SignificanceThese results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-κB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.

Phosphorylation and Ankyrin-G Binding of the C-terminal Domain Regulate Targeting and Function of the Ammonium Transporter RhBG

RhBG, a human member of the Amt/Mep/Rh/superfamily of ammonium transporters, has been shown to facilitate NH(3) transport and to be anchored to the basolateral plasma membrane of kidney epithelial cells, via ankyrin-G. We showed here that triple alanine substitution of the (419)FLD(421) sequence, which links the cytoplasmic C-terminal domain of RhBG to ankyrin-G, not only disrupted the interaction of RhBG with the spectrin-based skeleton but also delayed its cell surface expression, decreased its plasma membrane stability, and abolished its NH(3) transport function in epithelial cell lines. Similarly, we demonstrated that both anchoring to the membrane skeleton and ammonium transport activity are regulated by the phosphorylation status of the C-terminal tail of RhBG. Tyrosine 429, which belongs to the previously reported YED basolateral targeting signal of RhBG, was demonstrated to be phosphorylated in vitro using purified Src and Syk kinases and ex vivo by analyzing the effect of pervanadate treatment on wild-type RhBG or Y429A mutants. Then, we showed that Y429D and Y429E mutations, mimicking constitutive phosphorylation, abolished NH(3) transport and enhanced Triton X-100 solubilization of RhBG from the cell membrane. In contrast, the nonphosphorylated/nonphosphorylatable Y429A and Y429F mutants behaved the same as wild-type RhBG. Conversely, Y/A or Y/F but not Y/E or Y/D mutations of residue 429 abolished the exclusive basolateral localization of RhBG in polarized epithelial cells. All these results led to a model in which targeting and ammonium transport function of RhBG are regulated by both phosphorylation and membrane skeleton binding of the C-terminal cytoplasmic domain.

Molecular biology of Rh proteins and relevance to molecular medicine

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

The RHCE allele ceSL: the second example for D antigen expression without D‐specific amino acids

The example of ceRT proved that the expression of some D epitopes does not require D-specific amino acids. This allele denoted as RHce(R154T) caused the "false-positive" reactions that were observed in ccddee blood donors who typed positive for the D antigen with some monoclonal anti-D. No other example exposing a similar molecular mechanism was known. Eleven donor and 1 patient ccddee samples were collected in Switzerland that typed "false-positive" with some monoclonal anti-D in bromelain technique. Their RHCE alleles were determined by nucleotide sequencing from genomic DNA and by a polymerase chain reaction with sequence-specific priming. The D epitope profile was compared to ceRT. The population frequencies were estimated in Switzerland and Germany by serology or at the molecular level, respectively. The "false-positive" reactions were caused by the RHCE allele RHce(S122L) occurring in the cde haplotype. Its ceSL phenotype expressed few D epitopes that belonged to the D epitope 6 group. The frequency of ceSL among D- donors was about 1:675 in the region of Bern, Switzerland. No ceSL donors were found elsewhere in Switzerland or in southwestern Germany. ceSL represented the second molecular mechanism for D antigen expression without any D-specific amino acids. ceSL and ceRT were useful to delineate the molecular mechanisms of D expression by RhCE proteins carrying amino acids not representative for the RhD proteins. The ceSL population frequencies differed significantly among three Swiss and German populations.

Molecular basis of weak D in Taiwanese.

Two genes, RHD and RHCE, encode the antigens of the RH blood group system. The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, resulting in distinct serologic phenotypes and anti-D immunization. We analyzed seven weak D phenotypes excluding D(el), using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods to detect the changes of all ten RHD exons. The results show that there are four types of weak D in Taiwanese: one case each for CGG to CAG mutation at codon 10, GTG to ATG mutation at codon 174, and GTG to GAG mutation at codon 270, and four cases for GGT to GAT mutation at codon 282. In conclusion, we present the first data of a molecular basis of weak D in Taiwanese, which suggest a clinically relevant potential for anti-D immunization and may improve transfusion strategy in weak D Taiwanese patients.

Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

In most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein

AbstractIn most cases, the lack of Rh in Rhnull red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter–based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Weak D alleles express distinct phenotypes

The weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, raising the possibility of distinct serologic phenotypes and of anti-D immunizations in weak D. We reported 6 new RHD alleles, D category III type IV, DIM, and the weak D types 4.1, 4.2.1, 4.2.2, and 17. The immunohematologic features of 18 weak D types were examined by agglutination and flow cytometry with more than 50 monoclonal anti-D. The agglutination patterns of the partial D phenotypes DIM, DIII type IV, and DIVtype III correlated well with the D epitope models, those of the weak D types showed no correlation. In flow cytometry, the weak D types displayed type-specific antigen densities between 70 and 4000 RhD antigens per cell and qualitatively distinct D antigens. A Rhesus D similarity index was devised to characterize the extent of qualitative changes in aberrant D antigens and discriminated normal D from all tested partial D, including D category III. In some rare weak D types, the extent of the alterations was comparable to that found in partial Ds that were prone to anti-D immunization. Four of 6 case reports with anti-D in weak D represented auto-anti-D. We concluded that, in contrast to previous assumptions, most weak D types, including prevalent ones, carry altered D antigens. These observations are suggestive of a clinically relevant potential for anti-D immunizations in some, but not in the prevalent weak D types, and were used to derive an improved transfusion strategy in weak D patients.

Weak D alleles express distinct phenotypes

AbstractThe weak D phenotype is caused by many different RHD alleles encoding aberrant RhD proteins, raising the possibility of distinct serologic phenotypes and of anti-D immunizations in weak D. We reported 6 new RHD alleles, D category III type IV, DIM, and the weak D types 4.1, 4.2.1, 4.2.2, and 17. The immunohematologic features of 18 weak D types were examined by agglutination and flow cytometry with more than 50 monoclonal anti-D. The agglutination patterns of the partial D phenotypes DIM, DIII type IV, and DIVtype III correlated well with the D epitope models, those of the weak D types showed no correlation. In flow cytometry, the weak D types displayed type-specific antigen densities between 70 and 4000 RhD antigens per cell and qualitatively distinct D antigens. A Rhesus D similarity index was devised to characterize the extent of qualitative changes in aberrant D antigens and discriminated normal D from all tested partial D, including D category III. In some rare weak D types, the extent of the alterations was comparable to that found in partial Ds that were prone to anti-D immunization. Four of 6 case reports with anti-D in weak D represented auto-anti-D. We concluded that, in contrast to previous assumptions, most weak D types, including prevalent ones, carry altered D antigens. These observations are suggestive of a clinically relevant potential for anti-D immunizations in some, but not in the prevalent weak D types, and were used to derive an improved transfusion strategy in weak D patients.

Weak D and partial D in Slovenian population through serology and genotyping.

Weak D red cell phenotype (formerly Du) exhibits weaker serological reaction with anti-D antibodies. Weak D occurs in 0.2% to 1% of whites and is caused by qualitatively altered RhD proteins called partial D or normal, only weakly expressed RhD proteins that are called weak D. Partial D genes are hybrid alleles between RHD an RHCE genes. 23 partial RHD alleles are described. Weak D phenotypes with reduced expression are likely to posses the normal RHD gene, but the latest findings indicate that weak D alleles carry at least one point mutation. The aim of the present work was to answer an important question how to approach partial and weak D identification in diagnostic use and if it is possible to distinguish between partial D and weak D using commercially available anti-D reagents for routine use. We also wanted to evaluate D-screen kit for partial D identification. We compared phenotypes identified by serological testing and genotypes identified by RHD Multiplex PCR and DVII specific ASPA PCR. Our results showed that it is not possible to distinguish between partial and weak D using commercially available anti-D reagents for routine use. D-screen proved to be useful for DVI and DVII identification, whereas for partial DDFR identification we must look for another set of monoclonal antibodies or simply use genotyping methods. In 44 samples with not interpretable serological results out of 80 we found all RHD specific exons present and we classified the samples as weak D. Fourteen types of weak D with at least one point mutation were recently proposed. Designing of allele specific PCRs for identification of proposed types of weak D is in progress.

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.