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The Rh system is one of the most important and complex blood group systems because of the large number of antigens and the serious complications for the fetus of a woman sensitized by transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes and with functional evidence that the Rh blood group proteins belong to an ancient family of membrane proteins involved in ammonia transport.
The arrangement and configuration of the genes at the RH locus promotes genetic exchange, generating new antigens. Importantly, RH genetic testing can now be applied to clinical transfusion medicine and prenatal practice. This includes testing for RHD zygosity, confirmation or resolution of D antigen status, and detection of altered RHD and RHCE genes in individuals at risk for producing antibodies to high incidence Rh antigens, particularly sickle cell disease patients. The Rh proteins form a core complex that is critical to the structure of the erythrocyte membrane, and may play a physiologically role in the sequestration of blood ammonia. The Rh family of proteins now includes non-erythroid Rh homologs present in many other tissues, and comparative genomics reveals Rh homologs in all domains of life.
The history of the Rh system began in 1941 with its discovery as the cause of severe jaundice and fetal demise, i.e. erythroblastosis fetalis. The syndrome had been observed for many years to complicate pregnancies. The fact that it was due to an immune reaction to a paternal antigen was only realized with the delivery of a stillborn fetus, and an adverse reaction of the mother to a blood transfusion from the father.1 The syndrome, referred to as hemolytic disease of the newborn (HDN), was principally caused by maternal sensitization to Rh, specifically to the D antigen. HDN due to D incompatibility was prevalent in Caucasians, who have the highest incidence of the D-negative phenotype (15-17%), but was rare in other ethnic groups. The incidence of HDN was dramatically altered when it was realized that ABO incompatibility between a mother and the fetus had a partial protective effect against immunization to D. This suggested the rationale for the development of Rh immune globulin. By the 1960's, a mere 20 years after the discovery of Rh incompatibility, HDN due to anti-D could be effectively prevented. 2,3
The Rh system has long been known to be one of the most complex blood group systems. In addition to the presence or absence of the D antigen, other common Rh antigens include the allelic C or c, and E or e. However, over fifty different Rh antigens have been identified by investigating the specificity of antibodies produced after blood transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes. The arrangement and configuration of the RH locus result in gene conversion events that generate hybrid proteins, which represent additional Rh antigens. Importantly, genetic testing for RH can now be used in prenatal and clinical transfusion medicine to detect the presence of a recessive D-negative allele or to determine the inheritance of RH genes carrying mutations that encode altered Rh proteins. The latter is especially relevant for SCD patients who, because of genetic disparity with Caucasian donors, are at high risk for becoming alloimmunized.
The Rh complex is critical to the structure of the membrane. Rhnull erythrocytes, which lack Rh proteins, are stomatocytic and spherocytic, and affected individuals have hemolytic anemia.4 Recent findings indicate that Rh proteins mediate key interactions with the underlying cytoskeleton through protein 4.2 and ankyrin.5-7
The erythrocyte Rh blood group proteins are well known because of their importance in blood transfusion, but recent functional studies and structural modeling reveal that the Rh blood group proteins are members of an ancient family of proteins involved in ammonia transport.8-11 Non-erythroid Rh proteins have now been found in other tissues including the kidney, liver, brain, and skin 12-15, in locations where ammonia production and elimination occurs. The family of Rh proteins has now been expanded significantly through comparative genomics and structure-function studies that reveal the presence of Rh homologs in all domains of life. Current investigations can be compared to an “archeological dig” to uncover relationships between the Rh family members to gain insight into the mechanism of transport.
Rh terminology distinguishes between the antigens, genes, and the proteins. The antigens are referred to by the letter designations, D, C, c, E, e, etc. The RH genes are designated by capital letters, with or without italics, and include erythroid RHD, RHCE, and RHAG, as well as the non-erythroid homologs expressed in other tissues, RHBG and RHCG. The different alleles of the RHCE gene are designated RHce, RHCe, RHcE, according to which antigens they encode. The proteins are indicated as RhD, RhCE (or according to the specific antigens they carry Rhce, RhCe, or RhcE), and include erythroid RhAG and those found in other tissues, RhBG and RhCG.
Two genes (RHD, RHCE) in close proximity on chromosome 1 encode the erythrocyte Rh proteins, RhD and RhCE; one carries the D antigen, and the other carries CE antigens in various combinations (ce, Ce, cE, or CE), (Fig.1A).16-19 The genes each have ten exons, are 97% identical, and arose via gene duplication. RhD and RhCE proteins differ by 32-35 of 416 amino acids (Fig. 1B shown as circles on RhD). This is in contrast to most blood group antigens that are encoded by single genes with alleles that differ by only one or a few amino acids. Individuals who lack RhD protein, “Rh or D negative”, most often have a complete deletion of the RHD gene (Fig. 1A). An important consideration in the immunogenicity of a protein is the degree of foreignness to the host. The large number of amino acid changes explains why exposure to RhD can result in a potent immune response in a D-negative individual.
RHCE, expressed in all but rare D- - individuals, encodes both C/c and E/e antigens on a single protein. C and c antigens differ by four amino acids, but only the amino acid change Ser103Pro is extracellular (Fig. 1B). The E and e antigens differ by one amino acid, Pro226Ala, located on the fourth extracellular loop of the protein.
The RH genes and proteins detailed in Fig.1 and Fig. 4A are typical for the majority of individuals, and commercial antibody reagents detect expression of these “conventional” D, C, c, E and e antigens shown. The proximity of the two RH genes, and their inverted orientation (Fig. 1A), augments opportunity for genetic exchange.20 Many RH genes carry point mutations, or have rearrangements and exchanges between RHD and RHCE that result from gene conversion events. The latter encode hybrid proteins that have RhCE-specific amino acids in RhD, or RhD-specific residues in RhCE. These can generate new antigens in the Rh blood group system, and alter or weaken expression of the conventional antigens.
Numerous mutations in RHD (greater than 100) are currently known. Most encode single amino acid changes, and many of these simply alter the quantity of RhD protein in the membrane, while others alter RhD membrane topology and D-epitopes. The latter are responsible for the enigmatic individual with D-positive RBCs who presents with anti-D following transfusion or pregnancy.
Mutations in both RHD and RHCE are found in Blacks, and other ethnically diverse groups, and alter D, C, or e antigen expression (detailed below). The incidence of variant RH genes in patients with sickle cell disease underlies the complex incompatibilities that can arise following transfusion in this patient population. Commercial typing reagents are not available to detect red cells that express variant Rh proteins, and individuals at risk go undetected until they produce antibodies reactive with all, or most, conventional Rh antigens. Finding compatible blood then becomes a serious and potentially life-threatening problem. Understanding of RH gene variation can now be applied to assess alloimmunization risk in SCD, because patients who inherit variant RH genes can now be identified at the genomic level.
D-negative is prevalent in Caucasians (15%-17%), but less common in Black Africans (5%) and Asians (3%).21 The D-negative phenotype has arisen numerous times, and multiple genetic events are responsible for loss of RhD expression in different populations. Caucasians have a deletion of the entire RHD gene,22 but Africans and Asians often have an inactive or silent RHD. Approximately 66% of South African, D-negative, Black persons have RHD with a 37-bp internal duplication that causes a premature stop codon and does not encode functional protein, while 15% carry a hybrid RHD-CE-D.23 This hybrid D-CE-D does not express D antigen and encodes an altered C antigen (Fig. 4B). In Asians, 10-30% of D-negative phenotypes are Del and have very low levels of D antigen not detectable by standard typing (see below).
Serologic testing for RBC expression of D, C/c, and E/e can only predict the likelihood that a sample is homozygous (D/D) or heterozygous (D/−). However, RHD zygosity can now be determined by assaying for the presence of a recessive D-negative allele. In the prenatal setting, paternal RHD zygosity testing is important to predict fetal D status when the mother has anti-D. The ethnic background of the parents is important to the design of the assay because different genetic events are responsible for D-negative phenotypes. In addition to testing for the absence of RHD, zygosity assays include detection of the region generated by deletion of RHD (arrows in Fig.1), the 37-bp insert RHD pseudogene, and the D-negative RHD-CE-D hybrid gene.23,24
If the father is RHD homozygous, monitoring of the pregnancy will be required, but if the father is heterozygous, the D-status of the fetus should be determined. The goal is to prevent invasive and costly monitoring of the pregnancy when the infant is D-negative. Fetal DNA can be obtained by amniocentesis or villus sampling. However, cell-free, fetal-derived DNA is present in maternal plasma by approximately five weeks, which allows maternal plasma to be tested as a non-invasive sample source.25, 26 The assay detects the presence or absence of RHD, and because cell-free fetal DNA will be present in low amount, positive controls for isolation of sufficient fetal DNA is critical to validate negative results. Y chromosome markers are useful when the fetus is a male, but when the fetus is female, proper controls present a challenge and polymorphic paternal markers are then needed.27 Testing of samples from the parents also limits the possibility of misinterpretation due to inheritance of rare or familial RHD inactivating mutations or rearranged hybrid genes.
The majority of D-positive individuals inherit RHD as shown in Fig.1, but exceptions are encountered, and these can complicate determination of D status.
Reduced expression of D antigen occurs in an estimated 0.2%-1% of Caucasians.28 Historically, RBCs that react with anti-D only after extended testing with the indirect antiglobulin test (IAT) are called weak D. However, the number of samples classified as weak D depends on the characteristics of the typing reagent. Weak D expression primarily results from single point mutations in RHD that encode amino acid changes predicted to be intracellular or in the transmembrane regions of RhD (Fig. 2A).29 These affect the efficiency of insertion, and, therefore, the quantity of RhD protein in the membrane, reflected in the reduced number of D antigen sites on the RBCs. Over 50 different mutations, the most common being a Val270Gly designated Type 1, cause weak D expression (Fig. 2A). Mutations are catalogued on the RhesusBase and blood group mutation websites and are updated regularly.30,31
Del red cells express very low levels of D antigen that cannot be detected by routine testing but these adsorb and elute anti-D, hence the name. Del are often found in Asian ethnic groups (10-30% of D-negative) and result from a mutation causing reduced synthesis of the 3′ region of RhD (Fig. 2C). Del are less frequently in Europeans (0.027%) and result from an amino acid change, M295I.29,32,33
RBCs with partial D antigen type as D-positive (some in direct tests, and others by IAT), but individuals often produce anti-D when stimulated by transfusion or pregnancy. Some partial D, similar to weak D, result from point mutations in RHD that cause single amino acid changes. But, in contrast to weak D, these changes are located on the extracellular regions and alter or create new epitopes (Fig. 2B). Many partial D result from hybrid genes that have regions of RHD replaced by the corresponding regions of RHCE. Some examples are shown in Fig. 3. These replacements can involve short regions encompassing several codons, entire exons, or large regions of the gene, and the novel sequence of amino acids that result from RhD joined with RhCE can generate new Rh antigens (e.g., DAK, Goa, Evans, Dw, BARC, FPTT, Rh32 etc) (Fig. 3).
Of clinical concern, particularly when determining the D status of a woman of child-bearing age, is the distinction between a partial D and weak D phenotypes. The former may make anti-D, whereas the latter are unlikely to do so. It is impossible to distinguish between these with commercial serologic reagents. The extensive history of transfusing patients who have weak D RBCs with D-positive blood strongly suggests that weak D Types 1, 2, and 3 (Fig. 2A), which comprise 90 percent of weak D individuals, do not make anti-D and can safely receive D-positive blood. In contrast, patients with partial D RBCs often make anti-D and should receive D-negative blood and be considered candidates for RhIG. This is a consideration for only performing a direct test to determine the D type in prenatal and transfusion practice. It is important to note that the current monoclonal FDA-licensed anti-D typing reagents are formulated to not react in direct tests with partial DVI RBCs. DVI is the most common partial D in Caucasians,34 and anti-D produced by partial DVI has resulted in fatal HDN. 35 Elimination of the IAT will better serve women with DVI RBCs by classifying them as D-negative for transfusion and RhIG prophylaxis.
As donors, weak D and partial D RBCs can stimulate the production of anti-D in a D-negative recipient. Therefore donor centers are required to use extended methods that detect and label these as D-positive. This can result in an individual who is appropriately labeled D-positive as a donor, but classified D-negative as a transfusion recipient. Communication may be required to avoid confusion for the patient or donor, the physician, or the nursing staff. In the age of genomics, with the potential for future health care and treatment options based on genetic polymorphisms, RHD polymorphisms that result in altered D antigen expression should be part of a patient's medical history.
Lastly, since there are no serologic reagents that detect Del RBCs, these will go undetected, type as D-negative, and be labeled D-negative as donors. Whether it is important to use genetic screening to eliminate donors with Del red cells from the D-negative donor pool has been debated. 36-38 Although Del RBCs have stimulated anti-D in two cases39,40, more data are needed to assess the potential risk in communities with large Asian donor populations. Of relevance, in approximately 10-13% of cases in Asia, D-negative individuals have been transfused with Del blood.
Multiple factors can complicate the determination of the D status. These include the different methods used in various laboratories, the different monoclonal antibodies in FDA-licensed reagents that can react differently with variant D antigens, and the large number of different RHD genes, which can affect both the level of expression and, potentially, the structure of the molecule and D-epitopes.
The discovery that some Rhce proteins carry D-specific amino acids or express a D-like epitope that reacts with some monoclonal anti-D reagents further complicates serologic determination of D status. Two examples, DHAR, found in individuals of German ancestry, and Crawford, ceCF , found in African-Americans, are often responsible for serologic D typing discrepancies in the U.S. 41. These two are notable because of their strong reactivity (3+/4+) with some FDA-licensed monoclonal reagents and complete lack of reactivity with others.
In conclusion, because all of these lack RhD, they can readily be sensitized44(and our observations) if not appropriately classified as D-negative.
Altered RHce genes are prevalent in Blacks and mixed ethnic backgrounds. Individuals with these variant alleles type as e-positive, but they often make alloantibodies with e-like specificities. Many altered RHce genes have now been characterized (Fig. 4C). The amino acid changes from conventional RHce are shown, and most carry a Tryptophan.. Cysteine at amino acid position 16 (W16C) encoded in exon 1, as well as additional changes, primarily in exon 5. Individuals homozygous for these alleles lack high incidence Rh antigens (hrB and hrS). The ceS alleles are associated with RBCs that are hrB-, and numerous alleles (ceMO, ceEK, etc) are associated with RBCs that are hrS- (Fig. 4C). Importantly, because of the multiple and varied changes responsible for hrS- and hrB- phenotypes, the antibodies produced by these individuals are not all compatible with RBCs from the other variants.
As an additional complication, these variant RHce are often linked to the D-negative hybrid RHD-CE-D (Fig. 4B) that encodes an altered C antigen, or are linked to a variant RHD (DIII, DAU, DAR etc.). Patients with these alleles are at risk of producing alloantie, −C, and also anti-D, in spite of having RBCs that are positive for these Rh antigens by serologic typing. This is not a rare occurrence; for example, the hybrid RHD-CE-D allele encoding altered C has a frequency of 25% in African-American Blacks.45 Genetic testing for RH can now be used to identify patients homozygous for these altered alleles. It is very difficult to find compatible donors for these patients after they become alloimmunized, and the challenge is to develop a registry of donors that are genotyped for these variants.
Transfusion of patients with sickle cell disease (SCD) represents a significant challenge in clinical transfusion medicine. SCD may be the single disease for which transfusion therapy may increase in the next decade as a result of the stroke prevention trial in sickle cell anemia (STOP). This clinical trial was halted before its scheduled closure because of the significant benefit of chronic red cell transfusion in reducing the risk of stroke.46 STOP II further confirmed the markedly lower risk of stroke in participants receiving blood transfusions. 47 Nevertheless, serious complications of chronic transfusion include iron overload and alloimmunization, and these risks must be considered in medical decision-making regarding treatment. The recent availability of oral iron chelation agents is predicted to make transfusion a more acceptable treatment option 48, and genetic testing for RH has the potential to reduce alloimmunization.
The approach to management of alloimmunization in SCD varies, and although the goal is to provide blood with maximal survival, how best to accomplish this has been the subject of debate.49,50 Some programs aim to prevent or reduce the risk and incidence of alloantibody production by transfusing RBCs that are phenotype-matched, i.e. antigen-matched for D, C, E and Kell. Less often, Duffy (Fya/b) and Kidd (Jka/b) blood groups are also included. In addition to antigen-matching, some programs actively recruit and supply RBCs from African-American donors to SCD patients whenever possible. These approaches clearly reduce the risk and incidence of alloantibody production. Others provide antigen-negative blood only after a patient has made an antibody, arguing that it is logistically difficult and expensive to provide these units prophylactically, and not all patients will become sensitized and require antigen-matched products. Both approaches have strong proponents, and the community resources and donor center support are often the deciding factor in implementation of a specific approach in the absence of consensus.
Knowledge of RH gene variation sheds light on immunization in SCD patients. Although transfusion of antigen-matched units reduces the incidence of alloantibody production, some SCD patients still become sensitized. Many of the antibodies present as autoantibodies, and most represent complex, high incidence specificities within the Rh system. The prevalence in the sickle cell population of RH alleles that encode variant or altered e, C, or D antigens explains why these patients become immunized despite conventional antigen-matching, and why these incompatibilities are complex to resolve and compatible blood is difficult to obtain. Importantly, RH genotyping can identify SCD patients who are homozygous for variant alleles and at risk for production of “apparent auto” and alloantibodies to high incidence Rh antigens. RH genotyping of SCD patients, partnered with RH genotyping of donors, would have a positive impact to reduce alloimmunization in SCD and would optimize the use of minority donations, as not all SCD patients require blood from minority donors.
The Rh blood group proteins are well known because of their importance in blood transfusion. However, the mammalian family of Rh proteins has been expanded with the discovery of Rh-associated glycoprotein, RhAG, in erythrocytes, and the related proteins, RhBG and RhCG, in other tissues. Erythrocyte RhAG is not polymorphic, and while it is not associated with any blood group antigen, it is important for targeting RhCE and RhD to the membrane, as mutations in RHAG are responsible for loss of Rh antigen expression (Rhnull).51,52
Protein sequences with similarities to the mammalian Rh proteins were first found in C. elegans, and these homologs, in turn, showed similarity to the ammonium transporters from bacteria, yeast (MEP), and plants (AMT).53 This provided the first testable hypothesis for the function for these proteins. The relationship of the Rh-glycoproteins to the AMT/MEP ammonium transporters from these other organisms has now been substantiated by functional transport data 9,54-57 and structural modeling 10,11,58. The Rh proteins reveal the power of comparative genomics and proteomics, in which sequence analysis and homology modeling can give important insight into mammalian protein function.
The non-erythroid Rh glycoproteins, RhBG and RhCG, are present in the kidney, liver, brain, and skin12,13 where ammonia production and elimination occur. In the kidney collecting segment and collecting duct, RhBG and RhCG are found on the basolateral and apical membranes, respectively, of the intercalated cells59 where they mediate transepithelial movement of ammonium from the interstitium to the lumen. 60,61 In the liver, RhBG is found on the basolateral membrane of perivenous hepatocytes, where it may function in ammonia uptake. RhCG is also present in bile duct epithelial cells, where it is positioned to contribute to ammonia secretion into the bile fluid.14
The mechanism of ammonia transport by Rh-glycoproteins is an active area of investigation. Expression of Rh-glycoproteins in heterologous systems indicated that mammalian transport is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient.9,55,56 Importantly, this mechanism effectively exchanges NH4+ for H+ in a process that results in transport of net NH3. Functional studies of the kidney, liver, and brain Rh homologs, along with the erythrocyte RhAG/Rh proteins, promises to lead to development of a unifying hypothesis of ammonia transport in mammals by the Rh family of proteins.
The function of the more recently evolved erythrocyte blood group proteins, RhCE and RhD, has not yet been determined. When expressed in heterologous systems, they do not directly transport ammonia.62 Importantly, RhCE/D lack the highly conserved Histidine residues located in the membrane pore that are critical for transport.11,62 An attractive alternative hypothesis is that the RhCE/D may mediate movement of the uncharged molecule CO2, however, this remains to be determined. Evidence that RhCE/D may be evolving a new function in the RBC membrane comes from DIVERGE analysis (our observations) and evidence from others 63,64 that indicate the RhCE/D proteins are rapidly evolving, suggesting their function may be changing.
A structural role for the erythrocyte Rh proteins is suggested from the RBC shape defects, fragility, and the resulting anemia seen in patients with Rhnull disease.4,65 Rh/RhAG are part of a membrane protein complex that includes CD47, also known as integrin-associated protein (IAP), glycophorin B, and LW (ICAM-4).66,67 Band 3 may also be associated with the complex. 68 The Rhnull defect suggests the presence of a membrane-cytoskeletal attachment site in RBC mediated by Rh, RhAG, or a member of the Rh complex. Recent studies reveal that the Rh complex is linked to the membrane skeleton through CD47-protein 4.2 interactions 69, and through a novel Rh/RhAG-ankyrin cytoskeleton connection.7 RBC membrane protein-cytoskeleton and protein-protein interactions are an active area of investigation. Future studies are needed to determine the protein-protein associations and the dynamics of the assembly of the Rh-membrane complex to understand the Rhnull defect and to determine the functional role of the RhCE/D proteins.
The genetic basis of the Rh blood group proteins has been intensely investigated in the past decade, and the polymorphisms responsible for most of the antigens have now been determined. Presently, routine testing for RH is hampered because of the large number of Rh polymorphisms, as over one hundred RHD and forty-two RHCE gene variants have been reported 30, and additional variants are still being discovered. Currently, RH genetic testing is a powerful adjunct to serologic methods. It is being used to determine RHD zygosity, to type patients who are multiply transfused, to resolve D typing discrepancies, and to identify compatible donors for patients with antibodies to high incidence Rh antigens, primarily in SCD patients. The challenge is to develop automated platforms that sample numerous regions of the RH genes to detect the many hybrid genes, and to develop algorithms for unequivocal interpretation.
Ideally, tests to determine D status would distinguish those with RBCs that lack, or have altered, D-epitopes and are at risk of sensitization to conventional D from those with reduce expression levels of D who are not at risk of producing anti-D. Unfortunately, serologic reagents cannot discriminate between these RBCs. These limitations suggest that genetic testing to determine D status may be commonplace in the future.
Understanding of RH gene variation can now be applied to more accurately assess alloimmunization risk for SCD patients. The diversity of RH genes in African-Americans often underlies development of allo- and auto-antibodies in this ethnic group, despite implementation of antigen- or phenotype-matching for transfusion. This suggests that identification of individuals homozygous for variant RH genes and at risk of alloimmunization, will improve transfusion management for these patients, and much more efficiently utilize minority donors.
The relationship of Rh and Rh-glycoproteins to the AMT/MEP ammonium transporters from bacteria, yeast, and plants has now been substantiated by functional transport data and structural modeling. The Rh proteins reveal the power of comparative genomics and proteomics, in which sequence analysis and homology modeling can give important insight into mammalian protein function.
The structural and physiologic role of Rh/RhAG in the RBCs remains to be more firmly established, but a role for the Rh complex in ammonia sequestration in RBCs, at the same time contributing to membrane shape and flexibility, can be imagined. The uncharged molecule NH3 is very toxic to brain cells and mitochondria,70 and the Rh/RhAG complex may play a role in keeping the total blood ammonia level low by trapping ammonium inside the RBC. In support, plasma total ammonia levels are low (0.1-0.2 mM), but total erythrocyte ammonia levels are three times greater. 71
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