The Rh blood group system is one of the most polymorphic and immunogenic systems known in humans. In the past decade, intense investigation has yielded considerable knowledge of the molecular background of this system. The genes encoding 2 distinct Rh proteins that carry C or c together with either E or e antigens, and the D antigen, have been cloned, and the molecular bases of many of the antigens and of the phenotypes have been determined. A related protein, the Rh glycoprotein is essential for assembly of the Rh protein complex in the erythrocyte membrane and for expression of Rh antigens. The purpose of this review is to provide an overview of several aspects of the Rh blood group system, including the confusing terminology, progress in molecular understanding, and how this developing knowledge can be used in the clinical setting. Extensive documentation is provided to enable the interested reader to obtain further information.

The Rh blood group system was first described 60 years ago. A woman had a severe transfusion reaction when she was transfused with blood from her husband following delivery of a stillborn child with erythroblastosis fetalis. Her serum agglutinated red blood cells (RBCs) from her husband and from 80% of Caucasian ABO-compatible donors.1 The following year, Landsteiner and Wiener2 found that sera from rabbits (and later guinea pigs) immunized with RBCs from Macaca mulatta (Macacus rhesus in the original paper) agglutinated 85% of human RBC samples. Initially, it was thought that the animal and human antibodies identified a common factor, Rh, on the surface of rhesus and human RBCs. It was soon realized that this was not the case.3 Therefore, the original terms (Rh factor and anti-Rh) coined by Landsteiner and Wiener, although being misnomers, have continued in common usage. The heteroantibody was renamed anti-LW (after Landsteiner and Wiener), and the human alloantibody was renamed anti-D.4 

The Rh blood group system is the most polymorphic of the human blood groups, consisting of at least 45 independent antigens and, next to ABO, is the most clinically significant in transfusion medicine. The ability to clone complementary DNA (cDNA) and sequence genes encoding the Rh proteins has led to an understanding of the molecular bases associated with some of the Rh antigens. Serologic detection of polymorphic blood group antigens and of phenotypes provides a valuable source of appropriate blood samples for study at the molecular level. This review summarizes our present understanding of the complexities of Rh blood group expression and how this knowledge impacts on clinical situations that arise through Rh blood group incompatibility.

Terminology

Several nomenclatures have been used to describe antigens, proteins, and genes in the Rh system. Throughout this review, we will use traditional terminology recommended by the International Society of Blood Transfusion (ISBT) committee for terminology of blood group antigens.5 The numeric portion of the ISBT terminology for Rh antigens is based on the nomenclature described by Rosenfield et al.6-9 RH30 and RH50 have been used to describe genes encoding Rh proteins (Rh30) and Rh glycoprotein (Rh50), respectively, where the numbers relate to the apparent molecular mass of the proteins on a SDS-polyacrylamide gel. Because Rh30 and Rh50 also relate to Goa and FPTT antigens, respectively, we will useRH as a generic term for genes encoding either the RhD protein or the RhCcEe (also known as RhCE) protein and use RHAG for the gene encoding the Rh-associated glycoprotein (RhAG). The common Rh antigens: D, C or c, and E or e, were originally written in alphabetical order (CDE) but later, when it was recognized that C and E antigens are inherited en bloc, the order was changed to DCE. Although d antigen, which was thought to be antithetical to D, does not exist, the letter “d” is used to indicate the D-negative phenotype. The most frequently occurring forms of RHCE and RHD encode 8 haplotypes: Dce, dce, DCe, dCe, DcE, dcE, DCE, and dCE, known in short, respectively, as R0, r, R1, r′, R2, r″, Rz, and ry. The uppercase “R” is used when the D antigen is expressed, lowercase “r” when it is not. This notation has practical value in transfusion medicine as a means to communicate the Rh phenotype of a patient or donor. Rare deletion phentoypes use dashes in the notation to indicate a lack of antithetical antigens; eg, Dc−. RBCs lack E and e antigens, and D−− RBCs lack C, c, E, and e antigens. RBCs with the Rhnullphenotype do not express any of the Rh antigens.

The Rh complex

Biochemical studies, protein purification, and amino acid sequencing of Rh and RhAG are beyond the scope of this article but have been reviewed elsewhere.10-16 

The Rh proteins carry Rh antigens but are only expressed on the erythrocyte surface if RhAG is also present. The amino acid sequence homology (approximately 40%) of the Rh and RhAG proteins indicates an ancestral relationship, and collectively they are referred to as the “Rh protein family.” Hydrophobicity profiles, immunochemical analyses, and data obtained through site-directed mutagenesis imply that Rh and RhAG proteins have 12 transmembrane spans with both the N-terminus and C-terminus oriented to the cytoplasm (Figure1).17-24 

Fig. 1.

Model of topology for RhAG, RhCE, and RhD.

RhAG (Mr 50 000) consists of 409 amino acids and is encoded by RHAG on chromosome 6p11-p21.1. RhCE and RhD (Mr 30 000) are predicted to have a similar topology and are encoded by RHCE and RHD, which are adjacent on chromosome 1p34-p36. The domain of the RhD protein encoded by each exon is depicted by numbered boxes, which represent the start and finish of each exon. Of the D-specific amino acids, 8 are on the exofacial surface (yellow ovals), and 24 are predicted to reside in the transmembrane and cytoplasmic domains (black ovals). Red ovals represent amino acids that are critical for C/c (Ser103Pro) and E/e (Pro226Ala) antigens; purple ovals represent Ser103 and Ala226 on RhD. The zigzag lines represent the Cys-Leu-Pro motifs that are probably involved in the palmitoylation sites. The N-glycan on the first loop of RhAG is indicated by the branched structure of red circles.

Fig. 1.

Model of topology for RhAG, RhCE, and RhD.

RhAG (Mr 50 000) consists of 409 amino acids and is encoded by RHAG on chromosome 6p11-p21.1. RhCE and RhD (Mr 30 000) are predicted to have a similar topology and are encoded by RHCE and RHD, which are adjacent on chromosome 1p34-p36. The domain of the RhD protein encoded by each exon is depicted by numbered boxes, which represent the start and finish of each exon. Of the D-specific amino acids, 8 are on the exofacial surface (yellow ovals), and 24 are predicted to reside in the transmembrane and cytoplasmic domains (black ovals). Red ovals represent amino acids that are critical for C/c (Ser103Pro) and E/e (Pro226Ala) antigens; purple ovals represent Ser103 and Ala226 on RhD. The zigzag lines represent the Cys-Leu-Pro motifs that are probably involved in the palmitoylation sites. The N-glycan on the first loop of RhAG is indicated by the branched structure of red circles.

“Rh accessory proteins” is a collective term for other glycoproteins that are associated with the Rh protein family as defined by their absence or deficiency from Rhnull RBCs (see below and Table 1).25 Together, the association of the Rh protein family and the Rh accessory proteins is called the “Rh complex.”

Table 1.

Proteins in the Rh Complex in Normal RBC Membranes That May Be Absent or Reduced in Rhnull RBC Membranes

Protein  Antigens  Gene Location  Mr Accession Numbers for cDNAs  
Rh protein family  
 RhD  1p36.13-p34.3180 30-32 kd  X63094, X63097,U66341 
 RhCcEe  Ce, CE, ce, cE  1p36.13-p34.3180 32-34 kd  X54534, M34015, U66340 
 RhAG  Carries MB2D10 epitope58 6p21.1-p1117,75,174  45-100 kd X64594 
Rh accessory proteins  
 LW  LW 19p13.3181 37-47 kd  L27670, L27671 
 IAP None known  3q1355 47-52 kd  Z25521 
 GPB ‘N,’ S, s, U  4q28-q31  20-25 kd  J02982 
 Band 3 Diego  17q12-q21182 90-100 kd  X77738,M27819 
Protein  Antigens  Gene Location  Mr Accession Numbers for cDNAs  
Rh protein family  
 RhD  1p36.13-p34.3180 30-32 kd  X63094, X63097,U66341 
 RhCcEe  Ce, CE, ce, cE  1p36.13-p34.3180 32-34 kd  X54534, M34015, U66340 
 RhAG  Carries MB2D10 epitope58 6p21.1-p1117,75,174  45-100 kd X64594 
Rh accessory proteins  
 LW  LW 19p13.3181 37-47 kd  L27670, L27671 
 IAP None known  3q1355 47-52 kd  Z25521 
 GPB ‘N,’ S, s, U  4q28-q31  20-25 kd  J02982 
 Band 3 Diego  17q12-q21182 90-100 kd  X77738,M27819 

Rh protein family

The complex of the Rh protein family is estimated by density ultracentrifugation to be 170 000 daltons26 and to consist of a tetramer with 2 RhAG molecules and 2 RhCcEe or RhD protein molecules stabilized by both N-terminal and C-terminal domain associations.18,19,26,27 The mode of association of this core complex with Rh-accessory proteins, some of which interact directly with the membrane skeleton, remains undefined.

RhD and RhCcEe proteins.

The RhD protein expresses the D antigen, while the RhCcEe protein carries either C or c antigens (involving the second extracellular loop) together with E or e antigens (involving the fourth extracellular loop) on the same protein.19,28-30 Characteristics of the RhD protein (synonyms: Rh30, Rh30B, Rh30D, D30, Rh30 polypeptide [30 kd], RhXIII, Rh13) and of the RhCcEe protein (synonyms: Rh30, Rh30A, Rh30C [RhCE], Rh30 polypeptide [32 kd], RhIXb cDNA, [RhcE], Rh21 cDNA [RhcE], R6A32, Rhce, RhCe, RhcE, RhCE, CcEe) are summarized in Table 1 and depicted in Figure 1. Analysis of the primary amino acid sequences (inferred from cDNAs) shows that the first 41 N-terminal amino acids of RhD and RhCE/e are identical.20,31-33 and that RhD differs from the common forms of RhCE by only 30 to 35 amino acids along the entire protein.20,28,31-33,35,36 Despite the high degree of homology, the various RhCcEe proteins do not express any D epitopes, and RhD protein does not express C or e antigens.

The Rh proteins are thought to interact with the membrane bilayer by palmitoylation,26,37 where acylated palmitic acid residues are attached to cysteine side chains. These cysteine residues are predicted to be at the boundary of the cytosol and lipid bilayer (Figure 1). Cys-Leu-Pro motifs, flanked by charged amino acids (2 are on RhD and 3 are on RhCcEe) are likely candidates although 2 other cysteine residues (315 and 316) may be alternative sites.20,26 This interaction may explain why alteration of membrane cholesterol concentration affects the accessibility of the D antigen.38 The ability to label Rh proteins with3H-palmitate26,37 indicates that a reversible coenzyme A and adenosine triphosphate (ATP)-dependent acylation-deacylation cycle occurs in mature RBC membranes, which is of unknown significance.

Rh-associated glycoprotein.

The characteristics of RhAG (synonyms: Rh50, Rh glycoprotein Rh50A, D50, MB-2D10 protein, R6A45, GP50, GP50A) are summarized in Table 1 and depicted in Figure 1. One of the 2 potential N-glycan sites is glycosylated. A third site is predicted to be cytoplasmic and, therefore, not accessible for glycosylation.17,39 The N-glycan carries ABH antigens,12 but RhAG is not known to possess a protein-based blood group polymorphism. Based on the predicted amino acid sequence, RhAG shares 39.2% and 38.5% amino acid sequence identity with, respectively, the Rhce and RhD proteins.17,20,31-33 

Expression of Rh proteins and RhAG during erythropoiesis.

Rh antigens appear early during erythropoietic differentiation. Anti-D binds to approximately 3% of BFU-E (burst-forming unit, erythroid), 68% of CFU-E (colony-forming unit, erythrocyte), and to all of the more mature erythroid cells. However, the binding of anti-D to proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and normoblasts was, respectively, 25%, 50%, 66%, and 75% compared with mature RBCs.33 RhAG protein is detectable on CD34 progenitors isolated from cord blood, after culture for 3 to 5 days, while RhCcEe appears after 5 to 7 days, and RhD appears after 9 to 11 days of culture.40 In the fetus, Rh antigens are expressed on RBCs from the 6-week conceptus.41 

Possible function of Rh protein family.

The function of the Rh complex remains unclear. Rh proteins have approximately 20% homology to the methylamine permease (Mep) transporters and ammonium transporters (Amt) in yeast, bacteria, and simple plants.42 This family of transporters are uniporters that have evolved to concentrate ammonium salts from the surrounding environment. Higher animals use more complex nitrogen sources, and they eliminate toxic ammonium via the urea cycle and transport it in the form of glutamine and alanine. The role of the Rh complex as a dedicated ammonium transporter is unlikely, but the complex could cotransport ammonium with other cations; however, further study is needed. Matassi et al43 report that RHAG shares greatest homology to MEP2, which behaves as an ammonium sensor and transporter in yeast.44 Furthermore, the presence of RhAG homologs in Caenorhabditis elegans and Geodia cydonium infers they have roles that are not confined to RBCs.

Rh accessory proteins

The blood group antigens associated with the Rh family of proteins, the gene location, their molecular mass, number of copies per RBC, and selected accession numbers are summarized in Table1.

LW glycoprotein.

The LW glycoprotein (synonym: ICAM-4) is a single pass (type I) membrane protein with homology to intercellular adhesion molecules (ICAMs), which are ligands for β2 integrins. LW has been reported to be a ligand for the integrin LFA-1 (synonyms: αLβ2, CD11a/CD18).45 

While the LW glycoprotein is absent from RBCs of LW(a−b−) and Rhnull individuals, expression of Rh antigens is normal on LW(a−b−) RBCs. LW antigens are more abundant on D-positive RBCs than on D-negative RBCs from adults, which led to the initial interpretation that anti-D and anti-LW were the same.46,47 It is possible that the LW glycoprotein interacts preferentially with RhD as compared with RhCcEe; however, the nature of such an interaction awaits definition. Interestingly, LW antigens are expressed equally well on D-positive and D-negative RBCs from fetuses and newborns and more strongly than on RBCs from adults.48,49 

Integrin-associated protein.

Isoform 2 of integrin-associated protein (IAP; synonyms: CD47, BRIC 125 glycoprotein, AgOAB, 1D8) is present in the RBC membrane, where it is predicted to pass through the RBC membrane 5 times and have 6 potential N-glycan motifs.50,51 IAP carries ABH antigens but no known protein-based blood group antigen. IAP occurs as different isoforms in various tissues where it binds to β3 integrins.50,52 The IAP isoform in RBCs does not bind integrins but does bind thrombospondin53 and may be involved in calcium transport, possibly as a gated channel.54 While the amount of IAP is reduced in RBC membranes from Rhnull and D−− people, it is present in normal levels in lymphoblastoid cell lines from these people.55-57 

Glycophorin B.

Glycophorin B (GPB; synonyms: Ss sialoglycoprotein [SGP], δ-SGP, PAS-3) is a type I membrane glycoprotein that has several O-glycans but no N-glycan. The Rh complex appears to aid, but is not essential for, the correct insertion of GPB in RBC membranes. In S−s−U− RBCs that lack GPB, the Rh proteins are apparently normal, but RhAG has increased glycosylation, suggesting a slower migration through the endoplasmic reticulum and Golgi apparatus.39 An interaction of GPB and RhAG may be required for full expression of the U antigen58,59 and, to a lesser extent, S and s antigens (Table 2). Further, the known ability of GPB to form heterodimers with glycophorin A (GPA) may bridge the Rh complex with the band 3/GPA complex, forming a large unit in the RBC membrane.

Table 2.

Comparison of amorph Rhnull, regulator Rhnull, and Rhmod RBCs

Phenotype  Rh Proteins/Antigens  RhAG  LW  IAP GPB Protein/Antigens  Obligate Heterozygotes Altered Gene  
Rhnull-amorph  Absent Reduced (20%)  Absent  Reduced by 90%  Reduced by 50% Express one Rh haplotype* RHCE (RHDdeleted)  
     S/s Normal  
     U Weak  
Rhnull-regulator  Absent Absent  Absent  Reduced  Reduced by 70%183 Express both Rh haplotypes  RHAG 
     S/s weak  
     U Absent 
Rhmod Reduced (variable)  Absent or reduced (variable)  Absent or reduced  Reduced (variable)  Reduced (variable)  Express both Rh haplotypes  RHAG 
     S/s Normal  
     U Normal/Weak 
Phenotype  Rh Proteins/Antigens  RhAG  LW  IAP GPB Protein/Antigens  Obligate Heterozygotes Altered Gene  
Rhnull-amorph  Absent Reduced (20%)  Absent  Reduced by 90%  Reduced by 50% Express one Rh haplotype* RHCE (RHDdeleted)  
     S/s Normal  
     U Weak  
Rhnull-regulator  Absent Absent  Absent  Reduced  Reduced by 70%183 Express both Rh haplotypes  RHAG 
     S/s weak  
     U Absent 
Rhmod Reduced (variable)  Absent or reduced (variable)  Absent or reduced  Reduced (variable)  Reduced (variable)  Express both Rh haplotypes  RHAG 
     S/s Normal  
     U Normal/Weak 
*

That is, appear as homozygotes.

Fy glycoprotein.

A possible association between the Fy glycoprotein (synonyms: Duffy, DARC) and the Rh complex is indicated by the Fy5 antigen, which is absent from Fy(a−b−) and RhnullRBCs.60 However, Rhnull RBCs have normal Fya, Fyb, Fy3, and Fy6 antigens, and Fy(a−b−) RBCs have normal Rh antigens. The specific requirements for expression of the Fy5 antigen remain unknown.

Band 3.

Band 3 (synonyms: AE1, anion exchanger, solute carrier family 4 anion exchanger member 1) is a glycosylated protein that is predicted to pass through the RBC membrane 12 or 14 times and is the major anion transporter.61,62 Unlike the proteins described above, band 3 is apparently normal in Rhnull RBCs; however, based on hemagglutination studies, antigens on Rh proteins and on band 3 are decreased in South-East Asian ovalocytic RBCs.63 The molecular defect associated with South-East Asian ovalocytic RBCs results from a deletion of a segment of DNA encoding 9 amino acids located at the boundary of the cytoplasmic N-terminal domain and membrane domain of band 3.64-67 Recent evidence that the expression of endogenous and retrovirally expressed Rh antigens were enhanced following transduction of K562 cells with band 3 suggests that band 3 and Rh proteins associate in erythroid cells.68 

Structure of RH and RHAG genes

The genes encoding RhD and RhCcEe are highly homologous, while the gene encoding RhAG is almost 40% homologous. The 3 genes are each composed of 10 exons; RHCE and RHD in tandem encompass 69 kilobases (kb) of DNA (Figure 2), whileRHAG encompasses 32 kb. The RhD protein is encoded byRHD (synonyms: RH30, RH30B, RH30D, RHXIII, RH13); the RhCcEe protein is encoded by RHCE (synonyms: RH30, RH30A, RH30C (RHCE),RHIXB, RH21); and the RHAG glycoprotein is encoded by RHAG (synonyms: RH50, RH50A).

Fig. 2.

RHCE-RHD gene organization.

Organization of exons (E) and introns of RHCE and RHDis shown. Exon sizes are indicated above the line as number of nucleotides, and intron sizes are indicated below the line. A c-specific short tandem repeat (STR) is located in intron 2 ofRHCE and another in intron 6 of RHD. The information used to compile this figure came from the database accession numbers given in the figure.

Fig. 2.

RHCE-RHD gene organization.

Organization of exons (E) and introns of RHCE and RHDis shown. Exon sizes are indicated above the line as number of nucleotides, and intron sizes are indicated below the line. A c-specific short tandem repeat (STR) is located in intron 2 ofRHCE and another in intron 6 of RHD. The information used to compile this figure came from the database accession numbers given in the figure.

The intron-exon boundaries of the RHCE gene21 and the complete nucleotide sequences of some RHCE and RHDintrons have been described.69-74 Selected GenBank accession numbers for cDNA are listed in Table 1, and those for introns are given in Figure 2. The intron-exon structure of the RHAGgene also has been defined and is remarkably similar to RHCEand RHD.22,75-77 Several mutations in RHAGhave been described that cause the regulator type of Rh deficiency syndrome (see below).

Evolution of the RH gene family

It was thought that Rh proteins were erythroid-specific and confined to higher vertebrates. However, the discovery of sequence-relatedRHAG homologs in invertebrates suggests otherwise. These homologs have been found as 2 different RHAG-like genes inCaenorhabditis elegans (a nematode; GenBank accession U64 847 and Z74 026)78 and as at least 1 in Geodia cydonium (a marine sponge; GenBank accession Y12 397).79 These genes are predicted to encode proteins with remarkably high (respectively, 46%, 39%, and 47%) amino acid identity to human RhAG. The highest homology is within the transmembrane domains, suggesting a conserved functional role for the RhAG protein family. Recent work has also demonstrated the presence ofRHAG counterparts in mouse (GenBank accession AF065 395; AF057 524-27, AF012 430), macaque (AF058 917) and RHorthologs in chimpanzee (L37 048-50), gorilla (L37 052, L37 053), orangutan (AF012 425), gibbon (L37 051), baboon (AF012 426), macaque (L37 054 570 343), New World monkeys (AF012 427-9, AF021 845) and cow (U59 270).77,80,81 

As the invertebrate homologs more strongly resemble human RHAGthan human RH, it is likely that an ancient gene duplication event, estimated to have occurred 250 million to 346 million years ago, caused divergence of RH from RHAG.75Subsequent to the gene duplication, RH and RHAGunderwent different evolutionary pathways.43 A second gene duplication event, being the origin of the human RHCE andRHD genes, occurred much later in a primate ancestor 5 million to 12 million years ago. Based on the evolutionary rates ofRHAG and RH genes in different species, it appears thatRHAG evolved some 2.6 times slower than RH,suggesting that RhAG has a more important functional role than Rh proteins.22,73,77,82 

The order of the Rh genes on chromosome 1 is probablyRHCE-RHD.83 (After submission of this manuscript, a paper was published that questions the order of the Rh genes on chromosome 1. Sequencing the intergenic region of the two RH genes suggests that the order may in fact beRHD-RHCE.211) The primordial human Rh haplotype is believed to be Dce, and the other 7 common Rh haplotypes most likely each arose from this gene complex by a single genetic event. The predominant Caucasian D-negative haplotype (dce) probably arose by a deletion of the RHD gene84 from theRHce/RHD gene complex, whereas the DCe haplotype (the most common D-positive haplotype in Caucasians) arose by gene conversion with exon 1 and 2 from RHD replacing the same exons of RHce. The remaining haplotypes arose through point mutations (eg, the E/e polymorphism) or rare recombination events of the various haplotypes.83 

Molecular basis of Rh antigens

Since the first descriptions of Rh cDNAs,20,31-33 much effort has been expended in differentiating the molecular bases underlying the antigens of the Rh system. The different genetic mechanisms that give rise to the major clinically relevant Rh antigens are described within this section. These include gene deletion (D-negative phenotype); gene conversion (C/c polymorphism); antithetical missense mutations (E/e); and other missense mutations (VS and V). The RH genes appear to be a source of massive diversity, and combinations of these different genetic rearrangements abound among all racial groups. We have selected examples of Rh polymorphisms that are of clinical significance and have been defined at the molecular level. Figures 3-6 detail the molecular basis of published examples of Rh variants. Enthusiastic readers requiring more data regarding Rh variants should consult references.16,25,85,86 

D antigen

The D antigen is a collection of conformation-dependent epitopes along the entire RhD protein. While in most D-negative Caucasians there is a deletion of RHD, in other populations (notably Japanese and African blacks) the D-negative phenotype is associated with a grossly normal RHD, and the reason for the lack of expression of the D antigen is not known (except in Africans; see later). Figure 3 depicts the molecular basis of some D-negative phenotypes.70,74,84,87-89 

Fig. 3.

Rearrangements at the Rh locus giving rise to D-negative and Rh deletion haplotypes.

The structures of the RH locus (located at 1p34-36) that has been defined in various D-negative phenotypes and rare Rh antigen deletion phenotypes are depicted. Each RH gene is represented as 10 boxes, each box representing an exon, where RHCE is shown as gray, RHD as black. Crosshatched boxes depict silent RHDalleles (eg, RHD Q41 X ). The positions of microinsertions or deletions of DNA that cause or are indicative of D-negative phenotypes are shown as triangles. Because exon 8 ofRHCE and RHD are of identical sequence and their origins are not possible to define, they are shaded according to the gene loci position. The significance of these rearrangements, and their impact in particular on molecular genotyping, is discussed within the text. Sources for the information in this figure: DCW − (AM)184; DCW − (Glo)185; D−− (LM)186; D−− (Gou)186; D−− (SH)72; D−− and Evans+ D•• (JD)187; Evans+ D•• (AT)69; Evans+ D•• (Dav)186; Dc− (Bol)186; Dc− (LZ)188; Ce70,74,149; r”G(SF)105,140; (Ce)Ce184; (C)ceS VS+ (Donor 1077) V−r'S 29;98;104; Amorph Rhnull (BK/DR)176,189; Amorph Rhnull (DAA)177; CML.179 

Fig. 3.

Rearrangements at the Rh locus giving rise to D-negative and Rh deletion haplotypes.

The structures of the RH locus (located at 1p34-36) that has been defined in various D-negative phenotypes and rare Rh antigen deletion phenotypes are depicted. Each RH gene is represented as 10 boxes, each box representing an exon, where RHCE is shown as gray, RHD as black. Crosshatched boxes depict silent RHDalleles (eg, RHD Q41 X ). The positions of microinsertions or deletions of DNA that cause or are indicative of D-negative phenotypes are shown as triangles. Because exon 8 ofRHCE and RHD are of identical sequence and their origins are not possible to define, they are shaded according to the gene loci position. The significance of these rearrangements, and their impact in particular on molecular genotyping, is discussed within the text. Sources for the information in this figure: DCW − (AM)184; DCW − (Glo)185; D−− (LM)186; D−− (Gou)186; D−− (SH)72; D−− and Evans+ D•• (JD)187; Evans+ D•• (AT)69; Evans+ D•• (Dav)186; Dc− (Bol)186; Dc− (LZ)188; Ce70,74,149; r”G(SF)105,140; (Ce)Ce184; (C)ceS VS+ (Donor 1077) V−r'S 29;98;104; Amorph Rhnull (BK/DR)176,189; Amorph Rhnull (DAA)177; CML.179 

People whose RBCs have an altered form of RhD protein (partial D) may make alloanti-D. Such RBCs, depending on which D epitopes are altered, are agglutinated by a proportion of anti-D reagents. Figure4 summarizes the molecular changes that are associated with partial D antigens.

Fig. 4.

Molecular bases of partial D phenotypes.

The different alleles of RHD that cause partial D phenotypes are depicted here graphically. The genetic structure of each partial DRHD 10-exon gene is shown, as are associated low-incidence antigen(s) and the estimated gene frequency. RHD (ie, wild type) exons are shown as black boxes; where they have been replaced byRHCE equivalents is shown as white boxes. Missense mutations are indicated within the exon where they occur. We have used the original Roman numeral notation (ie, DII to DVII) and the more recent 3-letter notation (eg, DFR, DBT) for the different D categories. Where partial D phenotypes have identical (or very similar) serologic profiles but different genetic backgrounds, we have adapted the classification originally described by Mouro et al190 to describe different DVIphenotypes (types I and II). Thus, we depict DIV types I to IV, DV types I to VI; DVI types I to III, and DFR types I and II. We use DVa to indicate the presence of the DW antigen and DV to represent samples that have a similar molecular background but that either do not express the DW antigen or have not been tested for this antigen.Few = 1 to 10 examples. Many = 11 or more examples as indicated by serological testing. DVII is common (1 in 900) in the German population.191 Under “Ethnic Origin,” B = black, C = Caucasian, and J = Japanese. The information used for the point mutations used in this figure are as follows: D+G−106; DNU and DII192; DHMi92; DVII193; DVa71,194 DFW195; DHR.196 The information used for the rearrangements in this figure was obtained from the following: DIIIa197; DIIIb106; DIIIc 198; DIVa type I 194; DIVbtype II194; DIVb type III92; DIVb type IV195; DVa type I194; DVa type II194; DV type III102; DVa type IV156; DV type V156; DVtype VI156; DVI type I199,200; DVI type II190; DVI type III71; DFR type I194; DFR type II201; DBT type I202; DBT type II203; ARRO-1204; DCS205.

Fig. 4.

Molecular bases of partial D phenotypes.

The different alleles of RHD that cause partial D phenotypes are depicted here graphically. The genetic structure of each partial DRHD 10-exon gene is shown, as are associated low-incidence antigen(s) and the estimated gene frequency. RHD (ie, wild type) exons are shown as black boxes; where they have been replaced byRHCE equivalents is shown as white boxes. Missense mutations are indicated within the exon where they occur. We have used the original Roman numeral notation (ie, DII to DVII) and the more recent 3-letter notation (eg, DFR, DBT) for the different D categories. Where partial D phenotypes have identical (or very similar) serologic profiles but different genetic backgrounds, we have adapted the classification originally described by Mouro et al190 to describe different DVIphenotypes (types I and II). Thus, we depict DIV types I to IV, DV types I to VI; DVI types I to III, and DFR types I and II. We use DVa to indicate the presence of the DW antigen and DV to represent samples that have a similar molecular background but that either do not express the DW antigen or have not been tested for this antigen.Few = 1 to 10 examples. Many = 11 or more examples as indicated by serological testing. DVII is common (1 in 900) in the German population.191 Under “Ethnic Origin,” B = black, C = Caucasian, and J = Japanese. The information used for the point mutations used in this figure are as follows: D+G−106; DNU and DII192; DHMi92; DVII193; DVa71,194 DFW195; DHR.196 The information used for the rearrangements in this figure was obtained from the following: DIIIa197; DIIIb106; DIIIc 198; DIVa type I 194; DIVbtype II194; DIVb type III92; DIVb type IV195; DVa type I194; DVa type II194; DV type III102; DVa type IV156; DV type V156; DVtype VI156; DVI type I199,200; DVI type II190; DVI type III71; DFR type I194; DFR type II201; DBT type I202; DBT type II203; ARRO-1204; DCS205.

Analysis of genes encoding the weak D phenotype (previously known as DU) showed a normal RHD sequence but a severely reduced expression of RHD messenger RNA (mRNA), suggesting a defect at the level of transcription or pre-mRNA processing.70,90 More recently, RHD transcripts from people whose RBCs express a weak form of the D antigen were found to have missense mutation(s) within the predicted transmembrane or cytoplasmic domains of RhD (Figure5).91,92 RBCs with some weak D antigens may not be agglutinated by all monoclonal anti-D. People whose RBCs express this type of weak D antigen do not make anti-D.

Fig. 5.

Molecular basis of weak D phenotypes.

This figure depicts missense mutations in the RHD gene associated with weak D phenotypes.92,153 The locations of these mutations on the predicted topology of the RhD protein are depicted as checkered ovals; the D-specific amino acids are shown as open ovals. Most of the missense mutations are located within nonconserved membrane spans (gray) and cytoplasmic regions. Regions of conserved Rh protein family sequence are indicated as black rectangles.

Fig. 5.

Molecular basis of weak D phenotypes.

This figure depicts missense mutations in the RHD gene associated with weak D phenotypes.92,153 The locations of these mutations on the predicted topology of the RhD protein are depicted as checkered ovals; the D-specific amino acids are shown as open ovals. Most of the missense mutations are located within nonconserved membrane spans (gray) and cytoplasmic regions. Regions of conserved Rh protein family sequence are indicated as black rectangles.

CcEe antigens

The RhC/c and RhE/e polymorphisms are caused by nucleotide substitutions in RHCE.28,93 While 6 nucleotide substitutions causing 4 amino acid changes (Cys16Trp; Ile60Leu; Ser68Asn; Ser103Pro) are associated with the C to c polymorphism (Figure 6), only the Ser103Pro polymorphism strictly correlates with C/c antigenicity.94 However, Pro102 appears to be a critical part of the c antigen.95,96The presence of 2 adjacent proline residues (102 and 103) would be expected to form a relatively rigid structure that is resistant to changes in nearby amino acid residues and may explain the relatively low number of c variants as compared with other Rh antigens. It has been generally accepted that a single nucleotide substitution is sufficient for expression of the E to e polymorphism (Pro226Ala). However, variants of the e antigen have been described,97showing that the requirements for expression of the e antigen are not fully understood. For example, the presence of Val at residue 245 instead of Leu,29,98,99 a deletion of Arg at amino acid residue 229,100 or the presence of Cys (instead of Trp) at amino acid residue 16 101 affects the expression of the e antigen. The molecular basis of partial E antigens (categories I, II, and III, and DV type III) has been determined and are shown in Figures 4 and 6.102,103 

Fig. 6.

Changes in RHCE.

Amino acids encoded by RHCE are shown by gray boxes, and those encoded by exons from RHD are shown by black boxes. The amino acids associated with E/e and C/c antigens28,93 are shown at the top, and single amino acid changes associated with variant forms of RhCE are shown in the middle. The bottom portion of the figure shows rearrangements of the RHCE and associated antigens. Polymorphism that does not have a 100% correlation with expression of c and C antigens. The information depicted in this figure was obtained from the following sources: Point Mutations35,98,99,104,185,206; Rearrangements DHAR 207; rG208;R¯¯N90; E Cat II and III185; and Variant e100,101.

Fig. 6.

Changes in RHCE.

Amino acids encoded by RHCE are shown by gray boxes, and those encoded by exons from RHD are shown by black boxes. The amino acids associated with E/e and C/c antigens28,93 are shown at the top, and single amino acid changes associated with variant forms of RhCE are shown in the middle. The bottom portion of the figure shows rearrangements of the RHCE and associated antigens. Polymorphism that does not have a 100% correlation with expression of c and C antigens. The information depicted in this figure was obtained from the following sources: Point Mutations35,98,99,104,185,206; Rearrangements DHAR 207; rG208;R¯¯N90; E Cat II and III185; and Variant e100,101.

VS and V antigens

The imultaneous presence of 2 low-incidence antigens (VS and V) occurs with a single amino acid substitution (Leu245Val) that is predicted to be within a transmembrane domain (Figure6).104 The V antigen (in the presence of VS) is not expressed when another transmembrane amino acid substitution is present at residue 336 (Gly→Cys) (Figure 3).98,104 The membrane location of residues 245 and 336 illustrate that Rh antigen expression is affected significantly by nonexofacial amino acids and suggests that the prediction of some Rh epitope expression cannot be based solely on externalized residues.

G antigen

RhD and RhC proteins carry the G antigen, which is associated with residues in the second extracellular loop encoded by exon 2.105,106 In DVIcE (DVI type I) RBCs, which are predicted to have a hybrid RhD (exons 1-3)–RHCE (exons 4 and 5)–RhD (exons 6-10) protein, the G antigen was not detected by 1 of 2 monoclonal anti-G.107 Thus, it would appear that the G antigen is conformation-dependent and not solely dependent on the second external domain of RhC(e/E) or RhD proteins.

Rh variants

Rh-variant phenotypes arise through at least 4 mechanisms: (1) rearrangements of the tandemly arrangedRHCE and/or RHD (Figures 3, 4, and 6); (2) point mutation(s) in either gene causing amino acid change(s), with subsequent loss of some epitopes and/or expression of a low-incidence antigen; (3) nonsense mutations, and (4) deletion of nucleotides causing a frameshift and premature stop codon. There is some evidence that there are recombination hot spots due to Alu IV elements in the RH genes.72,108 

Rearranged RHCE genes, associated with D−− and D••, ablate expression of C, c, E, and e antigens, while the D antigen expression is exalted to the extent that immunoglobulin (Ig) G anti-D can agglutinate the RBCs in saline.109 It is now clear that this increased expression is due to a large insert ofRHD into RHCE in tandem with a RHD gene (Figure3). In DCW− and Dc− phenotypes, the region of the RHCE gene encoding the E/e antigen is replaced by an RhD equivalent with loss of E/e antigenicity (Figure 3). While these appear as RHCE deletion phenotypes at the protein level, they are encoded by rearranged RHCE and thus are RHCE-depleted.

Low-incidence antigens associated with partial D antigens.

Low-incidence antigens associated with some partial D phenotypes are due to novel structures on the RBC surface and are useful markers for the identification of the partial D (Figure 4).110 A few low-incidence antigens are associated with more than 1 molecular background, eg, the FPTT (Rh50) antigen is expressed on DFR, RoHar, and DIVa(C)− phenotype RBCs; the Rh32 antigen is expressed on DBT andR¯¯NRBCs.The Evans antigen is expressed on D••, and a weak form of Evans is present on DIVb RBCs. RBCs expressing Rh23 or Rh32 possess an antigen (Rh23/32) present on both phenotypes.111In these cases, it is likely that external surfaces of the altered proteins have localized similarities.

RhD epitope mapping

Partial D antigens were classically identified by testing the RBCs with well-characterized polyclonal anti-D made by other people with partial D phenotypes and, also, by testing the patient's anti-D against RBCs with known partial D antigens. Human monoclonal antibodies are now being used to classify partial D antigens in terms of expressed epitopes. The original model consisted of 8- and 9-epitope D (epD)112,113 but has been expanded to consist of 16,110 30,114 and 37 epitopes.115 When using monoclonal anti-D to define D epitopes, it is important to perform the testing at the correct pH, temperature, ionic strength, and antibody concentration; to use RBCs that have been stored appropriately; and to include controls.110,114 Most D epitopes are conformation-dependent and may be influenced by other proteins and lipids in the RBC membrane. Indeed, only 1 monoclonal anti-D has been described that reacts strongly by immunoblotting, implying that the epitope it recognizes may be linear.116 

Predictions as to the location of various D epitopes have been based on which epitopes are absent from RBCs with a partial D for which the molecular basis is known.117,118 However, the absence of a D epitope may not always be a direct result of the change in molecular structure, and the presence of Rh proteins encoded by cis andtrans genes can effect the binding of certain monoclonal anti-D. For example, R0Har and DVado not have any RHD exons in common, but they have overlapping reactivity with monoclonal antibody anti-D, demonstrating the difficulty of correctly defining the molecular basis of D epitopes. A model proposed by Chang and Siegel119 suggests that anti-D are essentially similar in that they react with the basic footprint of the D protein. In this model, a change in the footprint, induced by an amino acid substitution or a hybrid protein, is predicted to interfere with binding of anti-D. The involvement of certain residues for binding of monoclonal anti-D has been investigated by site-directed mutagenesis (SDM), which showed that incorporation of 3 D-specific amino acids (Asp350, Gly353, and Ala354) into an RhcE construct generated some epD3 and epD9 expression,120,121 and incorporation of 9 exofacial D residues generated epitopes that were recognized by 40 of 50 monoclonal anti-D.122 These data argue that at least some D epitopes are spatially distinct. However, SDM studies have not yet addressed the impact of amino acids located within the lipid bilayer or on the cytoplasmic side of the RBC membrane. Accurate determination of the contact points of interaction(s) between antigen and antibody awaits crystallographic data.

Clinical aspects

Clinical complications result from RBC destruction due to the interaction of an alloantibody with RBCs carrying the corresponding antigen. The D antigen is highly immunogenic and induces an immune response in 80% of D-negative persons when transfused with 200 mL of D-positive blood.123 For this reason, in most countries D typing is performed routinely on every blood donor and transfusion recipient so that D-negative patients receive D-negative RBC products. Consequently, clinical complications due to mismatched transfusions are infrequent. In contrast, despite the use of immunosuppressive therapy with anti-D immunoglobulin prophylaxis, D alloimmunization in pregnancy still occurs.

Alloantibodies

Alloantibodies that recognize Rh antigens are usually IgG and react by the indirect antiglobulin test. This is a test in which RBCs are incubated in serum, washed to remove free immunoglobulin, and then exposed to an antiglobulin reagent that is formulated to detect the cell-bound IgG. The end point of the test is hemagglutination. Alloantibodies in the Rh blood group system can cause destruction of transfused RBCs and of fetal RBCs in hemolytic disease of the newborn (HDN). People whose RBCs have a rare deleted Rh phenotype (Rhnull, D−−) readily make alloantibodies. People with the Rhnull phenotype of amorph or regulator type can make anti-Rh29 (an antibody to “total” Rh), anti-Rh17 (an antibody to the RhCc/Ee protein), anti-D, anti-C, or a mixture of specificities. Transfusion of a patient with anti-Rh29 is a problem because only Rhnull RBCs will be compatible: People with the Rhnull phenotype are not only rare, but they have a compensated hemolytic anemia and are therefore unlikely to meet predonation criteria.124 People with either the D−−, D••, DCW−, or Dc− phenotype make anti-Rh17. A patient with anti-Rh17 also represents a transfusion conundrum because only RBCs with a deleted phenotype will be compatible.

Autoantibodies

An autoantibody is one that reacts with an antigen on the antibody maker's own RBCs. Autoantibodies that react optimally at 37°C are present in the serum of about 80% of patients with warm autoimmune hemolytic anemia.125 Although most of these autoantibodies appear to be “nonspecific,” many have specificity to an Rh antigen, notably to e. Rarely is the specificity clear-cut, but the autoantibody commonly reacts more weakly with antigen-negative RBCs than with antigen-positive RBCs; however, in these cases, transfused antigen-negative RBCs only rarely survive better than antigen-positive RBCs.123 Autoantibodies in serum from patients with warm autoimmune hemolytic anemia may be nonreactive only with Rhnull and D−− RBCs (autoanti-Rh17), or only with Rhnull RBCs (autoanti-Rh29). In such cases, antigen-negative blood will not be available, and transfusion with antigen-positive RBCs should not be withheld if the patient has life-threatening anemia.125,126 In most cases, the autoantibody is equally reactive with all RBCs tested—whether from donors or antibody detection/identification kits. Thus, in the clinical setting, it is important to perform tests to ensure that the patient's serum does not have potentially clinically significant alloantibodies underlying the autoantibodies before transfusing incompatible RBCs. Detection and identification of such antibodies is required to prevent transfusion reactions but is beyond the scope of this review. For more information, see a current textbook on laboratory aspects of transfusion medicine.125-127 

Partial and weak D phenotypes

As described earlier, people whose RBCs have a weak D phenotype (quantitative D variant) do not make anti-D, whereas people whose RBCs have a partial D phenotype (qualitative D variant with or without weakening of the D antigen) can make alloanti-D. This presents a different problem depending on whether the person is a donor or a patient. For donors, detection of weak and partial D antigens would eliminate the possibility of immunization should such blood be transfused to a true D-negative patient. However, historical data show that weakly expressed D antigens are most unlikely to be immunogenic. For transfusion recipients and pregnant women, it is common practice to use a procedure that will classify RBCs with a weak D antigen or some partial D antigens as D-negative. Thus, blood donated from such a person should be labeled as D-positive (Rh-positive), but the same person should be listed as D-negative (Rh-negative) when they are recipients in need of transfusion. The transfusion recipient will receive D-negative RBC products, and the pregnant woman will receive prophylactic Rh immunoglobulin, thereby preventing alloimmunization. Although a pregnant woman with the DVI partial phenotype may make alloanti-D, this has rarely caused a clinical problem to a D-positive fetus.128 In the autologous transfusion setting (in which the person is both the donor and patient), the above policy can cause confusion because partial D RBCs may be typed as D-positive at the donor center but D-negative at the hospital. In practice, it is difficult to distinguish RBCs with the DVI phenotype from other weak D; however, this now can be accomplished by immunoblotting with the unique anti-D, LOR-15C9.129 

Rh and hemolytic disease of the newborn

HDN is caused by maternal IgG antibody crossing the placenta, binding to the fetal antigen-positive RBCs, and initiating their destruction, thereby causing anemia. Prior to the use of prophylactic Rh immunoglobulin, anti-D frequently caused fetal brain damage due to increased levels of bilirubin (kernicterus) and even death (erythroblastosis fetalis). Despite the widespread use of prophylactic Rh immunoglobulin, a significant number of women still become alloimmunized during pregnancy for a variety of reasons, including nonadministration of Rh immunoglobulin, unrecognized miscarriage, leakage of fetal RBCs into the maternal circulation late in pregnancy, and exposure to maternal D-positive RBCs while in utero (grandmother effect).130 

The D antigen accounts for about 50% of cases of maternal alloimmunization; the remainder is due mainly to incompatibility to K, c, C/G, E, and Fya antigens and to low incidence antigens in Rh, MNS, and Diego blood group systems.131-133Therefore, feto-maternal Rh incompatibility still represents the major cause of HDN. Ultraviolet phototherapy and, occasionally, exchange transfusion or even intrauterine transfusion may be required. Invasive procedures are used as a “last option” in monitoring and treating HDN, because they may cause further leakage of fetal RBCs into the maternal circulation. Measures such as determination of the optical density of amniotic fluid and functional assays (ADCC, MMA, chemiluminescence) have been used to monitor at-risk pregnancies and to identify cases requiring treatment (for review, see Zupanska134). With current molecular technology, it is possible to perform analyses on fetally derived DNA to predict the blood type of a fetus.

Interestingly, a fetus that is ABO incompatible with the maternal anti-A/B is less likely to have HDN due to anti-D, presumably due to rapid removal of the ABO-incompatible RBCs by the naturally occurring anti-A/B. Also, because the number of copies of the D antigen per RBC is higher in the R2 haplotype (range, 14 000 to 16 000) than in the R1 haplotype (range, 9000 to 14 600), fetuses whose RBCs are R2 have more severe anemia than their R1counterparts.123 There is also evidence that male fetuses have more severe HDN than female fetuses.135 

Rh immunoglobulin prophylaxis in the prevention of HDN.

The immunologic mechanism responsible for preventing production of maternal anti-D following administration of prophylactic Rh immunoglobulin may be due, at least in part, to antigen blocking and central inhibition of the immune response by negative feedback in the spleen (for review, see Bowman130). In some instances, recommendations have been made to administer anti-D to partial D-phenotype mothers (eg, DVI and DBT phenotypes) following the birth of D-positive babies.110,136 In Europe, anti-D reagents are selected to deliberately type DVI mothers as D-negative and, thus, ensure that such mothers would automatically receive prophylactic Rh immunoglobulin therapy following pregnancy.

Prophylactic Rh immunoglobulin preparations for this purpose are usually for intramuscular injection. However, products approved also for intravascular injection are used for the treatment of idiopathic thrombocytopenia.137,138 

Legislative restrictions for immunization of D-negative volunteers with accredited D-positive RBCs are partly responsible for the declining source of polyclonal anti-D for prophylaxis. Thus, clinical trials have explored the possibility of using human monoclonal anti-D to prevent anti-D alloimmunization;139 however, the in vivo use of monoclonal antibodies derived from EBV-transformed cells remains controversial. It is possible that recombinant forms of anti-D can be prepared as an injectable prophylactic product.

Prenatal Rh genotyping

When a pregnant woman has a potentially clinically significant alloantibody and the father of the fetus is phenotypically heterozygous for the gene encoding the corresponding antigen (or is unknown), prenatal determination can be considered. The potential benefits of identifying a fetus whose RBCs are predicted to be antigen-negative is enormous in that the need for further invasive techniques is diminished. Fetal DNA can be obtained from amniocytes, chorionic villi, vaginal swabs, and mother's blood (see later). Following cloning and sequencing of RHCE and RHD, many polymerase chain reaction (PCR)-based tests to analyze DNA prepared from amniocytes have been reported (for recent review, see Flegel86). However, the genetic diversity of the Rh genes, particularly among blacks and Japanese, has reduced the clinical utility of this approach because false-negative and false-positive results can occur. Prenatal diagnosis of fetal RHD status exploits structural differences between theRHD and RHCE genes and is based on the assumption that D-negative individuals have a deleted RHD gene. As the knowledge regarding the molecular basis of partial D antigens evolved, use of multiplex,70,86,140,141heteroduplex,142,143 and multiple sequence-specific PCR reactions144,145 have replaced the single exon genotyping assays32,146,147 in an attempt to avoid “false-negative” typing of a fetus with a partial D antigen. However, because HDN in a fetus whose RBCs have a partial D antigen is rare,148 the clinical value of RHD multiplex analysis may only have marginal added value.

All current RhD genotyping assays will mistype people whose RBCs are D-negative and yet carry an intact, nonfunctional RHD. Such people have been described in Caucasians (rare),70 African blacks (common),89,144 and Asians (common).74,144 Molecular genotyping will have limited clinical utility in populations where the presence of nonexpressedRHD is frequent. The molecular backgrounds of these D-negative phenotypes are beginning to emerge: In 2 Caucasians expressing the dCe phenotype, 1 had an in-frame stop codon in exon 1 of the RHDgene70 and the other a deletion of 4 nucleotides in exon 4.149 Very recently, the molecular basis of the major silent RHD allele (namedRHDψ) found in persons of African ancestry has been defined.150,RHDψ has a 37–base pair insertion of DNA, being a duplication of the intron 3/exon 4 boundary, and has missense mutations in exon 5 and a nonsense and missense mutation in exon 6. The Del phenotype (ie, D antigen is detectable only by adsorption-elution tests) was thought to have a deletion ofRHD;151,152 however, a deletion of 1013 base pairs encompassing intron 8, exon 9, and intron 9 has been observed (Figure5).153 

Clearly, knowledge of the ethnic group of both parents is helpful in the selection of appropriate genotyping tests. Wherever possible, to limit the gene pool, concurrent analyses of maternal and paternal blood group phenotypes and genotypes should be performed. It is worth noting that samples that have been used for clinical automated instruments are often contaminated with blood from previous tests.144,154 

As molecular analysis becomes more common, it is worth remembering that some D variants may be more common than previously thought. An example of this is the hybrid gene encoding the DIIIa phenotype, which has recently been shown to be present in 18% of blacks in New York and 28% of blacks from Brazil.155 Furthermore, a similar pattern of reactivity may be obtained with monoclonal anti-D in tests with RBCs from people with different Rh genes. This is illustrated by the large number of molecular events associated with DVa (or DV-like) samples as defined by the pattern of reactivity with monoclonal anti-D.156 Not all of the molecular events give rise to the DW antigen, whose presence on RBCs is required for DVacategorization.110 

Although hemolytic disease due to Rh antibodies other than anti-D is less frequent, PCR-based tests have been designed to defineRHCE alleles using fetally derived DNA.157-159 Most of these are relatively straightforward; however, genotyping C in the presence of D is difficult because RHC(E/e) and RHDhave identical sequences in exons 1 and 2. RhC typing is possible by exploitation of a polymorphism in intron 2 of RHCE, which involves a 109-base pair insert of DNA in RHC(E/e) but notRhc(E/e) or RHD.94,159 

Noninvasive prenatal Rh genotyping.

It is now possible to obtain fetally derived DNA using noninvasive procedures. Fetally derived RHD has been detected using nested PCR analysis on genomic DNA (gDNA) extracted from maternal peripheral blood or plasma160-163 or from transcervical samples.164-166 An alternative approach uses cDNA templates derived by reverse transcriptase–PCR from maternal peripheral blood and detection of fetal RhD mRNA targets.167 All noninvasive procedures have limited value because there is no suitable way to assess the presence of fetal cells in a given sample and, thus, negative results cannot be interpreted with confidence. Nevertheless, the fact that fetally derived Rh mRNAs and gDNA can be detected in maternal blood indicates that this area of prenatal diagnosis may soon have an impact. However, it is possible that fetal-nucleated RBCs are the most pertinent target cell type for noninvasive diagnosis168,169 because other fetally derived CD34+ cells have been detected in maternal blood for as long as 27 years postpartum169 and thus could interfere with analyses in women who have had multiple pregnancies.

Rh and other disease states

Rhnull disease.

RBCs from people who have the Rhnull phenotype (synonyms: Rhnull syndrome, Rhnull disease) lack Rh proteins and, thus, Rh antigens. This phenotype is rare (approximately 1 in 6 × 106 individuals)170 and most often results from a consanguineous mating. The syndrome is associated with stomatocytosis, spherocytosis, increased osmotic fragility, altered phospholipid asymmetry, altered cell volume, defective cation fluxes, and elevated Na+/K+ ATPase activity.13,14,171-173 Rhnull RBCs may have a shortened in vivo survival, and the person may have a mild compensated hemolytic anemia.

There are 2 types of Rhnull, amorph and regulator, that historically were classified based on their pattern of inheritance. It is now known that the amorph type is the result of a molecular change in RHCE in tandem with a deleted RHD (Figure 3),whereas the regulator type is associated with a molecular defect inRHAG (Figure7).22,76,174-177 Table 2summarizes the characteristics of the 2 types of Rhnullphenotypes and compares them with the Rhmodphenotype, in which the Rh antigens are suppressed. While RhAG is apparently critical for the correct assembly of the Rh proteins in the RBC membrane, RhAG by itself can form stable complexes, albeit in reduced quantity, in the absence of Rh proteins.22,58 

Fig. 7.

Localization of molecular defects on RhAG.

The regulator type of Rhnull is associated with 2 mutantRHAG genes (homozygote or double heterozygote). The mutations include splice site/frameshift alterations and missense mutations (gray circles). The missense changes predominantly occur within conserved Rh protein family domains (black rectangles), within membrane-spanning regions. It is thought that missense mutations affect either RhAG-RhAG associations/RhAG-Rh protein assocations, resulting in an absence of the Rh protein family from mature RBC membranes. The Rhmodphenotype is associated with missense mutations (crosshatched circles), which lead to a marked reduction of the RhAG-Rh protein complex in mature RBC membranes. The initials refer to the probands. The information used in this figure was obtained from the following: SM209; SF, JL174; AL177; YT27,175; VL174; HT 210; TT, AC22; TB174; WO210.

Fig. 7.

Localization of molecular defects on RhAG.

The regulator type of Rhnull is associated with 2 mutantRHAG genes (homozygote or double heterozygote). The mutations include splice site/frameshift alterations and missense mutations (gray circles). The missense changes predominantly occur within conserved Rh protein family domains (black rectangles), within membrane-spanning regions. It is thought that missense mutations affect either RhAG-RhAG associations/RhAG-Rh protein assocations, resulting in an absence of the Rh protein family from mature RBC membranes. The Rhmodphenotype is associated with missense mutations (crosshatched circles), which lead to a marked reduction of the RhAG-Rh protein complex in mature RBC membranes. The initials refer to the probands. The information used in this figure was obtained from the following: SM209; SF, JL174; AL177; YT27,175; VL174; HT 210; TT, AC22; TB174; WO210.

Myeloid leukemia

Patients with acute or chronic myeloid leukemia, myeloid metaplasia, polycythemia, or myelofibrosis occasionally have 2 populations of RBCs of different Rh type. In some cases, a loss of Rh antigens is associated with chromosome aberrations.178 Recent analysis of blood from a D-positive patient with CML who became D-negative for the 3 years that she was studied revealed a single base deletion in exon 4 of RHD that occurred by somatic mutation.179 

Discussion

Considerable progress has been made in our understanding of the molecular basis of Rh and other blood group antigens in the past 10 years. Despite this, our knowledge concerning the function of many of the components in the RBC remains speculative. The Rh protein complex is a prime example of this; it is a major red cell protein of considerable clinical importance, yet our understanding of its functional significance in human RBCs and other animals relies almost entirely on circumstantial evidence.

The Rh blood group system consists of numerous antigens that are located on variant forms of RhD and RhCE proteins. These proteins form a core complex in the erythrocyte membrane with a glycosylated homolog (RhAG) and are only expressed when it is present. RhD, RhCE, and RhAG associate with other membrane proteins (LW, IAP, GPB, Duffy, and band 3) to form a large complex. Although the function(s) of these proteins has not been defined, it is possible that the complex forms a concerted transporter. The genes encoding the Rh proteins (RHCE andRHD) are highly homologous and adjacent on the short arm of chromosome 1, while the gene encoding RhAG (RHAG) is nearly 40% homologous and is located on the short arm of chromosome 6. Although the molecular basis associated with many of the Rh antigens is known, the actual epitopes have not been defined, but it is apparent that most of the Rh antigens are conformation-dependent. The molecular knowledge is increasingly being used in the clinical setting. However, the allelic diversity in this system is a potential problem for reliable genotyping by PCR-based assays. Hemagglutination is still a powerful, practical, and economical test with a specificity and sensitivity that is appropriate for clinical applications. However, the use of hemagglutination in conjunction with molecular techniques undoubtedly will lead to insights that can enhance approaches for the treatment of Rh incompatibility. Further understanding of the immunologic responses to the Rh antigens will be of importance in the treatment of hemolytic disease, and detailed epitope maps involving serologic, molecular, and protein crystallographic studies of the Rh proteins will contribute to this objective.

Acknowledgments

We thank Christine Lomas-Francis, Narla Mohandas, Olga Blumenfeld, Geoff Daniels, Michael J. A. Tanner, Jill Storry, and Karina Yazdanbakhsh for reading the manuscript and giving helpful suggestions. Neil Avent thanks Willy Flegel, Giorgio Matassi, and Tim Kemp for providing manuscripts prior to publication. We also thank Robert Ratner for preparing the manuscript and cataloging the references.

Supported in part by a National Institutes of Health Specialized Center of Research (SCOR) grant in transfusion medicine and biology HL54 459.

Reprints:Marion E. Reid, Immunochemistry Laboratory, New York Blood Center, 310 East 67th St, New York, NY 10021; e-mail:mreid@nybc.org.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Levine
P
Stetson
RE
An unusual case of intragroup agglutination.
JAMA.
113
1939
126
127
2
Landsteiner
K
Wiener
AS
An agglutinable factor in human blood recognized by immune sera for rhesus blood.
Proc Soc Exp Biol Med.
43
1940
223
3
Fisk
RT
Foord
AG
Observations on the Rh agglutinogen of human blood.
Am J Clin Pathol.
12
1942
545
552
4
Levine
P
Celano
MJ
Wallace
J
Sanger
R
A human ‘D-like’ antibody.
Nature.
198
1963
596
597
5
Daniels
GL
Anstee
DJ
Cartron
J-P
et al. 
Blood group terminology 1995: ISBT working party on terminology for red cell surface antigens.
Vox Sang.
69
1995
265
279
6
Rosenfield
RE
Allen
FH
Jr
Swisher
SN
Kochwa
S
A review of Rh serology and presentation of a new terminology.
Transfusion.
2
1962
287
312
7
Rosenfield
RE
Allen
FH
Jr
Rubinstein
P
Genetic model for the Rh blood-group system.
Proc Natl Acad Sci U S A.
70
1973
1303
1307
8
Allen
FH
Jr
Rosenfield
RE
Review of Rh serology: eight new antigens in nine years.
Haematologia.
6
1972
113
120
9
Rosenfield
RE
Allen
FH
Jr
Swisher
SN
Kochwa
S
Rh nomenclature.
Transfusion.
19
1979
487
10
Moore
S
Woodrow
CF
McClelland
DB
Isolation of membrane components associated with human red cell antigens Rh(D), (c), (E) and Fy.
Nature.
295
1982
529
531
11
Gahmberg
CG
Molecular identification of the human RhO (D) antigen.
FEBS Lett.
140
1982
93
97
12
Moore
S
Green
C
The identification of specific Rhesus polypeptide blood group ABH-active glycoprotein complexes in the human red-cell membrane.
Biochem J.
244
1987
735
741
13
Agre
P
Cartron
J-P
Molecular biology of the Rh antigens.
Blood.
78
1991
551
563
14
Anstee
DJ
Tanner
MJ
Biochemical aspects of the blood group Rh (rhesus) antigens.
Baillieres Clin Haematol.
6
1993
401
422
15
Moore
S
Gahmberg
CG
Identification of Rh polypeptide and Rh polypeptide/Rh glycoprotein complexes.
Biotest Bull.
5
1997
409
413
16
Huang
C-H
Molecular insights into the Rh protein family and associated antigens.
Curr Opin Hematol.
4
1997
94
103
17
Ridgwell
K
Spurr
NK
Laguda
B
MacGeoch
C
Avent
ND
Tanner
MJ
Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression.
Biochem J.
287
1992
223
228
18
Eyers
SA
Ridgwell
K
Mawby
WJ
Tanner
MJ
Topology and organization of human Rh (rhesus) blood group-related polypeptides.
J Biol Chem.
269
1994
6417
6423
19
Avent
ND
Liu
W
Warner
KM
et al. 
Immunochemical analysis of the human erythrocyte Rh polypeptides.
J Biol Chem.
271
1996
14,233
14,239
20
Avent
ND
Ridgwell
K
Tanner
MJA
Anstee
DJ
cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.
Biochem J.
271
1990
821
825
21
Chérif-Zahar
B
Le Van Kim
C
Rouillac
C
Raynal
V
Cartron
J-P
Colin
Y
Organization of the gene (RHCE) encoding the human blood group RhCcEe antigens and characterization of the promoter region.
Genomics.
19
1994
68
74
22
Huang
C-H
The human Rh50 glycoprotein gene—structural organization and associated splicing defect resulting in Rhnull disease.
J Biol Chem.
273
1998
2207
2213
23
Avent
ND
Butcher
SK
Liu
W
et al. 
Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.
J Biol Chem.
267
1992
15,134
15,139
24
Hermand
P
Mouro
I
Huet
M
et al. 
Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.
Blood.
82
1993
669
676
25
Cartron
JP
Bailly
P
Le Van Kim
C
et al. 
Insights into the structure and function of membrane polypeptides carrying blood group antigens.
Vox Sang.
74(suppl 2)
1998
29
64
26
Hartel-Schenk
S
Agre
P
Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex.
J Biol Chem.
267
1992
5569
5574
27
Huang
CH
Liu
Z
Cheng
GJ
Chen
Y
Rh50 glycoprotein gene and Rhnull disease: a silent splice donor is trans to a Gly279—>Glu missense mutation in the conserved transmembrane segment.
Blood.
92
1998
1776
1784
28
Mouro
I
Colin
Y
Chérif-Zahar
B
Cartron
J-P
Le Van Kim
C
Molecular genetic basis of the human Rhesus blood group system.
Nature Genet.
5
1993
62
65
29
Blunt
T
Daniels
G
Carritt
B
Serotype switching in a partially deleted RHD gene.
Vox Sang.
67
1994
397
401
30
Smythe
JS
Avent
ND
Judson
PA
Parsons
SF
Martin
PG
Anstee
DJ
Expression of RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of Rh blood group antigens.
Blood.
87
1996
2968
2973
31
Le Van Kim
C
Mouro
I
Chérif-Zahar
B
et al. 
Molecular cloning and primary structure of the human blood group RhD polypeptide.
Proc Natl Acad Sci U S A.
89
1992
10,925
10,929
32
Arce
MA
Thompson
ES
Wagner
S
Coyne
KE
Ferdman
BA
Lublin
DM
Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals.
Blood.
82
1993
651
655
33
Chérif-Zahar
B
Bloy
C
Le Van Kim
C
et al. 
Molecular cloning and protein structure of a human blood group Rh polypeptide.
Proc Natl Acad Sci U S A.
87
1990
6243
6247
34
Suyama
K
Goldstein
J
Aebersold
R
Kent
S
Regarding the size of Rh proteins.
Blood.
77
1991
411
35
Mouro
I
Colin
Y
Sistonen
P
Le Pennec
PY
Cartron
J-P
Le Van Kim
C
Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities.
Blood.
86
1995
1196
1201
36
Kajii
E
Umenishi
F
Iwamoto
S
Ikemoto
S
Isolation of a new cDNA clone encoding an Rh polypeptide associated with the Rh blood group system.
Hum Genet.
91
1993
157
162
37
de Vetten
MP
Agre
P
The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein.
J Biol Chem.
263
1988
18,193
18,196
38
Basu
MK
Flamm
M
Schachter
D
Bertles
JF
Maniatis
A
Effects of modulating erythrocyte membrane cholesterol on Rho(D) antigen expression.
Biochem Biophys Res Commun.
95
1980
887
893
39
Ridgwell
K
Eyers
SA
Mawby
WJ
Anstee
DJ
Tanner
MJ
Studies on the glycoprotein associated with Rh (rhesus) blood group antigen expression in the human red blood cell membrane.
J Biol Chem.
269
1994
6410
6416
40
Southcott
MJG
Tanner
MJ
Anstee
DJ
The expression of human blood group antigens during erythropoiesis in a cell culture system.
Blood.
93
1999
4425
4435
41
Chown
B
On a search for Rhesus antibodies in very young foetuses.
Arch Dis Child.
30
1955
232
233
42
Marini
AM
Urrestarazu
A
Beauwens
R
André
B
The Rh (rhesus) blood group polypeptides are related to NH4+ transporters.
Trends Biochem Sci.
22
1997
460
461
43
Matassi
G
Chérif-Zahar
B
Pesole
G
Raynal
V
Cartron
JP
The members of the RH gene family (RH50 and RH30) followed different evolutionary pathways.
J Mol Evol.
48
1999
151
159
44
Lorenz
MC
Heitman
J
The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae.
EMBO J.
17
1998
1236
1247
45
Bailly
P
Tontti
E
Hermand
P
Cartron
JP
Gahmberg
CG
The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins.
Eur J Immunol.
25
1995
3316
3320
46
Issitt
PD
Anstee
DJ
Applied Blood Group Serology.
1998
Montgomery Scientific Publications
Durham, NC
47
Daniels
G
Human Blood Groups.
1995
Blackwell Science Ltd
Oxford, England
48
Giles
CM
The LW blood group: a review.
Immunol Commun.
9
1980
225
242
49
Mallinson
G
Martin
PG
Anstee
DJ
et al. 
Identification and partial characterization of the human erythrocyte membrane component(s) that express the antigens of the LW blood-group system.
Biochem J.
234
1986
649
652
50
Brown
E
Hooper
L
Ho
T
Gresham
H
Integrin-associated protein: a 50-kD plasma membrane antigen physically and functionally associated with integrins.
J Cell Biol.
111
1990
2785
2794
51
Mawby
WJ
Holmes
CH
Anstee
DJ
Spring
FA
Tanner
MJ
Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin- associated protein (IAP) and the ovarian tumour marker OA3.
Biochem J.
304
1994
525
530
52
Reinhold
MI
Lindberg
FP
Plas
D
Reynolds
S
Peters
MG
Brown
EJ
In vivo expression of alternatively spliced forms of integrin-associated protein (CD47).
J Cell Sci.
108
1995
3419
3425
53
Gao
AG
Lindberg
FP
Finn
MB
Blystone
SD
Brown
EJ
Frazier
WA
Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin.
J Biol Chem.
271
1996
21
24
54
Schwartz
MA
Brown
EJ
Fazeli
B
A 50-kDa integrin-associated protein is required for integrin-regulated calcium entry in endothelial cells.
J Biol Chem.
268
1993
19,931
19,934
55
Miller
YE
Daniels
GL
Jones
C
Palmer
DK
Identification of a cell-surface antigen produced by a gene on human chromosome 3 (cen-q22) and not expressed by Rhnull cells.
Am J Hum Genet.
41
1987
1061
1070
56
Avent
N
Judson
PA
Parsons
SF
et al. 
Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes: one group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.
Biochem J.
251
1988
499
505
57
Gardner
B
Anstee
DJ
Mawby
WJ
Tanner
MJ
von dem Borne
AE
The abundance and organization of polypeptides associated with antigens of the Rh blood group system.
Transfus Med.
1
1991
77
85
58
Mallinson
G
Anstee
DJ
Avent
ND
et al. 
Murine monoclonal antibody MB-2D10 recognizes Rh-related glycoproteins in the human red cell membrane.
Transfusion.
30
1990
222
225
59
Ballas
SK
Reilly
PA
Murphy
DL
The blood group U antigen is not located on glycophorin B.
Biochim Biophys Acta.
884
1986
337
343
60
Colledge
KI
Pezzulich
M
Marsh
WL
Anti-Fy5, and antibody disclosing a probable association between the Rhesus and Duffy blood group genes.
Vox Sang.
24
1973
193
199
61
Tanner
MJ
Molecular and cellular biology of the erythrocyte anion exchanger (AE1).
Semin Hematol.
30
1993
34
57
62
Fujinaga
J
Tang
X-B
Casey
JR
Topology of the membrane domain of human erythrocyte anion exchange protein, AE1.
J Biol Chem.
274
1999
6626
6633
63
Booth
PB
Serjeantson
S
Woodfield
DG
Amato
D
Selective depression of blood group antigens associated with hereditary ovalocytosis among Melanesians.
Vox Sang.
32
1977
99
110
64
Tanner
MJ
Bruce
L
Martin
PG
Rearden
DM
Jones
GL
Melanesian hereditary ovalocytes have a deletion in red cell band 3.
Blood.
78
1991
2785
2786
65
Schofield
AE
Reardon
DM
Tanner
MJ
Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells.
Nature.
355
1992
836
838
66
Liu
S-C
Palek
J
Yi
SJ
et al. 
Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton.
Blood.
86
1995
349
358
67
Mohandas
N
Winardi
R
Knowles
D
et al. 
Molecular basis for membrane rigidity of hereditary ovalocytosis: a novel mechanism involving the cytoplasmic domain of band 3.
J Clin Invest.
89
1992
686
692
68
Beckmann
R
Smythe
JS
Anstee
DJ
Tanner
MJA
Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythroleukemia cells: band 3 enhances the cell surface reactivity of Rh antigens.
Blood.
92
1998
4428
4438
69
Huang
C-H
Chen
Y
Reid
M
Ghosh
S
Genetic recombination at the human RH locus: a family study of the red cell-Evans phenotype reveals a transfer of exons 2-6 from the RHD to the RHCE gene.
Am J Hum Genet.
59
1996
825
833
70
Avent
ND
Martin
PG
Armstrong-Fisher
SS
et al. 
Evidence of genetic diversity underlying Rh D-, weak D (Du), and partial D phenotypes as determined by multiplex polymerase chain reaction analysis of the RHD gene.
Blood.
89
1997
2568
2577
71
Wagner
FF
Gassner
C
Müller
TH
Schönitzer
D
Schuhnter
F
Flegel
WA
Three molecular structures cause rhesus D category VI phenotypes with distinct immunohematological features.
Blood.
91
1998
2157
2168
72
Kemp
TJ
Poulter
M
Carritt
B
A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D—phenotype.
Am J Hum Genet.
59
1996
1066
1073
73
Westhoff
CM
Wylie
DE
Investigation of the RH locus in gorillas and chimpanzees.
J Mol Evol.
42
1996
658
668
74
Okuda
H
Kawano
M
Iwamoto
S
et al. 
The RHD gene is highly detectable in RhD-negative Japanese donors.
J Clin Invest.
100
1997
373
379
75
Matassi
G
Chérif-Zahar
B
Raynal
V
Rouger
P
Cartron
JP
Organization of the human RH50A gene (RHAG) and evolution of base composition of the RH gene family.
Genomics.
47
1998
286
293
76
Iwamoto
S
Omi
T
Yamasaki
M
Okuda
H
Kawano
M
Kajii
E
Identification of 5' flanking sequence of RH50 gene and the core region for erythroid-specific expression.
Biochem Biophys Res Commun.
243
1998
233
240
77
Kitano
T
Sumiyama
K
Shiroishi
T
Saitou
N
Conserved evolution of the Rh50 gene compared to its homologous Rh blood group gene.
Biochem Biophys Res Commun.
249
1998
78
85
78
Wilson
R
Ainscough
R
Anderson
K
et al. 
2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.
Nature.
368
1994
32
38
79
Seack
J
Pancer
Z
Muller
IM
Muller
WE
Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium.
Immunogenetics.
46
1997
493
498
80
Apoil
PA
Blancher
A
Sequences and evolution of mammalian RH gene transcripts and proteins.
Immunogenetics.
49
1999
15
25
81
Apoil
PA
Roubinet
F
Blancher
A
Gorilla RH-like genes and antigens.
Immunogenetics.
49
1999
125
133
82
Blancher
A
Klein
J
Socha
WW
Molecular Biology and Evolution of Blood Group and MHC Antigens in Primates.
1997
Springer-Verlag
Berlin
83
Carritt
B
Kemp
TJ
Poulter
M
Evolution of the human RH (rhesus) blood group genes: a 50 year old prediction (partially) fulfilled.
Hum Mol Genet.
6
1997
843
850
84
Colin
Y
Chérif-Zahar
B
Le Van Kim
C
Raynal
V
Van Huffel
V
Cartron
J-P
Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis.
Blood.
78
1991
2747
2752
85
Avent ND. The Rhesus blood group system: insights from recent advances in molecular biology. Transfus Med Rev. In press.
86
Flegel
WA
Wagner
FF
Müller
TH
Gassner
C
Rh phenotype prediction by DNA typing and its application to practice.
Transfus Med.
8
1998
281
302
87
Umenishi
F
Kajii
E
Ikemoto
S
Molecular analysis of Rh polypeptides in a family with RhD-positive and RhD-negative phenotypes.
Biochem J.
299
1994
207
211
88
Hyland
CA
Wolter
LC
Saul
A
Three unrelated Rh D gene polymorphisms identified among blood donors with Rhesus CCee (r'r') phenotypes.
Blood.
84
1994
321
324
89
Daniels
G
Green
C
Smart
E
Differences between RhD-negative Africans and RhD-negative Europeans.
Lancet.
350
1997
862
863
90
Rouillac
C
Gane
P
Cartron
J
Le Pennec
PY
Cartron
JP
Colin
Y
Molecular basis of the altered antigenic expression of RhD in weak D (Du) and RhC/e in RN phenotypes.
Blood.
87
1996
4853
4861
91
Legler
TJ
Maas
JH
Blaschke
V
et al. 
RHD genotyping in weak D phenotypes by multiple polymerase chain reactions.
Transfusion.
38
1998
434
440
92
Wagner
FF
Gassner
C
Müller
TH
Schönitzer
D
Schunter
F
Flegel
WA
Molecular basis of weak D phenotypes.
Blood.
93
1999
385
393
93
Simsek
S
de Jong
CA
Cuijpers
HT
et al. 
Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of of E/e and C/c polymorphisms.
Vox Sang.
67
1994
203
209
94
Avent
ND
Daniels
GL
Martin
PG
Green
CA
Finning
KM
Warner
KM
Molecular investigation of the Rh C/c polymorphism [abstract].
Transfus Med.
7(suppl 1)
1997
18
95
Blancher
A
Socha
WW
The Rhesus system.
Molecular Biology and Evolution of Blood Group and MHC Antigens in Primates.
Blancher
A
Klein
J
Socha
WW
1997
147
218
Springer-Verlag
Berlin
96
Westhoff
CM
Silberstein
LE
Wylie
DE
Apoil
P
Blancher
A
Characterization of the Rh locus of the New World capuchin (Cebus apella) monkey [abstract].
Transfusion
38(suppl)
1998
102S
97
Issitt
PD
An invited review: the Rh antigen e, its variants, and some closely related serological observations.
Immunohematology.
7
1991
29
36
98
Faas
BHW
Beckers
EAM
Wildoer
P
et al. 
Molecular background of VS and weak C expression in blacks.
Transfusion.
37
1997
38
44
99
Steers
F
Wallace
M
Johnson
P
Carritt
B
Daniels
G
Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA.
Br J Haematol.
94
1996
417
421
100
Huang
C-H
Reid
ME
Chen
Y
Novaretti
M
Deletion of Arg229 in RhCE polypeptide alters expression of RhE and CE-associated Rh6 [abstract].
Blood.
90(suppl 1)
1997
272a
101
Westhoff
CM
Silberstein
LE
Sipherd
B
et al. 
Altered “e” antigen expression associated with 16Cys in exon 1 of the RHce gene [abstract].
Transfusion.
38(Suppl)
1998
64S
102
Avent
ND
Finning
KM
Liu
W
Scott
ML
Molecular biology of partial D phenotypes.
Transfus Clin Biol.
3
1996
511
516
103
Noizat-Pirenne
F
Mouro
I
Gane
P
et al. 
Heterogeneity of blood group RhE variants revealed by serological analysis and molecular alteration of the RHCE gene and transcript.
Br J Haematol.
103
1998
429
436
104
Daniels
GL
Faas
BHW
Green
CA
et al. 
The VS and V blood group polymorphisms in Africans: a serological and molecular analysis.
Transfusion.
38
1998
951
958
105
Faas
BHW
Beckers
EAM
Simsek
S
et al. 
Involvement of Ser103 of the Rh polypeptides in G epitope formation.
Transfusion.
36
1996
506
511
106
Rouillac
C
Le Van Kim
C
Blancher
A
Roubinet
F
Cartron
J-P
Colin
Y
Lack of G blood group antigen in DIIIb erythrocytes is associated with segmental DNA exchange between RH genes.
Br J Haematol.
89
1995
424
426
107
Lomas
C
Mougey
R
Rh antigen D: variable expression in DVI phenotypes; a possible subdivision of category VI by a low frequency antigen [abstract].
Transfusion.
29(suppl)
1989
14S
108
Matassi
G
Chérif-Zahar
B
Mouro
I
Cartron
JP
Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the DVI phenotype.
Am J Hum Genet.
60
1997
808
817
109
Race
RR
Sanger
R
Selwyn
JG
A probable deletion in a human Rh chromosome.
Nature.
166
1950
520
521
110
Tippett
P
Lomas-Francis
C
Wallace
M
The Rh antigen D: partial D antigens and associated low incidence antigens.
Vox Sang.
70
1996
123
131
111
Reid
ME
Sausais
L
Zaroulis
CG
Mohandas
K
Coghlan
G
Lomas-Francis
C
Two examples of an inseparable antibody that reacts equally well with DW+ and Rh32+ red blood cells.
Vox Sang.
75
1998
230
233
112
Lomas
C
Tippett
P
Thompson
KM
Melamed
MD
Hughes-Jones
NC
Demonstration of seven epitopes on the Rh antigen D using human monoclonal anti-D antibodies and red cells from D categories.
Vox Sang.
57
1989
261
264
113
Lomas
C
McColl
K
Tippett
P
Further complexities of the Rh antigen D disclosed by testing category DII cells with monoclonal anti-D.
Transfus Med.
3
1993
67
69
114
Jones
J
Scott
ML
Voak
D
Monoclonal anti-D specificity and Rh D structure: criteria for selection of monoclonal anti-D reagents for routine typing of patients and donors.
Transfus Med.
5
1995
171
184
115
Scott
M
Rh serology—Coordinator's report.
Transfus Clin Biol.
3
1996
333
337
116
Apoil
PA
Reid
ME
Halverson
G
et al. 
A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblotting.
Br J Haematol.
98
1997
365
374
117
Cartron
J-P
Rouillac
C
Le Van Kim
C
Mouro
I
Colin
Y
Tentative model for the mapping of D epitopes on the RhD polypeptide.
Transfus Clin Biol.
3
1996
497
503
118
Scott
ML
Voak
D
Jones
JW
et al. 
A structural model for 30 Rh D epitopes based on serological and DNA sequence data from partial D phenotypes.
Transfus Clin Biol.
3
1996
391
396
119
Chang
TY
Siegel
DL
Genetic and immunological properties of phage-displayed human anti-Rh(D) antibodies: implications for Rh(D) epitope topology.
Blood.
91
1998
3066
3078
120
Liu
W
Smythe
JS
Scott
ML
Jones
JW
Voak
D
Avent
ND
Site-directed mutagenesis of the human D antigen: definition of D epitopes on the sixth external domain of the D protein expressed on K562 cells.
Transfus.
39
1999
17
25
121
Zhu
A
Haller
S
Li
H
Chaudhuri
A
Blancher
A
Suyama
K
Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes.
J Biol Chem.
274
1999
5731
5737
122
Liu W, Avent ND, Jones JW, Scott ML, Voak D. D epitope localisation using site directed mutagenesis and expression of Rh mutant constructs in K562 cells. Blood. In press.
123
Mollison
PL
Engelfriet
CP
Contreras
M
Blood Transfusion in Clinical Medicine.
1997
Blackwell Science
Oxford, England
124
Standards Committee of American Association of Blood Banks
Standards for Blood Banks and Transfusion Services.
1999
American Associations of Blood Banks
Bethesda, MD
125
Petz
LD
Garratty
G
Acquired Immune Hemolytic Anemias.
1980
Churchill Livingstone
New York
126
Sloan
SR
Silberstein
LE
Transfusion in the face of autoantibodies.
Red Cell Transfusion: A Practical Guide.
Reid
ME
Nance
SJ
1998
55
70
Humana Press
Totowa, NJ
127
Vengelen-Tyler
V
Technical Manual.
1999
American Association of Blood Banks
Bethesda, MD
128
Lacey
PA
Caskey
CR
Werner
DJ
Moulds
JJ
Fatal hemolytic disease of a newborn due to anti-D in an Rh-positive Du variant mother.
Transfusion.
23
1983
91
94
129
Reid
ME
Halverson
GR
Roubinet
F
Apoil
PA
Blancher
A
Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DVI antigen from those with other partial D antigens or weak D antigens.
Immunohematology.
14
1998
89
93
130
Bowman
JM
The development and use of polyclonal prophylactic anti-D IgG.
Biotest Bull.
5
1997
503
510
131
Giblett
ER
Blood group alloantibodies: an assessment of some laboratory practices.
Transfusion.
4
1977
299
308
132
Hoeltge
GA
Domen
RE
Rybicki
LA
Schaffer
PA
Multiple red cell transfusions and alloimmunization: experience with 6996 antibodies detected in a total of 159,262 patients from 1985 to 1993.
Arch Pathol Lab Med.
119
1995
42
45
133
Heddle
NM
Klama
L
Frassetto
R
O'Hoski
P
Leaman
B
A retrospective study to determine the risk of red cell alloimmunization and transfusion during pregnancy.
Transfusion.
33
1993
217
220
134
Zupanska
B
Assays to predict the clinical significance of blood group antibodies.
Curr Opin Hematol.
5
1998
412
416
135
Ulm
B
Svolba
G
Ulm
MR
Bernaschek
G
Panzer
S
Male fetuses are particularly affected by maternal alloimmunization to D antigen.
Transfusion.
39
1999
169
173
136
Lubenko
A
Contreras
M
Habash
J
Should anti-Rh immunoglobulin be given D variant women?
Br J Haematol.
72
1989
429
433
137
Bussel
JB
Graziano
JN
Kimberly
RP
Pahwa
S
Aledort
LM
Intravenous anti-D treatment of immune thrombocytopenic purpura: analysis of efficacy, toxicity, and mechanism of effect [comments].
Blood.
77
1991
1884
1893
138
Scaradavou
A
Woo
B
Woloski
BMR
et al. 
Intravenous anti-D treatment of immune thrombocytopenic purpura: experience in 272 patients.
Blood.
89
1997
2689
2700
139
Kumpel
BM
Goodrick
MJ
Pamphilon
DH
et al. 
Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers.
Blood.
86
1995
1701
1709
140
Maaskant-van Wijk
PA
Faas
BH
de Ruijter
JA
et al. 
Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons.
Transfusion.
38
1998
1015
1021
141
Pope
J
Navarrete
C
Warwick
R
Contreras
M
Multiplex PCR analysis of RhD gene.
Lancet.
346
1995
375
376
142
Stoerker
J
Hurwitz
C
Rose
NC
Silberstein
LE
Highsmith
WE
Heteroduplex generator in analysis of Rh blood group alleles.
Clin Chem.
42
1996
356
360
143
Rose
NC
Hurwitz
C
Silberstein
L
Andovalu
R
Stoerker
J
Prenatal analysis of rhesus CcDEe blood groups by heteroduplex generator.
Am J Obstet Gynecol.
176
1997
1084
1089
144
Aubin
JT
Kim
CL
Mouro
I
et al. 
Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification.
Br J Haematol.
98
1997
356
364
145
Gassner
C
Schmarda
A
Kilga-Nogler
S
et al. 
RHD/CE typing by polymerase chain reaction using sequence-specific primers.
Transfusion.
37
1997
1020
1026
146
Bennett
PR
Le Van Kim
C
Colin
Y
et al. 
Prenatal determination of fetal RhD type by DNA amplification.
N Engl J Med.
329
1993
607
610
147
Wolter
LC
Hyland
CA
Saul
A
Rhesus D: genotyping using polymerase chain reaction.
Blood.
82
1993
1682
1683
148
Mayne
K
Bowell
P
Woodward
T
Sibley
C
Lomas
C
Tippett
P
Rh immunization by the partial D antigen of category DVa.
Br J Haematol.
76
1990
537
539
149
Andrews
KT
Wolter
LC
Saul
A
Hyland
CA
The RhD-trait in a white patient with the RhCCee phenotype attributed to a four-nucleotide deletion in the RHD gene.
Blood.
92
1998
1839
1840
150
Singleton
BK
Green
CA
Avent
ND
et al. 
An RHD pseudogene containing a 37 bp duplication and a nonsense mutation is present in most Africans with the Rh D-negative blood group phenotype.
Blood.
95
2000
12
18
151
Fukumori
Y
Hori
Y
Ohnoki
S
et al. 
Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers.
Transfus Med.
7
1997
227
231
152
Sun
C-F
Chou
CS
Lai
NC
Wang
WT
RHD gene polymorphisms among RhD-negative Chinese in Taiwan.
Vox Sang.
75
1998
52
57
153
Chang
JG
Wang
JC
Yang
TY
et al. 
Human RhDel is caused by a deletion of 1,013 bp between introns 8 and 9 including exon 9 of RHD gene.
Blood.
92
1998
2602
2604
154
Reed
W
Lee
T-H
Busch
MP
Vichinsky
EP
Sample suitability for the detection of minor leukocyte populations by polymerase chain reaction (PCR) [abstract].
Transfusion.
37(suppl)
1997
107S
155
Rios
M
Storry
JR
Hue-Roye
K
Reid
ME
Castilho
LL
Pelegrino
JJ
Incidence of partial D, DIIIa, in Black donors as determined by PCR-RFLP analysis [abstract].
Transfusion.
38(suppl)
1998
63S
156
Omi
T
Takahashi
J
Tsudo
N
et al. 
The genomic organization of the partial D category DVa: the presence of a new partial D associated with the DVa phenotype.
Biochem Biophys Res Commun.
254
1999
786
794
157
Le Van Kim
C
Mouro
I
Brossard
Y
Chavinie
J
Cartron
J-P
Colin
Y
PCR-based determination of Rhc and RhE status of fetuses at risk of Rhc and RhE haemolytic disease.
Br J Haematol.
88
1994
193
195
158
Faas
BHW
Simsek
S
Bleeker
PM
et al. 
Rh E/e genotyping by allele-specific primer amplification.
Blood.
85
1995
829
832
159
Poulter
M
Kemp
TJ
Carritt
B
DNA-based rhesus typing: simultaneous determination of RHC and RHD status using the polymerase chain reaction.
Vox Sang.
70
1996
164
168
160
Lo
YM
Bowell
PJ
Selinger
M
et al. 
Prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers.
Lancet.
341
1993
1147
1148
161
Lo
YMD
Corbetta
N
Chamberlain
PF
et al. 
Presence of fetal DNA in maternal plasma and serum.
Lancet.
350
1997
485
487
162
Lo
YMD
Hjelm
NM
Fidler
C
et al. 
Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma.
N Engl J Med.
339
1998
1734
1738
163
Faas
BH
Beuling
EA
Christiaens
GC
von dem Borne
AE
van der Schoot
CE
Detection of fetal RHD-specific sequences in maternal plasma.
Lancet.
352
1998
1196
164
Adinolfi
M
Sherlock
J
Kemp
T
et al. 
Prenatal detection of fetal RhD DNA sequences in transcervical samples.
Lancet.
345
1995
318
319
165
Bennett
PR
Overton
TG
Lighten
AD
Fisk
NM
Rhesus D typing.
Lancet.
345
1995
661
662
166
Kingdom
J
Sherlock
J
Rodeck
C
Adinolfi
M
Detection of trophoblast cells in transcervical samples collected by lavage or cytobrush.
Obstet Gynecol.
86
1995
283
288
167
Hamlington
J
Cunningham
J
Mason
G
Mueller
R
Miller
D
Prenatal detection of rhesus D genotype.
Lancet.
349
1997
540
168
Cheung
M-C
Goldberg
JD
Kan
YW
Prenatal diagnosis of sickle cell anaemia and thalassaemia by analysis of fetal cells in maternal blood.
Nat Genet.
14
1996
264
268
169
Bianchi
DW
Zickwolf
GK
Weil
GJ
Sylvester
S
DeMaria
MA
Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.
Proc Natl Acad Sci U S A.
93
1996
705
708
170
Seidl
S
Spielmann
W
Martin
H
Two siblings with Rhnull disease.
Vox Sang.
23
1972
182
189
171
Ballas
SK
Clark
MR
Mohandas
N
et al. 
Red cell membrane and cation deficiency in Rh null syndrome.
Blood.
63
1989
1046
1055
172
Nash
R
Shojania
AM
Hematological aspect of Rh deficiency syndrome: a case report and a review of the literature.
Am J Hematol.
24
1987
267
275
173
Kuypers
F
van Linde-Sibenius-Trip
M
Roelofsen
B
Tanner
MJ
Anstee
DJ
Op den Kamp
JA
Rhnull human erythrocytes have an abnormal membrane phospholipid organization.
Biochem J.
221
1984
931
934
174
Chérif-Zahar
B
Raynal
V
Gane
P
et al. 
Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency.
Nat Genet.
12
1996
168
173
175
Hyland
CA
Chérif-Zahar
B
Cowley
N
et al. 
A novel single missense mutation identified along the RH50 gene in a composite heterozygous Rhnull blood donor of the regulator type.
Blood.
91
1998
1458
1463
176
Huang
C-H
Chen
Y
Reid
ME
Seidl
C
Rhnull disease: the amorph type results from a novel double mutation in RhCe gene on D-negative background.
Blood.
92
1988
664
671
177
Chérif-Zahar
B
Matassi
G
Raynal
V
et al. 
Rh-deficiency of the regulator type caused by splicing mutations in the human RH50 gene.
Blood.
92
1998
2535
2540
178
Reid
ME
Bird
GW
Associations between human red cell blood group antigens and disease.
Transfus Med Rev.
4
1990
47
55
179
Chérif-Zahar
B
Bony
V
Steffensen
R
et al. 
Shift from Rh-positive to Rh-negative phenotype caused by a somatic mutation within the RHD gene in a patient with chronic myelocytic leukaemia.
Br J Haematol.
102
1998
1263
1270
180
Chérif-Zahar
B
Mattei
MG
Le Van Kim
C
Bailly
P
Cartron
J-P
Colin
Y
Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization.
Hum Genet.
86
1991
398
400
181
Hermand
P
Gane
P
Mattei
MG
Sistonen
P
Cartron
J-P
Bailly
P
Molecular basis and expression of the LWa/LWb blood group polymorphism.
Blood.
86
1995
1590
1594
182
Zelinski
T
Coghlan
G
White
L
Philipps
S
The Diego blood group locus is located on chromosome 17q.
Genomics.
17
1993
665
666
183
Dahr
W
Kordowicz
M
Moulds
J
Gielen
W
Lebeck
L
Krüger
J
Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes.
Blut.
54
1987
13
24
184
Huang
C-H
Alteration of RH gene structure and expression in human dCCee and DCW- red blood cells: phenotypic homozygosity versus genotypic heterozygosity.
Blood.
88
1996
2326
2333
185
Noizat-Pirenne
F
Mouro
I
Gane
P
et al. 
Molecular analysis of selected Rh variants.
Transfus Clin Biol.
3
1996
517
519
186
Chérif-Zahar
B
Raynal
V
Cartron
JP
Lack of RHCE-encoded proteins in the D—phenotype may result from homologous recombination between the two RH genes.
Blood.
88
1996
1518
1520
187
Cheng G-J, Chen Y, Reid ME, Huang C-H. Evans antigen: a new hybrid structure occurring on background of D•• andD—Rh complexes. Vox Sang. In press.
188
Huang C-H, Liu PZ, Cheng JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol. In press.
189
Chérif-Zahar
B
Matassi
G
Raynal
V
et al. 
Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type.
Blood.
92
1998
639
646
190
Mouro
I
Le Van Kim
C
Rouillac
C
et al. 
Rearrangements of the blood group RhD gene associated with the DVI category phenotype.
Blood.
83
1994
1129
1135
191
Flegel
WA
Wagner
FF
The frequency of RHD protein variants in Caucasians [abstract].
Transfus Clin Biol.
3
1996
10S
192
Avent
ND
Jones
JW
Liu
W
et al. 
Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein.
Br J Haematol.
97
1997
366
371
193
Rouillac
C
Le Van Kim
C
Beolet
M
Cartron
J-P
Colin
Y
Leu110Pro substitution in the RhD polypeptide is responsible for the DVII category blood group phenotype.
Am J Hematol.
49
1995
87
88
194
Rouillac
C
Colin
Y
Hughes-Jones
NC
et al. 
Transcript analysis of D category phenotypes predicts hybrid Rh D-CE-D proteins associated with alteration of D epitopes.
Blood.
85
1995
2937
2944
195
Wagner
FF
Gassner
C
Eicher
N
Lonicer
C
Flegel
WA
Characterization of D category IV type IV, DFW, and DNB [abstract].
Transfusion.
38(suppl)
1998
63S
196
Jones
JW
Finning
K
Mattock
R
et al. 
The serological profile and molecular basis of a new partial D phenotype, DHR.
Vox Sang.
73
1997
252
256
197
Huang
C-H
Chen
Y
Reid
M
Human DIIIa erythrocytes: RhD protein is associated with multiple dispersed amino acid variations.
Am J Hematol.
55
1997
139
145
198
Beckers
EA
Faas
BH
Ligthart
P
et al. 
Characterization of the hybrid RHD gene leading to the partial D category IIIc phenotype.
Transfusion.
36
1996
567
574
199
Avent
ND
Liu
W
Jones
JW
et al. 
Molecular analysis of Rh transcripts and polypeptides from individuals expressing the DVI variant phenotype: an RHD gene deletion event does not generate all DVIccEe phenotypes.
Blood.
89
1997
1779
1786
200
Huang
C-H
Human DVI category erythrocytes: correlation of the phenotype with a novel hybrid RhD-CE-D gene but not an internally deleted RhD gene.
Blood.
89
1997
1834
1835
201
Faas
BHW
Beckers
EAM
Maaskant-van Wijk
PA
Overbeeke
MAM
Van Rhenen
DJ
Von dem Borne
AEGK
Molecular characterization of qualitative Rh variants.
Biotest Bull.
5
1997
439
449
202
Beckers
EAM
Faas
BHW
Simsek
S
et al. 
The genetic basis of a new partial D antigen: DDBT.
Br J Haematol.
93
1996
720
727
203
Huang C-H, Chen Y, Reid ME, Okubo Y. Evidence for a separate genetic origin of the partial D phenotype DBT in a Japanese family. Transfusion. In press.
204
Hemker
M
Ligthart
PC
Faas
BHW
et al. 
ARRO-I: a new partial D phenotype involving exon 4 and 5 [abstact].
Vox Sang.
74(suppl 1)
1998
1331
205
Pisacka
M
Vytisková
J
Hejná
J
Gassner
C
A new variant of Rh(D) antigen: revealed by reactions of anti-ep12 monoclonal antibodies and lacking exon 5 D-specific reaction of exon-scanning RHD/CE PCR-SSP [abstract].
Vox Sang.
74(suppl 1)
1998
1332
206
Faas
BHW
Ligthart
PC
Lomas-Francis
C
Overbeeke
MAM
von dem Borne
AEG
van der Schoot
CE
Involvement of Gly96 in the formation of the Rh26 epitope.
Transfusion.
37
1997
1123
1130
207
Beckers
EAM
Faas
BHW
Von dem Borne
AEGK
Overbeeke
MAM
Van Rhenen
DJ
van der Schoot
CE
The R0HarRh:33 phenotype results from substitution of exon 5 of the RHCE gene by the corresponding exon of the RHD gene.
Br J Haematol.
92
1996
751
757
208
Mouro
I
Colin
Y
Gane
P
et al. 
Molecular analysis of blood group Rh transcripts from a rGr variant.
Br J Haematol.
93
1996
472
474
209
Huang
CH
Cheng
GJ
Reid
ME
Chen
Y
Rh mod syndrome: a family study of the translation-initiator mutation in the Rh50 glycoprotein gene.
Am J Hum Genet.
64
1999
108
117
210
Huang C-H, Cheng G, Liu Z, et al. Molecular basis for Rhnull syndrome: identification of three new missense mutations in the Rh50 glycoprotein gene. Am J Hematol. In press.
211
Okuda
H
Suganuma
H
Tsudo
N
Omi
T
Iwamoto
S
Kajii
E
Sequence analysis of the spacer region between the RHD and RHCE genes.
Biochem Biophys Res Commun.
263
1999
378
383