Rh, Kell, Duffy, and Kidd Antigens and Antibodies

Chapter 7

Rh, Kell, Duffy, and Kidd Antigens and Antibodies Connie M. Westhoff

●

Marion E. Reid

This chapter summarizes four clinically significant blood group systems that are defined by protein polymorphisms. The proteins that carry these blood group antigens were difficult to isolate because they are integral membrane proteins present as minor components of the total red blood cell (RBC) protein. Biochemical techniques developed in the 1970s and 1980s enabled their purification and partial amino acid sequencing. In the 1990s, the protein sequence data were used to construct nucleic acid probes for amplification and screening of bone marrow cDNA libraries to isolate the genes. In the past decade, there has been a considerable increase in the amount of information about these blood group antigens; principally, the development and use of the polymerase chain reaction (PCR) has resulted in the rapid elucidation of the molecular basis for the antigens and phenotypes. In addition, the structure and function of these membrane proteins is an active area of investigation. II 80

Rh BLOOD GROUP SYSTEM History The Rh system is second only to the ABO system in importance in transfusion medicine. Rh antigens, especially D, are highly immunogenic and can cause hemolytic disease of the newborn (HDN) and severe transfusion reactions. HDN was first described by a French midwife in 1609 in a set of twins, of whom one was hydropic and stillborn, and the other was jaundiced and died of kernicterus.1,2 In 1939, Levine and Stetson3 described a woman who delivered a stillborn fetus and suffered a severe hemolytic reaction when transfused with blood from her husband. Her serum agglutinated the RBCs of her husband and 80 of 104 ABO-compatible donors. Subsequently, in 1941, Levine and colleagues4 correctly concluded that the mother had been immunized by the fetus, which carried an antigen inherited from the father, and suggested that the cause of the erythroblastosis fetalis was maternal antibody in the fetal circulation. Attempts to immunize rabbits against this new antigen were not successful.1 Levine and colleagues did not name the antigen.5 Meanwhile, Landsteiner and Wiener, in an effort to discover additional blood groups, injected rabbits and guinea pigs with rhesus monkey RBCs. The antiserum agglutinated not only rhesus cells but also the RBCs of 85% of a group of white subjects from New York, whom the researchers called Rh positive; the remaining 15% were Rh negative.1 Because the anti-Rhesus appeared to have reactivity indistinguishable

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from the maternal antibody reported by Levine and Stetson, the antigen responsible for HDN was named Rh. Later it was realized that the rabbit antiserum was not recognizing the same antigen but was detecting an antigen found in greater amounts on Rh-positive than on Rh-negative RBCs.6 This antigen was named LW for Landsteiner and Wiener,1 and the original human specificity became known as anti-D. As early as 1941, it was obvious that Rh was not a simple single antigen system. Fisher named the C and c antigens (A and B had been used for ABO) on the basis of the reactivity of two antibodies that recognized antithetical antigens, and used the next letters of the alphabet, D and E, to define antigens recognized by two additional antibodies.1 Anti-e, which recognized the e antigen, was identified in 1945.7

Nomenclature The Rh system has long been acknowledged as one of the most complex blood group systems because of its large number of antigens (45) and the heterogeneity of its antibodies. The introduction of two different Rh nomenclatures reflected the differences in opinion concerning the number of genes that encoded these antigens. The Fisher-Race nomenclature was based on the premise that three closely linked genes — C/c, E/e, and D — were responsible, whereas the Wiener nomenclature (Rh-Hr) was based on the belief that a single gene encoded one agglutinogen that carried several blood group factors. Even though neither theory was correct (there are two genes, RHD and RHCE, correctly proposed by Tippett8), the Fisher-Race designation (CDE) for haplotypes is often preferred for written communication, and a modified version of Wiener’s nomenclature (the original form is nearly obsolete) is preferred for spoken communication (Table 7–1). A capital “R” indicates that D is present, and a lowercase “r” (or “little r”) indicates that it is not. The C or c and E or e Rh antigens carried with D are represented by subscripts: 1 for Ce (R1), 2 for cE (R2), 0 for ce (R0), and Z for CE (Rz). The CcEe antigens present without D (r) are represented by superscript symbols: “prime” for Ce (r’), “double-prime” for cE (r”) and “y” for CE (ry) (see Table 7–1). The “R” versus “r” terminology allows one to convey the common Rh antigens present on one chromosome in a single term (a phenotype). Dashes are used to represent missing antigens of the rare deletion (or CE-depleted) phenotypes; for example, D– – (referred to as D dash, dash) lacks C/c and E/e antigens. In 1962, Rosenfield and associates9 introduced numerical designations for the Rh antigens to more accurately represent

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Nomenclature and Prevalence for Rh Haplotypes Prevalence (%)

Haplotype Based on Antigens Present

Shorthand for Haplotype

White

DCe DcE Dce DCE ce Ce cE CE

R1 R2 R0 RZ r r’ r” ry

42 14 4 <0.01 37 2 1 <0.01

African American 17 11 44 <0.01 26 2 <0.01 <0.01

Asian 70 21 3 1 3 2 <0.01 <0.01

the serologic data, to be free of genetic interpretation, and to be more compatible for computer use (Table 7–2). However, this numerical nomenclature, with a few exceptions (Rh17, Rh32, Rh33), is not widely used in the clinical laboratory.

or lack of expression of, RhAG results in a lack of Rh antigen expression (Rh-null) or a marked reduction of Rh antigen expression (Rh-mod).17 RhAG has one N-glycan chain that also carries ABO and Ii specificities (Fig. 7–1).

Terminology

Rh-null and the Rh-Core Complex

Current Rh terminology distinguishes the genes and the proteins from the antigens, which are referred to by the letter designations, D, C, c, E, e, and so on. To indicate the RH genes, capital letters, with or without italics, are used (i.e., RHD, RHCE, and RHAG). The different alleles of the RHCE gene are designated RHce, RHCe, RHcE, according to which antigens they encode. In contrast, the proteins are designated RhD, RhCE (or according to the specific antigens they carry, Rhce, RhCe, or RhcE), and RhAG. RH haplotypes are designated Dce, DCe, DcE, etc., or ce, Ce, cE when referring to a specific CE haplotype.

Rh-null erythrocytes, which lack expression of Rh antigens, are stomatocytic and spherocytic, and affected individuals have variable degrees of anemia.18,19 The phenotype is rare, occurring on two different genetic backgrounds; the regulator type, caused by mutations in the RHAG gene, and the amorph type, which maps to the RH locus. The amorph Rh-null results from mutations in RHCE on a deleted RHD background.20 Regulator RBCs express no Rh and RhAG proteins, whereas amorph type RBCs have no Rh proteins but have reduced (20% of normal) RhAG. Rh and RhAG proteins are associated in the membrane, possibly as a tetramer consisting of two molecules of each as a core complex,21 although the precise configuration is not yet known. Evidence that other proteins interact with this Rh-core complex comes from observations that Rh-null RBCs have reduced expression of CD47 (an integrin-associated protein) and of glycophorin B (a sialoglycoprotein that carries S or s and U antigens). Rh-null RBCs also lack LW (ICAM-4), a glycoprotein of approximate Mr 42,000 that belongs to the family of intercellular adhesion molecules. Band 3 (the red cell anion exchanger AE1) may also be associated with the Rh complex.22 The Rh complex is linked to the membrane skeleton via Rh/RhAG–ankyrin interaction23 and CD47–protein 4.2 association.24 Some mutations underlying Rh-null disease involve potential contact sites between RhAG and RhCE/RhD and cytoskeleton ankyrin, which may explain the morphologic defect in these erythrocytes.23

Proteins Despite the clinical importance of the Rh blood groups, the RBC membrane proteins that carry them were identified only in the late 1980s.10,11 The extremely hydrophobic nature of these multipass transmembrane proteins made the biochemical isolation difficult and limited progress in their characterization until the genes were cloned. The Rh proteins, designated RhD and RhCE, are 417amino acid, nonglycosylated proteins; one carries the D antigen, and the other carries various combinations of the CE antigens (ce, cE, Ce, or CE).12–14 RhD differs from RhCE by 32 to 35 amino acids (depending on which form of RhCE is present), and both are predicted to span the membrane 12 times. They migrate in SDS-PAGE (sodium dodecyl sulfate– polyacrylamide gel electrophoresis) gels with an approximate molecular weight ratio (Mr) of 30,000 to 32,000, and hence are sometimes referred to as the Rh30 proteins. They are covalently linked to fatty acids (palmitate) in the lipid bilayer.15 A 409-amino acid glycosylated protein that coprecipitates with RhD and RhCE proteins and migrates with an approximate Mr of 40,000 to 100,000 is called RhAG (Rh-associated glycoprotein) or Rh50 glycoprotein to reflect its apparent molecular weight.16 RhAG (Rh50) shares 37% amino acid identity with the RhD and RhCE proteins and has the same predicted membrane topology. RhAG is not polymorphic and does not carry Rh antigens. It is important for targeting the RhD and RhCE to the membrane, because mutations in,

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RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

Table 7–1

7 81

Genes Two genes, designated RHD and RHCE, encode the Rh proteins. Rh-positive individuals have both genes, whereas most Rh-negative white people have only the RHCE gene.25 The genes are 97% identical. Each gene has 10 exons and is the result of a gene duplication on chromosome 1p34– p36.26 A comprehensive diagram of the intron–exon structure of RHD and RHCE can be found in the review by Avent and Reid.27 The single gene, RHAG, located at chromosome 6p11– p21.1 encodes RhAG. RHAG is 47% identical to the RH genes and also has 10 exons.16,28

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Table 7–2 Rosenfield Numerical Terminology for Rh Antigens Numerical Term

ISBT Symbol

Rh1 Rh2 Rh3 Rh4 Rh5 Rh6 Rh7 Rh8 Rh9 Rh10 Rh11 Rh12* Rh17 Rh18 Rh19 Rh20 Rh21 Rh22 Rh23* Rh26 Rh27 Rh28 Rh29 Rh30 Rh31 Rh32 Rh33 Rh34 Rh35 Rh36 Rh37 Rh39 Rh40 Rh41 Rh42 Rh43 Rh44 Rh45 Rh46 Rh47 Rh48 Rh49 Rh50 Rh51 Rh52 Rh53 Rh54 Rh55 Rh56

D C E c e ce or f Ce CW CX V EW G Hr0† Hr hrs VS CG CE DW c-like cE hrH Rh29 Goa hrB Rh32‡ Rh33§ HrB Rh35|| Bea Evans Rh39 Tar Rh41 Rh42 Crawford Nou Riv Sec Dav JAL STEM FPTT MAR BARC JAHK DAK LOCR CENR

*

Rh13 through Rh16, Rh24, and Rh25 are obsolete. High-incidence antigen; the antibody is made by D– –/D– and similar phenotypes. =N ‡ Low-incidence antigen expressed by R and DBT phenotypes. Har § =N Originally described on R0 phenotype, but also found on R and DVIa (C)−, R0JOH, and R1Lisa. || Low-incidence antigen on D(C)(e) cells. ISBT, International Society for Blood Transfusion. †

Antigens As previously described, the major Rh antigens are D, C, c, E, and e. The many other Rh antigens (see Table 7–2) define compound antigens in cis (e.g., f (ce), Ce, and CE), low-incidence antigens arising from partial D hybrid proteins (e.g., Dw, Goa, BARC), high-incidence antigens, and other variant antigens. The molecular bases of most Rh antigens have been determined.

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RhD Extracellular Lipid Bilayer Intracellular NH2

RhCE

417

C/c (Ser/Pro) 103

E/e (Pro/Ala) 226

NH2 417 RhAG Rh-associated glycoprotein (Rh50)

NH2

409 Figure 7–1 Predicted 12-transmembrane domain model of the RhD, RhCE, and RhAG proteins in the red blood cell membrane. The amino acid differences between RhD and RhCE are shown as symbols. The eight extracellular differences between RhD and RhCE are indicated as open circles. The zigzag lines represent the location of possible palmitoylation sites. Positions 103 and 226 in RhCE that are critical for C/c and E/e expression, respectively, are indicated as black circles. The Nglycan on the first extracellular loop of the Rh-associated glycoprotein is indicated by the branched structure.

Studies to estimate the number of D, C/c, and E/e antigen sites on RBCs found differences between Rh phenotypes. The number of D antigens ranges from 10,000 on Dce/ce RBCs to 33,000 on DcE/DcE. The number of C, c, and e antigens per RBC varies from 8500 to 85,000.6 Because C or c and E or e are carried on the same protein, their numbers should be equivalent. The equivalency was not always demonstrated in these studies, reflecting the inherent difficulty in the use of radiolabeled polyclonal antiserum to estimate antigen numbers. Results of tests with monoclonal antibodies to high-incidence Rh antigens suggest that the total number of Rh proteins (RhD and RhCE) per RBC is 100,000 to 200,000. The number of RhAG is also estimated to be 100,000 to 200,000,29 consistent with predictions that Rh and RhAG may be present in the membrane as a tetramer of two molecules of each.21 D Antigen Rh positive and Rh negative refer to the presence and absence, respectively, of the D antigen, which is the most immunogenic of the Rh antigens. The Rh-negative (D-negative) phenotype

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WEAK D (FORMERLY Du)

An estimated 0.2% to 1% of white persons (and a greater number of African Americans) have reduced expression of the D antigen, which is characterized serologically as failure of the RBCs to agglutinate directly with anti-D typing reagents and requires the use of the indirect antiglobulin test (IAT) for detection. The number of samples classified as weak D depends on the characteristics of the typing reagent. These weak D antigens were previously referred to as Du, a term that has been abolished. The molecular basis of weak D expression is heterogeneous. In a large study of samples from Germany,36 expression of weak D was found to be associated with the presence of point mutations in RHD. More than 70% of the samples had a Val270Gly amino acid change, but a total of 16 different mutations were found in the initial study. Since then, a large number of different mutations have been associated with the weak D phenotype (42 to date).37 Collectively, the weak D phenotypes have amino acid changes predicted to be intracellular or in the transmembrane regions of the RhD protein and not on the outer surface of the RBC,36 suggesting that the mutations affect the efficiency of insertion and, therefore, the quantity of RhD protein in the membrane but do not affect D epitopes. The majority of individuals with a weak D phenotype can safely receive D-positive blood and do not make anti-D. However, two weak D types (type 4.2.2 and type 15) have been reported to make anti-D.38 These weak D individuals would more accurately be classified as partial-D. The serologic differentiation of weak D from partial D is not unequivocal. Importantly, donor center typing procedures must detect and label weak D RBC components as D-positive. A very weak form of D, designated Del, detected by absorption and elution of anti-D, has a high incidence in Hong Kong Chinese and Japanese persons. The Del phenotype most often results from a splice site mutation or deletion that results in the absence of amino acids encoded by exon 9 of RHD39 in Asians or a M295I mutation in whites. These

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RBCs type as D negative (even when tested using the IAT), and they are usually only recognized if they stimulate production of anti-D in a D-negative recipient.40,41 PARTIAL D ANTIGENS (D CATEGORIES OR D MOSAICS)

The D antigen has long been described as a mosaic on the basis of the observation that some Rh-positive individuals make alloanti-D when exposed to normal D antigen. It was hypothesized that the RBCs of these individuals lack some part of RhD and that they can produce antibodies to the missing portion. Molecular analysis has shown that this hypothesis is correct, but what was not predicted is that the missing portions of RHD are replaced by corresponding portions of RHCE (Fig. 7–2).27,42 Some replacements involve entire exons, and the novel sequence of amino acids generates new antigens (e.g., Dw, BARC, Rh32) (Table 7–3). In addition, several exon rearrangements can give rise to the same partial D category (e.g., DVI can result from type I, II, or III rearrangements; see Fig. 7–2). Other partial D antigens result from multiple amino acid conversions between RHCE and RHD, and some are the result of single point mutations in RHD (DVI, DMH, DFW; see Table 7–3). These point mutations are predicted to be located on an extracellular loop or portion of the RhD protein, in contrast to the weak D antigens already described, which are predicted to have mutations located in cytoplasmic or transmembrane regions. Individuals with partial D antigens can make anti-D and therefore ideally should receive D-negative donor blood. In practice, however, most are typed as D positive and are recognized only after they have made anti-D.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

occurs in 15% to 17% of white persons but is not common in other ethnic populations. The absence of D in people of European descent is primarily the result of deletion of the entire RHD gene and occurred on a Dce (R0) haplotype because the allele most often carried with the deletion is ce. However, rare D-negative white persons carry an RHD gene that is not expressed because of a premature stop codon,30 a 4-base pair (bp) insertion at the intron 3/exon 4 junction,31 point mutations, or RHD/CE hybrids.32,33 Most of these are associated with the uncommon Ce (r⬘) or cE (r⬘⬘) haplotypes. D-negative phenotypes in Asian or African persons, however, are most often caused by inactive or silent RHD rather than a complete gene deletion. Asian D-negative individuals occur with a frequency of less than 1%, and most carry mutant RHD genes associated with Ce,34 indicating that the gene mutations probably originated on a DCe (R1) haplotype. Only 3% to 7% of South African black persons are D negative, but 66% of this group have RHD genes that contain a 37-bp internal duplication,35 which results in a premature stop codon. The 37-bp insert RHD-pseudogene was also found in 24% of D-negative African Americans. In addition, 15% of the D-negative phenotypes in Africans result from a hybrid RHD-CE-Ds gene characterized by expression of VS, weak C and e, and no D antigen.35 Only 18% of D-negative Africans and 54% of D-negative African Americans completely lack RHD.

ELEVATED D

Several phenotypes, including D– –, Dc−, and DCW−, have an elevated expression of D antigen and no, weak, and variant CE antigens, respectively.6 They are caused by replacement of portions of RHCE by RHD,27,42 analogous to the partial D rearrangements already described. The additional RHD sequences in RHCE along with a normal RHD may explain the enhanced D and accounts for the reduced or missing CE antigens. Immunized people with these CE-depleted phenotypes make anti-Rh17.

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D EPITOPES EXPRESSED ON RHCE PROTEINS (R0HAR, ceCF, ceRT)

Some Rhce proteins carry D-specific amino acids that react with some monoclonal anti-D reagents, adding further complexity to D antigen typing. Two examples, R0Har (DHAR), found in individuals of German ancestry,43 and Crawford (ceCF, Rh43), found in individuals of African ancestry, deserve attention because of their strong reactivity (3+4+) with some FDA-licensed monoclonal reagents and lack of reactivity with others (including the weak D test) (Table 7–4). Individuals with RoHar do not have a RHD gene, but exon 5 of the RHCE gene is from RHD (see Fig. 7–2). Those that carry a Crawford allele, ceCF, also do not have a RHD gene, but have a Rhce protein with three amino acid changes, Trp16Cys, Leu245Val, and Gln233Glu. The latter two are Dspecific residues responsible for reactivity with a monoclonal anti-D (see Table 7–4 and Fig. 7–2).44,45 Individuals with R0Har or Crawford phenotypes make anti-D when stimulated46 (our unpublished observations), and should be considered Rh negative for transfusion and Rh immune globulin prophylaxis.

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Origin of the common RHCE genes RHD

RHCE (ce) 226A

103P G antigen

16W 16C Exon 2 from RHD

Point mutation 226P

103S Ce 16C G antigen

cE

Examples of some RHCE rearrangements

Examples of some RHD rearrangements RHCE

rG

No RHD

DIIIc

R0HAR

No RHD

DVa Type II

RHCE DW

ceCF

No RHD

N

R

RHCE

DVI Type I 16C

Q233E L245V

RHD

RHCE

DVI Type II BARC

EII

RHD

RHCE

DBT Type I Rh32

Figure 7–2 Top panel, Diagram of the RHD and RHCE genes, indicating the changes that resulted in the common RhCE polymorphisms. The shared exon 2 of RHD and RHCE (shown as a black box) explains the expression of G antigen on RhCe and RhD proteins. Bottom panel, Examples of some RHCE and RHD rearrangements.

II

Table 7–3 84

Molecular Basis of Some Rh Antigens, Partial D, and Unusual Phenotypes

Molecular Basis

Gene

Phenotype/Antigen/Genotype

Single point mutations

RHD

Partial D: DMH, DVII, D+G−, DFW, DHR, DVa, DHMi, DNU, DII, DNB, DHO Weak D (previously called Du) CX, CW, Rh−26, E type I, IV, V+,VS+ Partial D: DIIIa, DIVa, DVa, DFR type I E type III, IV, V+VS+ Partial D: DIIIb, DIIIc, DIVb, DVa, DVI, DFR type II, DBT r”G, (Ce)Ce, (C)ces VS+V− =N DHar, rG, R E type II DCW− D− −, D••, Dc− D•• D••

Multiple mutations (gene conversions) Rearranged gene(s) RHD RHCE RHD: RHCE

RHCE RHD RHCE RHD-CE-D RHCE-D-CE RHD-CE RHD-CE: RHCE-D RHD: RHCE-D-CE RHD: RHD-CE RHCE-D: RHD-CE

Table 7–4 Composition (IgM and IgG Clones) and Reactivity of FDA-Licensed Anti-D Reagents with Some Rh Variant RBCs That Can Result in D Typing Discrepancies Reagent

IgM Monoclonal

IgG

DVI

DBT

DHAR (Whites)

Crawford (Blacks)

Gammaclone Immucor Series 4 Immucor Series 5 Ortho BioClone Ortho Gel (ID-MTS) Polyclonal

GAMA401 MS201 Th28 MAD2 MS201

F8D8 monoclonal MS26 monoclonal MS26 monoclonal Polyclonal

Neg/Pos* Neg/Pos Neg/Pos Neg/Pos Neg Neg/Pos

Pos Pos Pos Neg/Pos Pos Neg/Pos

Pos Pos Vary/Pos Neg/Neg Pos Neg/Neg

Pos Neg Neg Neg Neg Neg/Neg

*

Result following slash denotes anti-D test result by the IAT, as permitted by the manufacturer.

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C/c and E/e Antigens There are four major allelic forms of RHCE: ce, Ce, cE, and CE.14 C and c differ by four amino acids: Cys16Trp (cysteine at residue 16 replaced by tryptophan) encoded by exon 1, and Ile60Leu, Ser68Asn, and Ser103Pro encoded by exon 2. Only residue 103 is predicted to be extracellular; it is located on the second loop of RhCE (see Fig. 7–1). The amino acids encoded by exon 2 of RHC are identical to those encoded by exon 2 of RHD. At the genomic level, RHCe appears to have arisen from transfer of exon 2 from RHD into RHce (see Fig. 7–2). The shared exon 2 explains the expression of the G antigen on both RhD and RhC proteins. E and e differ by one amino acid, Pro226Ala. This polymorphism, predicted to reside on the fourth extracellular loop of the protein (see Fig. 7–1), is encoded by exon 5. A single point mutation in RHce resulted in RHcE (see Fig. 7–2).14,48 CW and CX antigens result from single amino acid changes, encoded by exon 1, which are predicted to be located on the first extracellular loop of the RhCE protein.49 The antigens V and VS are expressed on RBCs of more than 30% of black persons. They are the result of a Leu245Val substitution located in the predicted eighth transmembrane segment of Rhce.50 The close location of the e antigen, Ala226 on the fourth extracellular loop, suggests that Leu245Val causes a local conformation change responsible for the weakened expression of e antigen in many black persons who are V and VS positive. The V−VS+ phenotype results from a Gly336Cys change on the 245Val background,51 and these alleles are referred to as ceS (Fig. 7–3). Loss of VS expression (the V+VS− phenotype) is associated with additional amino acid changes and is characteristic of the ceAR haplotype (see Fig. 7–3).

Other modifications of RHCE, which are uncommon, are the hybrids rG, RN, and several E/e variants (see Fig. 7–2). RN RBCs are found in people of African origin and type as e-weak (or negative) with polyclonal reagents, but are indistinguishable from “normal” e-positive RBCs with some monoclonal anti-e. The E variants—EI, EII, and EIII—result either from a point mutation (EI) or from gene conversion events that lead to replacement of several extracellular RhcE amino acids with RhD residues (EII and EIII) and loss of some E epitope expression.52 Category EIV RBCs, which have an amino acid substitution in an intracellular domain, do not lack E epitopes but have reduced E expression.53 The very rare RH:-26 results from a Gly96Ser transmembrane amino acid change that abolishes Rh26 and weakens c expression.54 VARIATION IN

e EXPRESSION AND SICKLE CELL PATIENTS

Variation in expression of the e antigen is not uncommon and can result from several different mutations. Deletion of the codon for Arg229, which is close to the Ala226 residue found in normal e expression,55 and substitution of a Cys residue for Trp at position 16 in the Rhce protein56 weaken expression of the antigen. Individuals of African ancestry often have RHce genes that encode variant e antigens. The RBCs type as e positive, but they often make alloantibodies with e-like specificities. The antibodies, designated anti-hrS, -hrB, -RH18, and -RH34, are difficult to identify serologically, are clinically significant, and have caused transfusion fatalities.57 The prevalence of e variants in this population, together with the incidence of sickle cell disease requiring transfusion support often provided by white donors with conventional RHce, make the occurrence of alloanti-e in these patients not uncommon. Some of the RH genetic backgrounds have now been defined58 and include the RHCE haplotypes shown in Figure 7–3. All encode the Trp16Cys difference in exon 1, and have additional changes, primarily localized to exon 5. The ceS allele is associated with RBCs that are hrB−, whereas individuals homozygous for ceAR, ceMO, ceEK, and ceBI alleles lack

RHD

RHCE

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

Lastly, an Rhce protein with an R154T mutation, designated ceRT, demonstrates weak reactivity with some anti-D monoclonal reagents, and the reactivity is enhanced at lower temperatures. Interestingly, this variant does not carry any D-specific amino acid but mimics a D-epitope (epD6) structure.47

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Figure 7–3 Diagram of the RHD and RHCE genes indicating changes often found in African backgrounds that complicate transfusion in sickle cell patients.

ceS D/Ce/D (D-negative, C-positive)

W16C

L245V

G336C

ceMO

DIIIa

(hrS–)

W16C

N152T T201R F223V DIII Type 5

V223F ceEK (hrS–)

W16C M238V M267K R263G

L62F T201R F223V A137V N152T

ceBI (hrS–)

DIVa L62F N152T

D350H

W16C M238V A273V L378V I306V

DAR T201R F223V

ceAR

I342T W16C M238V M267K L245V R263G

DOL M170T

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(hrB–)

(hrS–)

F223V

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the high-incidence hrS antigen (see Fig. 7–3). Importantly, because of the multiple molecular backgrounds responsible for the hrB− and hrS− phenotypes, some of which are not yet elucidated, the antibodies produced are not all serologically compatible. This explains why it is difficult to find compatible blood for patients with these antibodies, and often only rare deleted D– – RBCs appear compatible. As an additional complication, these variant RHce can often be inherited with an altered RHD (e.g., DAR, DAU, or DIIIA), so they can also make anti-D (see Fig. 7–3).

Anti-c, clinically the most important Rh antibody after anti-D, may cause severe HDN. Anti-C, anti-E, and anti-e do not often cause HDN, and when they do, it is usually mild.6,59 Autoantibodies to high-incidence Rh antigens often occur in the sera of patients with warm autoimmune hemolytic anemia and in some cases of drug-induced autoimmune hemolytic anemia. These autoantibodies are nonreactive with Rh-null cells.60

Rh Genotyping

A large number of IgM, direct-agglutinating, anti-D monoclonals have been generated by immortalizing human B lymphocytes in vitro with Epstein-Barr virus. D-typing reagents licensed for use in the United States are a blend of monoclonal IgM reactive at room temperature along with monoclonal or polyclonal IgG reactive by the IAT for the determination of weak D. Four different FDA-licensed reagents are available for tube testing and one for gel (see Table 7–4). All but two contain different IgM clones. The reactivity of each with variant D antigens may result in D typing discrepancies. Importantly, the IgM anti-D component of these reagents has been selected to not react with DVI RBCs (see Table 7–4). DVI is the most common partial D found in white populations, and these individuals often make anti-D when exposed to conventional D. Most agree that they should be classified as Rh-negative for transfusion or Rh immune globulin. The IgG component in these reagents reacts with DVI RBCs in the IAT phase (see Table 7–4). DVI RBCs can stimulate production of anti-D in an Rh-negative recipient and must be typed as Rh-positive as donors. The composition of current FDAlicensed reagents has prompted the movement away from weak D testing in the hospital and prenatal setting to classify individuals with DVI RBCs, or some of the other partial D phenotypes (see Table 7–4), as Rh negative. Monoclonal anti-D has not yet been tested clinically for its ability to prevent immunization after pregnancy but has been shown to suppress D immunization in D-negative male volunteers.61

RH genotyping is a useful means to determine the Rh phenotype of patients who have been recently transfused or whose RBCs are coated with IgG. RH genotyping in the prenatal setting can be used to determine paternal RHD zygosity and to predict fetal D status to prevent invasive and expensive monitoring for the possibility of HDN. The ethnic background of the parents is important to the design of the assay, because the different molecular events responsible for Dnegative phenotypes must be considered. Testing of samples from the parents limits the possibility of misinterpretation. RH genotyping can aid resolution of D typing discrepancies. These often are the result of differences in manufacturers’ reagents, but in the donor setting they can be FDA reportable. Genotyping can determine a specific weak D type, partial D category, or the presence of Del. Genotyping to detect the inheritance of altered or variant e alleles, which are often linked to variant RHD, aids resolution of antibody specificities and is helpful to determine alloantibody versus autoantibody specificity. This can be of significance, especially in sensitized sickle cell patients. Molecular genotyping can aid in the selection of compatible blood for transfusion and ultimately improve long-term transfusion support. Currently, these investigations can sometimes be cumbersome, often requiring complete gene sequencing. The development of automated, high-throughput platforms that sample many regions of both RHD and RHCE, along with detailed algorithms for accurate interpretation, are needed.

Antibodies Most Rh antibodies are IgG, subclasses IgG1 and IgG3 (IgG2 and IgG4 have also been detected), and some sera have an IgM component.59 Rh antibodies do not activate complement, although two rare exceptions have been reported. The lack of complement activation by Rh antibodies is thought to be due to the distance between antigens, but is probably due to a lack of mobility.6 Reactivity of Rh antibodies is enhanced by enzyme treatment of the test RBCs, and most react optimally at 37°C. Anti-D can cause severe transfusion reactions and severe HDN. Approximately 80% to 85% of D-negative persons make anti-D after exposure to D-positive RBCs. The lack of response in 15% may be due to antigen dose, recipient HLADR alleles, and other as yet unknown genetic factors. AntiD was the most common Rh antibody, but its incidence has greatly diminished with the prophylactic use of Rh immune globulin for prevention of HDN. ABO incompatibility between the mother and the fetus has a partial protective effect against immunization to D; this finding suggested the rationale for development of Rh immune globulin.59

Ch07-F039816.indd 86

Anti-D reagents

Expression Northern blot analysis indicated that Rh and RhAG messenger RNA (mRNA) are restricted to cells of erythroid and myeloid lineage, but reverse transcriptase-PCR (RT-PCR) found Rh mRNA splicing isoforms in B and T lymphocytes and monocytes.62 The significance of this observation is not yet known. During erythropoiesis, RhAG appears early (on CD34+ progenitors), but the Rh proteins appear later—RhCE first, followed by RhD.63 Evolution The RHD and RHCE genes arose from an early duplication of the erythrocyte RHAG gene. RH and RHAG have been investigated in nonhuman primates64,65 and rodents,66–68 and most, with the exception of gorillas and chimpanzees, have a RHAG and only one RH gene. The RH gene duplicated in some common ancestor of gorillas, chimpanzees, and humans, leading to RHCE and RHD. Chimpanzees and some gorillas have three RH genes, indicating that a third duplication has taken place in these species. Function The predicted membrane structures of Rh and RhAG suggest that they are transport proteins, and the analysis of their

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Summary The molecular basis of many of the Rh antigens has now been elucidated. The revelation that RhD and RhCE proteins differ by 35 amino acids explains why D antigen is so immunogenic. In addition, exchanges between RHD and RHCE, mainly by gene conversion, have generated many Rh polymorphisms. The proximity of the two genes on the same chromosome probably affords greater opportunity for exchange. This finding finally explains the myriad of antigens observed in the Rh blood group system and gives interesting insight into the evolutionary history of duplicated genes and the interactions that can take place between them. The complexity hampers molecular genotyping, and additional Rh variants are still being discovered. The challenge is to develop automated platforms that sample several regions of the genes for unequivocal interpretation. The discovery that members of the Rh family of proteins, RhAG, RhBG, and RhCG, are involved in ammonia/ammonium transport and are ideally positioned in key tissues essential for ammonium elimination is a significant finding because it was long assumed that the high membrane permeability of ammonia would obviate the need for specific transport pathways in mammalian cells. RBC membrane protein–cytoskeleton and protein– protein interactions are an active area of investigation. Although the major attachment sites between the erythro-

Ch07-F039816.indd 87

cyte cytoskeleton and the lipid bilayer are understood to be through glycophorin C and band 3, an additional attachment site mediated by the Rh complex explains the Rh-null defect. Additional studies are needed to determine the protein–protein associations and the dynamics of the assembly of the Rh-membrane complex.

THE KELL AND Kx SYSTEM History The Kell blood group system was discovered in 1946, just a few weeks after the introduction of the antiglobulin test. The RBCs from a newborn baby who was thought to be suffering from HDN gave a positive reaction in the direct antiglobulin test.84 The serum of the mother reacted with RBCs from her husband, her older child, and 9% of random donors. The system was named from Kelleher, the mother’s surname, and the antigen is referred to as K (synonyms: Kell, K1). Three years later, the more common antigen, k (synonyms: Cellano, K2), which has a high incidence in all populations, was identified through the typing of large numbers of RBC samples with an antibody that had also caused a mild case of HDN.85 The Kell system remained a simple two-antigen system until 1957, when the antithetical Kpa and Kpb antigens were reported, as was the K0 (Kell-null) phenotype.6 Subsequently, the number of Kell antigens has grown to 25, making Kell one of the most polymorphic blood group systems known.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

amino acid sequence reveals distant similarity to ammonium transporters in bacteria, fungi, and plants.69 Evidence for ammonia transport by RhAG comes from yeast complementation experiments,70,71 expression studies in Xenopus oocytes,72 and direct evidence in RBCs.73 Rh and RhAG homologues have been found in many organisms, including the sponge (Geodia), the slime mold (Dictyostelium), the fruit fly (Drosophila), and the frog (Xenopus),74 indicating that they are conserved throughout evolution. Nonerythroid homologues, designated RhCG and RhBG, are found in the kidney,75 liver,76,77 testis, brain, gastrointestinal tract,78 and skin79,80 and are localized to regions where ammonium production and elimination are critical in mammalian tissues, strongly suggesting a role for these proteins in ammonia/ammonium homeostasis. When expressed in Xenopus oocytes, RhBG and RhCG also mediate transport of ammonia.81 In the kidney ammonium ions act as expendable cations that facilitate excretion of acids, and renal ammonium metabolism and transport are critical for acid-base balance. In the collecting segment and collecting duct, where large amounts of ammonia are excreted, RhBG and RhCG are found on the basolateral and apical membranes, respectively, of the intercalated cells.76 These localization studies suggest that RhBG and RhCG are ideally situated to mediate transepithelial movement of ammonium from the interstitium to the lumen of the collecting duct. In support, mouse collecting duct (mIMCD-3) cells, which show polarized expression of these proteins, demonstrate transportermediated movement of ammonia.82 The function of RhCE and RhD has not been determined. Co-expression of RhCE/RhAG did not influence the rate or total substrate accumulated in oocytes compared to that seen with expression of RhAG alone.83 Although further studies are necessary to determine if RhCE/D are involved in membrane transport, RhCE/D may have lost transport function and may have a structural role in the RBC membrane.

Proteins The Kell protein is a type II glycoprotein with an approximate Mr of 93,000. It has a 665-amino acid carboxyl terminal extracellular domain, a single 20-amino acid transmembrane domain, and a 47-amino acid N-terminal cytoplasmic domain.86 The protein has five N-glycosylation sites and 15 extracellular cysteine residues that cause folding through the formation of multiple intrachain disulfide bonds (Fig. 7–4). This explains why Kell blood group antigens are inactivated when RBCs are treated with reducing agents, such as dithiothreitol and aminoethylisothiouronium bromide, which disrupt disulfide bonds.87 All Kell system antigens are carried on this glycoprotein, which is present at 3500 to 17,000 copies per RBC.88,89 All but two (Jsa and Jsb) of the Kell antigens are localized in the N-terminal half of the protein before residue 550, strongly suggesting that the C-terminal domain does not tolerate change and is functionally important. Indeed, the Kell glycoprotein is a zinc endopeptidase, and the C terminus contains a zinc-binding domain that is the catalytic site.90,91 Kx is a 444-amino acid, 37-kD protein that is linked by a disulfide bond to the Kell protein.17 Kx is predicted to span the membrane 10 times (see Fig. 7–4), is not glycosylated, and may be a membrane transport protein. RBCs lacking Kx have the McLeod phenotype, which is characterized by a marked reduction of Kell antigens, acanthocytosis, and reduced in vivo RBC survival.

7 87

Genes The KEL gene has been localized on chromosome 7q33. It consists of 19 exons spanning approximately 21.5 kb.92,93 The Kell antigens result from nucleotide mutations that cause

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BLOOD BANKING

Catalytic domain 597 Jsb/Jsa Leu597Pro

Kpb/Kpa Arg281Trp

281

k/K Thr193Met

193

S

Extracellular

COOH Lipid Bilayer

Intracellular NH2 Kx

Kell

NH2

COOH Figure 7–4 Kell and Kx proteins. Kell is a single-pass protein, but Kx is predicted to span the red blood cell membrane 10 times. Kell and Kx are linked by a disulfide bond, shown as -S—. The amino acids that are responsible for the more common Kell antigens are shown. The N-glycosylation sites are shown as Y. The hollow Y represents the N-glycosylation site that is not present on the K (K1) protein.

very weak expression of Kell antigens. It is now evident that he lacked Kx, which is important for expression of Kell, and that lack of Kx is the basis for the McLeod syndrome. Males with the McLeod syndrome have muscular and neurologic defects, including skeletal muscle wasting, elevated serum creatine phosphokinase, psychopathology, seizures with basal ganglia degeneration, and cardiomyopathy.90,100 Most symptoms develop after the fourth decade of life. The syndrome is very rare. Approximately 60 males have been identified, and all but 2 have been white; however, because of the plethora of symptoms, the syndrome is probably underdiagnosed. Fifteen different mutations in the XK gene were found in a study of 17 families; these mutations involve major and minor deletions, point mutations, and splice site or frameshift mutations that result in the absence or truncation of Kx protein.17,100 At one time, chronic granulomatous disease (CGD) was thought to be related to the McLeod syndrome, but the gene controlling CGD is near the XK gene on the X chromosome, and the small minority of patients with CGD who have the McLeod phenotype have X-chromosome deletions encompassing both genes.6,101

Antigens The Kell system consists of five sets of high-incidence and low-incidence antigens, as follows (the names of the highincidence antigens appear in boldface): ●

II 88

single amino acid substitutions in the protein (Table 7–5). The lack of Kell antigens, K0, is caused by several different molecular defects, including nucleotide deletion, defective splicing, premature stop codons, and amino acid substitutions.91,94,95 The XK gene is on the short arm of the X chromosome at Xp21.96 XK has three exons, and mutations cause the McLeod syndrome, which, because the gene is X linked, affects males. Carrier females, because of X-chromosome inactivation, have two populations of RBCs (one of the McLeod phenotype and one normal), and the proportion varies from 5% McLeod:95% normal to 85% McLeod:15% normal.97,98

McLeod Syndrome When testing medical students in 1961, Allen and coworkers99 found that one of the students, Mr. McLeod, had RBCs with

Table 7–5 Molecular Basis of Antigens in the Kell Blood Group System Antigen

Amino Acid

Position

k(K2) K(K1) Kpa(K3) Kpb(K4) Kpc(K21) Jsa(K6) Jsb(K7) K11 K17 K14 K24 Ula

Threonine Methionine Tryptophan Arginine Glutamine Proline Leucine Valine Alanine Arginine Proline Glutamic acid→Valine

193

Ch07-F039816.indd 88

281 597 302 180 494

● ● ● ●

K and k Kpa, Kpb, and Kpc Jsa and Jsb K11 and K17 K14 and K24

In addition, 14 independently expressed antigens, 3 lowincidence (Ula, K23, VLAN), and 11 high-incidence (Ku, Km, K12, K13, K16, K18, K19, K22, Tou, KALT, KTIM), have been identified. The null phenotype, K0, lacks Kell antigens, and Kell-mod phenotypes have a weak expression of Kell antigens. Kell antigens show population variations (Table 7–6). K has an incidence of 9% in white persons but is much less common in people of other ethnic backgrounds.1,58 Kpa and K17 are also mainly found in white persons. Jsa is almost exclusively found in African Americans, with an incidence of 20%. Ula is found in Finnish and Japanese persons.1,58 The molecular basis of most of the Kell antigens has been determined (see Table 7–5).91,102 The K methionine substitution disrupts a glycosylation consensus sequence so that K has one less N-glycan than k.103 Jsa and Jsb are located within a cluster of cysteine residues,104 a finding that explains why they are more susceptible than other Kell antigens to treatment with reducing agents.105 No Kell haplotype has been found to express more than one low-incidence Kell antigen, not because of structural constraints, but because multiple expression would require more than one mutation encoding a recognized Kell system antigen to occur in the same gene.106 Weaker expression of Kell antigens is found when RBCs carry Kpa in cis.107–109 Weak expression of Kell antigens can be inherited or can be acquired and transient. Inherited weak expression occurs when the Kell-associated Kx protein is absent (McLeod phenotype), when glycophorins C and D are absent (Leach phenotype), or when a portion of the extracellular domain of glycophorin C and D, specifically exon 3, is

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Kell Phenotypes and Prevalence Prevalence (%)

Phenotype

White

African American

K−k+ K+k+ K+k− Kp(a+b−) Kp(a−b+) Kp(a+b+) Kp(a−b−c+) Js(a+b−) Js(a−b+) Js(a+b+) K11 K17 K14 K24 Ula

91 8.8 0.2 Rare 97.7 2.3 0.32 Japanese 0 100 Rare High incidence Low incidence High incidence Low incidence Low incidence (2.6% in Finns, 0.46% in Japanese)

98 2 Rare 0 100 Rare 0 1 80 19

deleted (some Gerbich-negative phenotypes).109,110 Transient depression of Kell system antigens has been associated with the presence of autoantibodies mimicking alloantibodies in autoimmune hemolytic anemia and with microbial infections. Kell expression was reduced in two cases of idiopathic thrombocytopenic purpura but returned to normal after remission.6

Antibodies Kell antigens are highly immunogenic, and anti K is common. However, because more than 90% of donors are K negative, it is not difficult to find compatible blood for patients with antiK. The other Kell system antibodies are less common but are also usually IgG, and they have caused transfusion reactions and HDN or neonatal anemia. In HDN due to Kell antibodies, neither maternal antibody titers nor amniotic bilirubin levels are good predictors of the severity of the disease. Reports demonstrate that Kell antigens are expressed very early during erythropoiesis63 and that Kell antibodies can cause suppression of erythropoiesis in vitro.111 This finding suggests that the low level of bilirubin observed, in the presence of neonatal anemia, is due to Kell antibodies that bind to erythroid progenitors and exert effects before hemoglobinization. Anti-Ku is the antibody made by immunized K0 individuals, and Ku represents the high-incidence or “total-Kell” antigen. McLeod males with CGD make anti-Kx+Km; this antibody reacts strongly with K0 cells, weaker with RBCs of common Kell phenotype, and not at all with McLeod phenotype RBCs. Anti-Km, made by McLeod persons who do not have CGD, reacts with RBCs of common Kell phenotypes but not with K0 or McLeod RBCs, suggesting that it detects one or more epitopes requiring both the Kell and Kx proteins. One case has been reported of a McLeod male without CGD who made an apparent anti-Kx without the presence of anti-Km.101

Expression Kell system antigens appear to be erythroid specific and are expressed very early during erythropoiesis.63 Kx mRNA is found in muscle, heart, brain, and hematopoietic tissue.17

Ch07-F039816.indd 89

Evolution Primate RBCs express the k antigen but not the K antigen, indicating that the K mutation appeared in human lineage. Chimpanzees are Js(a+b−),112 and Jsa is also present on RBCs from Old World monkeys,113 suggesting that Jsb also arose after human speciation. Kell protein is not found on immunoblots of RBCs from sheep, goat, cattle, rabbit, horse, mouse, donkey, cat, dog, or rat.114

Function The Kell glycoprotein is a member of the M13 or neprilysin family of zinc endopeptidases that cleave a variety of physiologically active peptides. One member of this family, endothelin converting enzyme 1 (ECE-1), is a membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1 (big ET-1).115 Like ECE-1, Kell protein can proteolytically cleave endothelins, specifically big ET-3 to generate ET-3, which is a potent vasoconstrictor.116 ET-3 is also involved in the development of the enteric nervous system and in migration of neural crest-derived cells. The biologic role of the endothelins is not yet completely elucidated, but they act on two G protein–coupled receptors, ETA and ETB, which are found on many cells. Kell-null individuals, who lack Kell protein, do not have any obvious defect, so they do not immediately give insight into the biologic function of the Kell protein. This lack of defect in Kell-null people may be because other enzymes probably also cleave ET-3,116 and determining whether Kell-null individuals have abnormal levels of plasma endothelins is an active area of investigation.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

Table 7–6

7

DUFFY (Fy) BLOOD GROUP SYSTEM

89

History The Duffy (Fya) blood group antigen was first reported in 1950 by Cutbush and associates,117 who described the reactivity of an antibody found in a hemophiliac male who had received multiple transfusions. This blood group system bears the patient’s surname, Duffy, the last two letters of which provide the abbreviated nomenclature (Fy). Fyb was found 1 year later.118 In 1975, Fy was identified as the receptor for the malarial parasite Plasmodium vivax.119 This discovery explained the predominance of the Fy(a−b−) (Fy-null) phenotype, which confers resistance to malarial invasion, in persons originating from West Africa.

Proteins The Fy protein is a transmembrane glycoprotein of 35 to 43 kD consisting of a glycosylated amino terminal region, which protrudes from the membrane and has seven transmembrane-spanning domains (Fig. 7–5).120,121 In 1993, it was realized that Fy was the erythrocyte chemokine receptor that could bind interleukin-8 and monocyte chemotactic peptide1 (MCP-1).122 The cloning of the FY gene123 confirmed that it belongs to the conserved family of chemokine receptors.

Gene The FY gene is located on the long arm of chromosome 1q22–q23124 and spans 1.5 kb. The gene, which has only two

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BLOOD BANKING

NH2

31

29

18

Fy6

Table 7–7

Prevalence (%)

Fya/Fyb Gly42Asp

40 42

Duffy Phenotypes and Prevalence

Phenotype

Caucasians

Blacks

Chinese Japanese Thai

Fy(a+b−) Fy(a−b+) Fy(a+b+) Fy(a−b−)* FyX

17 34 49 Rare 1.4

9 22 1 68 0

90.8 0.3 8.9 0 0

Fy3

Extracellular Lipid Bilayer

81.5 0.9 17.6 0 0

69 3 28 0 0

*

Fy(a−b−), incidence in Israeli Arabs 25%; Israeli Jews, 4%

Intracellular Fyx Arg89Cys COOH Figure 7–5 The predicted seven-transmembrane domain structure of the Duffy protein. The amino acid change responsible for the Fya/Fyb polymorphism, the mutation responsible for Fyx glycosylation sites, and the regions where Fy3 and Fy6 map are indicated.

exons, contains two ATG codons. The upstream exon contains the major start site.125,126 The same two-exon organization is also found in genes for other chemokine receptors.127

Antigens

II 90

The Fya and Fyb antigens are encoded by two allelic forms of the gene, designated FYA and FYB, and are responsible for the Fy(a+b−), Fy(a−b+), and Fy(a+b+) phenotypes.126 They differ by a single amino acid located on the extracellular domain (see Fig. 7–5). Fyx, which is a weak expression of Fyb, is found in white persons and is due to a single mutation in the FYB gene.128,129 The amino acid change, which is located in the first intracellular cytoplasmic loop, is associated with a decrease in the amount of protein in the membrane and results in diminished expression of Fyb, Fy3, and Fy6 antigens as well as reduced binding of chemokine.127,130,131 Fy3, as determined with one monoclonal anti-Fy3, is located on the third extracellular loop,132 whereas Fy6 maps to the amino terminal loop of the protein. The aspartic acid at amino acid 25 and glutamic acid at 26 are critical for antiFy6 binding.133 The Fy(a−b−) phenotype found in African Americans is caused by a mutation in the promoter region of FYB (T>C at position −46), which disrupts a binding site for the erythroid transcription factor GATA-1 and results in the loss of Fy expression on RBCs.134,135 The Fy protein has also been found on endothelial cells. Because the erythroid promoter controls expression only in erythroid cells, expression of Fy proteins on endothelium is normal in these Fy(a−b−). All African persons with a mutated GATA sequence to date have been shown to carry FYB; therefore, Fyb is expressed on their nonerythroid tissues. This finding explains why Fy(a−b−) individuals make anti-Fya but not anti-Fyb.136 It also is relevant in the selection of antigen-matched units for Fy(a−b−) patients with sickle cell disease, because they would not be expected to make anti-Fyb. Rare Fy(a−b−) who do make anti-Fyb should be investigated at the genetic level, because the molecular basis for this observation has not yet been explained. Lastly, a FYA allele with a mutated GATA sequence has been found in Papua New Guinea.137

Ch07-F039816.indd 90

The Fy(a−b−) phenotype in white persons is very rare (Table 7–7).1 One propositus, an Australian (AZ) woman, appears to be homozygous for a 14-bp deletion in FYA, which introduces a stop codon in the protein138; a Cree Indian female (Ye), a white female (NE), and a Lebanese Jewish male (AA) carry different Trp to stop codon mutations.139 Because these mutations would result in a truncated protein, these people would not be expected to express endothelial or erythroid Fy protein. All four people made anti-Fy3. There are 13,000 to 14,000 Fy antigen sites per RBC,140 and Fya, Fyb, and Fy6 antigens are sensitive to proteolytic enzyme treatment of antigen-positive RBCs, although Fy3 is resistant.

Antibodies Fy antigens are estimated to be 40 times less immunogenic than K antigens, and most Fy antibodies arise from stimulation by blood transfusion. They are mostly IgG, subclass IgG1, and only rarely are IgM. Anti-Fyb is less common than anti-Fya, and Fy antibodies are often found in sera with other antibodies. Anti-Fy3 is made by rare white Fy(a−b−) individuals.6 Anti-Fy6 is a mouse monoclonal antibody.127

Expression Duffy mRNA is present in kidney, spleen, heart, lung, muscle, duodenum, pancreas, placenta, and brain.123,127 Cells responsible for Fy expression in these tissues are the endothelial cells lining postcapillary venules,141–143 except in the brain, where expression is localized to the Purkinje cell neurons.144,145 The same polypeptide is expressed in endothelial cells and RBCs, but in brain, a larger, 8.5-kb mRNA is present. The function of Fy on neurons is an area of active investigation. In the fetus, Fy antigens can be detected at 6 to 7 weeks’ gestation and are well developed at birth.1 The expression of these antigens was found to occur late during erythropoiesis and RBC maturation.63

Evolution RBCs of monkeys and apes react with anti-Fyb, and the conserved GT repeat sequences in the 3 flanking region of the gene both suggest that FYB was the ancestral gene.146,147 The first human divergence occurred when a mutation in the erythroid promoter region of FYB resulted in the loss of Fyb expression on RBCs. This mutation conferred resistance to malaria infection in regions where P. vivax was endemic and selected for the high proportion of Fy(a−b−) in populations of African ancestry. Later, a single nucleotide change

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211

Jka/Jkb Asp280Asn

Extracellular Lipid Bilayer

Function The importance of Fy as a receptor for the malarial parasite P. vivax is well established, but its biologic role as a chemokine receptor on RBCs, endothelial cells, and brain is not yet clear. The chemokine receptors are a family of proteins that are receptors on target cells for the binding of chemokines. Chemokines are so named because they are cytokines that are chemotactic (cause cell migration), and their receptors are an active area of investigation.148 Chemokine receptors have been found principally on lymphocytes, where they are coupled to G proteins and activate intracellular signaling pathways that regulate cell migration into tissues. Unlike other chemokine receptors, Fy does not have a conserved amino acid DRY-motif in the second extracellular loop, and cells transfected with Fy and stimulation with chemokines do not mobilize free calcium.120 Also, Fy can bind chemokines from both the CXC (IL-8, MGSA) and the CC (RANTES, MCP-1, MIP-1) classes of chemokines.127 These features have led investigators to hypothesize that it may act as a scavenger or sink for excess chemokine release into the circulation.149,150 If the function of Fy on RBCs is to scavenge excess chemokine, it may be that Fy(a−b−) individuals would be more susceptible to septic shock143 or to cardiac damage after infarction. Renal allografts have been reported to have shorter survival in African American Fy(a−b−) recipients,151 and Fy has been shown to be upregulated in the kidney during renal injury.152 Abundant expression of Fy on high endothelial venules and sinusoids in the spleen,143 which is a site central to chemokine-induced leukocyte trafficking, suggests a role in leukocyte migration into the tissues. Fy is also similar to the receptor for endothelins (ETB), vasoactive proteins that strongly influence vascular biology and that may also be mitogenic.127

THE KIDD BLOOD GROUP SYSTEM History The Kidd blood group system was discovered in 1951, when a “new” antibody in the serum of Mrs. Kidd (who also had anti-K) caused HDN in her sixth child. Jk came from the initials of the baby (John Kidd), because K had been used previously for Kell.153 Anti-Jkb was found 2 years later in the serum of a woman who had a transfusion reaction (she also had anti-Fya).154 Kidd system antibodies are characteristically found in sera with other blood group antibodies, suggesting that Kidd antigens are not particularly immunogenic. However, Kidd antibodies induce a rapid and robust anamnestic response that is responsible for their reputation for causing severe delayed hemolytic transfusion reactions.

Proteins The Kidd glycoprotein has an approximate Mr of 46,000 to 60,000 and is predicted to span the membrane 10 times. The

Ch07-F039816.indd 91

Intracellular COOH

NH2 Figure 7–6 Predicted 10-transmembrane domain structure of the Kidd/urea transporter. The polymorphism responsible for the Kidd antigens and the site for the N-glycan are indicated.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

in the FYB gene caused the FYA polymorphism in people of European and Asian ancestry. Fy has been cloned from several nonhuman primates, including chimpanzees, squirrel monkeys, and rhesus monkeys, and from cows, pigs, rabbits, and mice.127

third extracellular loop is large and carries an N-glycan at Asn211 (Fig. 7–6). The protein has 10 cysteine residues, but only 1 is predicted to be extracellular, a fact that explains why the antigens are not sensitive to disulfide reagents. There is internal homology between the N-terminal and C-terminal halves of the protein, with each containing an LP box (LPXXTXPF) characteristic of urea transporters.155 Isolation of the Kidd protein from RBCs was elusive, and cloning of the Kidd gene was accomplished with primers complementary to the rabbit kidney urea transporter. A clone isolated from a human bone marrow library with the PCR product was confirmed by in vitro transcription–translation to encode a protein that carried Kidd blood group antigens.156 7

Gene The Kidd blood group gene (HUT11 or SLC14A1) is located at chromosome 18q11–q12.157,158 The HUT11 gene has 11 exons and spans approximately 30 kb. Two alternative polyadenylation sites generate transcripts of 4.4 and 2.0 kb, which appear to be used equally. Exons 4 to 11 encode the mature protein.17,159

91

Antigens Two antigens, Jka and Jkb, are responsible for the three common phenotypes—Jk(a+b−), Jk(a−b+), and Jk(a+b+). The two antigens are found with similar frequency in white persons but show large differences in other ethnic groups (Table 7–8).1 The Jk(a−b−) phenotype is rare but occurs with greater incidence in Asian and Polynesian people.

Table 7–8

Kidd Phenotypes and Prevalence Prevalence(%)

Phenotype

White

African

Asian

Jk(a+b−) Jk(a−b+) Jk(a+b+) Jk(a−b−)

26.3 23.4 50.3 Rare

51.5 8.1 40.8 Rare

23.2 26.8 49.1 0.9 (Polynesian)

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BLOOD BANKING

The Jka and Jkb polymorphism is located on the fourth extracellular loop and is caused by a single amino acid substitution (see Fig. 7–6).160 The null phenotype has been reported to arise on the following two genetic backgrounds: homozygous inheritance of a silent allele and inheritance of a dominant inhibitor gene, In(Jk), which is unlinked to JK (HUT11).1 The silent alleles were found to have acceptor or donor splice site mutations that cause skipping of exon 6 or exon 7.159,161 In Finnish persons, a single point mutation (Ser291Pro) was the only change associated with silencing of the expression of Jkb.161 The unlinked dominant inhibitor gene has not yet been identified. The Jk3 antigen (“total Jk”) is found on RBCs that are positive for Jka, Jkb, or both, but the specific amino acid residues responsible for Jk3 are unknown. There are approximately 14,000 Jka antigen sites on Jk(a+b−) RBCs.140

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Neither anti-Jka nor anti-Jkb is common, and anti-Jkb is found less often than anti-Jka. Once a Kidd antibody is identified, compatible blood is not difficult to find, because 25% of donors are negative for each antigen. Unfortunately, Kidd antibodies are often found in sera that contain other alloantibodies, so the situation is often more complicated. In addition, the antibodies are known to cause delayed hemolytic transfusion reactions and are responsible for at least one third of all cases of delayed hemolytic transfusion reactions.59 The antibodies often decrease in titer, react only with cells that are from persons homozygous for the antigen (the dosage phenomenon), or may escape detection altogether in the sensitized patient’s serum before transfusion. If the patient is transfused with antigen-positive RBCs, an anamnestic response results, with an increase in the antibody titer and hemolysis of the transfused RBCs.59 Because Kidd antibodies are mainly IgG, it was assumed that they must activate complement to cause such prompt RBC destruction. Kidd antibodies can be partially IgM, however, and the minor IgM component may be responsible for the pattern of destruction of incompatible RBCs in delayed hemolytic transfusion reactions. This theory is based on evidence that serum fractions containing only IgG anti-Jka do not bind complement.162 Kidd antibodies only rarely cause HDN; if they do, it is typically not severe. Anti-Jka has occasionally been found as an autoantibody, and the concurrent, temporary suppression of antigen expression has also been reported.59 Anti-Jk3, sometimes referred to as anti-Jkab, is produced by Jk(a−b−) individuals and reacts with RBCs that are positive for Jka, Jkb, or both.163

Expression In the fetus, Kidd antigens can be detected at 11 weeks’ gestation and are well developed at birth.6 Immunohistochemical and in situ hybridization have shown that Kidd/HUT11 is also expressed on endothelial cells of vasa recta in the medulla of the human kidney.17

Evolution Expression of RBC Kidd antigens has not been investigated in nonhuman primates, but the human RBC Kidd protein is clearly a member of a family of urea transporters, which

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have homologues in the human kidney and in nonhuman primates.

Function A first clue to the function of Kidd came from the observation in 1982 that the RBCs of a male of Samoan descent were resistant to lysis in 2M urea, which was being used to lyse RBCs for platelet counting.164 His RBCs were Jk(a−b−), and subsequent investigations revealed that movement of urea into Jk(a−b−) RBCs was equivalent to passive diffusion, in contrast to RBCs with normal Kidd antigens, with rapid urea influx. Fast urea transport in human RBCs is thought to be advantageous for RBC osmotic stability in the kidney, especially during transit through the vasa recta from the papilla to the isoosmotic renal cortex.165 Urea transport in the kidney contributes to urine concentration and water preservation. Two types of urea transporters have now been characterized, vasopressin-sensitive transporters and constitutive transporters. The Kidd/HUT11 in RBCs and kidney medulla is a constitutive transporter. A vasopressinsensitive transporter, HUT2, which is expressed in the collecting ducts of the kidney, has 62% sequence identity with Kidd/HUT11. HUT2 is also located on chromosome 18q12, suggesting that both transporters evolved from an ancestral gene duplication.166 Jk-null individuals do not suffer a clinical syndrome except for a reduced capacity to concentrate urine,167 suggesting that other mechanisms or other gene family members, such as HUT2, can compensate for the missing Kidd/HUT11 protein.

Summary The last decade has witnessed the rapid elucidation of the molecular basis for the various blood group antigens and phenotypes. In the next decade, research focus is turned to determination of the structure and function of the proteins carrying these blood group antigens. Null individuals (natural knockouts), persons who lack the antigens and the proteins that carry them, exist for all four of these blood group systems. Reminiscent of what is being found in many genetically engineered knockout mice, most of the individuals with null phenotypes do not have serious or any obvious defects. This observation is consistent with growing evidence that many genes have evolved as members of larger gene families, which appear to have overlapping abilities to substitute for the disrupted gene family member. Evidence is accumulating that genes encoding blood group proteins are also members of larger gene families. The mouse homologues of some of these blood group genes have been cloned, and knockout mice are being generated to study them. This process may aid in the further elucidation of the structure and function of the molecules carrying blood group antigens. REFERENCES 1. Race RR, Sanger R. Blood Groups in Man, 6th ed. Oxford, Blackwell Scientific, 1975. 2. Bowman JM. RhD hemolytic disease of the newborn [editorial; comment]. NEJM 1998;339:1775–1777. 3. Levine P, Stetson RE. An unusual case of intragroup agglutination. JAMA 1939;113:126–127. 4. Levine P, Burnham L, Katzin EM, Vogel P. The role of iso-immunization in the pathogenesis of erythroblastosis fetalis. AJOG 1941;42:925–937.

9/1/06 8:11:03 PM

Ch07-F039816.indd 93

33. Wagner FF, Frohmajer A, Flegel WA. RHD positive haplotypes in D negative Europeans. BMC Genetics 2001;2:10 34. Okuda H, Kawano M, Iwamoto S, et al. The RHD gene is highly detectable in RhD-negative Japanese donors. J Clin Invest 1997;100:373–379. 35. Singleton BK, Green CA, Avent ND, et al. The presence of an RHD pseudogene containing a 37-base pair duplication and a nonsense mutation in Africans with the Rh D-negative blood group phenotype. Blood 2000;95:12–18. 36. Wagner FF, Gassner C, Müller TH, et al. Molecular basis of weak D phenotypes. Blood 1999;93:385–393. 37. Blumenfeld OO, Patnaik SK. Allelic genes of blood group antigens: a source of human mutations and cSNPs docmented in the Blood Group Antigen Mutation Database. Hum Mutat 2004;23:8–16. Available at www.ncbi.nlm.nih.gov/projects/mhc/xslcgi.fcgi?cmd=bgumt/ home. 38. Wagner FF, Frohmajer A, Ladewig B, et al. Weak D alleles express distinct phenotypes. Blood 2000;95:2699–2708. 39. 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 1998;92:2602–2604. 40. Yasuda H, Ohto H, Sakuma S, Ishikawa Y. Secondary anti-D immunization by Del red blood cells. Transfusion 2005;45:1581–1584. 41. Wagner T, Kormoczi GF, Buchta C, et al. Anti-D immunization by DEL red blood cells. Transfusion 2005;45:520–526. 42. Huang C-H. Molecular insights into the Rh protein family and associated antigens. Curr Opin Hematol 1997;4:94–103. 43. Wallace M, Lomas-Francis C, Tippett P. The D antigen characteristic of RoHar is a partial D antigen. Vox Sang 1996;70:169–172. 44. Schlanser G, Moulds MK, Flegel WA, Wagner FF. Crawford (Rh43), a low-incidence antigen, is associated with a novel RHCE variant RHce allele (abstract). Transfusion 2003;43(Suppl):34A–35A. 45. Westhoff CM, Vege S, Nance S, et al. Determination of the molecular background of the Crawford antigen occurring with a weak C antigen (abstract). Vox Sang 2004;87(Suppl 3):42. 46. Beckers EAM, Porcelijn L, Ligthart P, et al. The R0Har antigenic complex is associated with a limited number of D epitopes and alloanti-D production: A study of three unrelated persons and their families. Transfusion 1996;36:104–108. 47. Wagner FF, Ladewig B, Flegel WA. The RHCE allele ceRT: D epitope 6 expression does not require D-specific amino acids. Transfusion 2003;43:1248–1254. 48. Mouro I, Colin Y, Chérif-Zahar B, et al. Molecular genetic basis of the human Rhesus blood group system. Nature Genet 1993;5:62–65. 49. Mouro I, Colin Y, Sistonen P, et al. Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities. Blood 1995;86:1196– 1201. 50. Faas BHW, Beckers EAM, Wildoer P, et al. Molecular background of VS and weak C expression in blacks. Transfusion 1997;37:38–44. 51. Daniels GL, Faas BHW, Green CA, et al. The VS and V blood group polymorphisms in Africans: a serological and molecular analysis. Transfusion 1998;38:951–958. 52. 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 1998;103:429–436. 53. Noizat-Pirenne F, Mouro I, Roussel M, et al. The molecular basis of a D(C)(E) complex probably associated with the RH35 low frequency antigen (abstract). Transfusion 1999;39(Suppl):103S. 54. Faas BHW, Ligthart PC, Lomas-Francis C, et al. Involvement of Gly96 in the formation of the Rh26 epitope. Transfusion 1997;37:1123–1130. 55. 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 1997;90(Suppl 1):272a. 56. Westhoff CM, Silberstein LE, Wylie DE, et al. 16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype. Br J Haematol 2001;113:666–671. 57. Noizat-Pirenne F, Lee K, Le Pennec P-Y, et al. Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety. Blood 2002;100:4223–4231. 58. Reid ME, Lomas-Francis C. Blood Group Antigen FactsBook, 2nd ed., San Diego, Academic Press, 2003. 59. Mollison PL, Engelfriet CP, Contreras M. Blood Transfusion in Clinical Medicine, 10th ed., Oxford, Blackwell Science, 1997. 60. Petz LD, Garratty G. Acquired Immune Hemolytic Anemias. New York, Churchill Livingstone, 1980. 61. Kumpel BM, Goodrick MJ, 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 1995;86:1701–1709.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

5. Rosenfield RE. Who discovered Rh? A personal glimpse of the LevineWiener argument. Transfusion 1989;29:355–357. 6. Daniels G. Human Blood Groups, 2nd ed. Oxford, Blackwell Science, 2002. 7. Mourant AE. A new rhesus antibody. Nature 1945;155:542. 8. Tippett P. A speculative model for the Rh blood groups. Ann Hum Genet 1986;50:241–247. 9. Rosenfield RE, Alen FHJr, Swisher SN, Kochwa S.A review of Rh Serology and presentation of a new terminology. Transfusion 1962;2:287–312. 10. Agre P, Saboori AM, Asimos A, Smith BL. Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. J Biol Chem 1987;262:17497–17503. 11. Bloy C, Blanchard D, Dahr W, et al. Determination of the N-terminal sequence of human red cell Rh(D) polypeptide and demonstration that the Rh(D), (c), and (E) antigens are carried by distinct polypeptide chains. Blood 1988;72:661–666. 12. Arce MA, Thompson ES, Wagner S, et al. Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhDnegative individuals. Blood 1993;82:651–655. 13. 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 USA 1990;87:6243–6247. 14. 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 1994;67:203–209. 15. Hartel-Schenk S, Agre P. Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex. J Biol Chem 1992;267:5569–5574. 16. Ridgwell K, Spurr NK, Laguda B, et al. Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression. Biochem J 1992;287:223–228. 17. 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 1998;74(Suppl 2):29–64. 18. Ballas SK, Clark MR, Mohandas N, et al. Red cell membrane and cation deficiency in Rh null syndrome. Blood 1989;63:1046–1055. 19. Sturgeon P. Hematological observations on the anemia associated with blood type Rhnull. Blood 1970;36:310–320. 20. 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 1998;92:664–671. 21. Eyers SA, Ridgwell K, Mawby WJ, Tanner MJ. Topology and organization of human Rh (rhesus) blood group-related polypeptides. J Biol Chem 1994;269:6417–6423. 22. 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 1998;92:4428–4438. 23. Nicolas V, Kim CL, Gane P, et al. Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rhnull-associated mutation. J Biol Chem 2003;278:25526–25533. 24. Dahl KN, Parthasarathy R, Westhoff CM, et al. Protein 4.2 is critical to CD47-membrane skeleton attachment in human red cells. Blood 2004;103:1131–1136. 25. Colin Y, Chérif-Zahar B, Le Van Kim C, et al. Genetic basis of the RhDpositive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 1991;78:2747–2752. 26. Chérif-Zahar B, Mattei MG, Le Van Kim C, et al. Localization of the human Rh blood group gene structure to chromosome region 1p34.3– 1p36.1 by in situ hybridization. Hum Genet 1991;86:398–400. 27. Avent ND, Reid ME. The Rh blood group system: a review. Blood 2000;95:375–387. 28. Huang C-H. The human Rh50 glycoprotein gene—Structural organization and associated splicing defect resulting in Rhnull disease. J Biol Chem 1998;273:2207–2213. 29. 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. Nature Genet 1996;12:168–173. 30. 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 1997;89:2568–2577. 31. 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 1998;92:1839–1840. 32. Huang C-H. Alteration of RH gene structure and expression in human dCCee and DCW-red blood cells: phenotypic homozygosity versus genotypic heterozygosity. Blood 1996;88:2326–2333.

7 93

9/1/06 8:11:03 PM

BLOOD BANKING

II 94

62. Kajii E, Umenishi F, Nakauchi H, Ikemoto S. Expression of Rh blood group gene transcripts in human leukocytes. Biochem Biophys Res Comm 1994;202:1497–1504. 63. Southcott MJG, Tanner MJ, Anstee DJ. The expression of human blood group antigens during erythropoiesis in a cell culture system. Blood 1999;93:4425–4435. 64. Westhoff CM, Wylie DE. Investigation of the human Rh blood group system in nonhuman primates and other species with serologic and Southern blot analysis. J Mol Evol 1994;39:87–92. 65. Salvignol I, Calvas P, Socha WW, et al. Structural analysis of the RHlike blood group gene products in nonhuman primates. Immunogenetics 1995;41:271–281. 66. Westhoff CM, Schultze A, From A, et al. Characterization of the mouse RH blood group gene. Genomics 1999;57:451–454. 67. Blancher A, Klein J, Socha WW (eds). Molecular Biology and Evolution of Blood Group and MHC Antigens in Primates. Berlin, SpringerVerlag, 1997. 68. 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 1998;249:78–85. 69. Marini AM, Urrestarazu A, Beauwens R, André B. The Rh (rhesus) blood group polypeptides are related to NH4+ transporters. Trends Biochem Sci 1997;22:460–461. 70. Marini AM, Matassi G, Raynal V, et al. The human rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast. Nature Genet 2000;26:341–344. 71. Westhoff CM, Siegel DL, Burd CG, Foskett JK. Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein. J Biol Chem 2004;279:17443–17448. 72. Westhoff CM, Ferreri-Jacobia M, Mak D-OD, Foskett JK. Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter. J Biol Chem 2002;277:12499–12502. 73. Ripoche P, Bertrand O, Gane P, et al. Human Rhesus-associated glycoprotein mediates facilitated transport of NH3 into red blood cells. Proc Natl Acad Sci USA 2004;101:17222–17227. 74. Huang C-H, Liu PZ, Cheng JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol 2000;37:150–165. 75. Eladari D, Cheval E, Quentin F, et al. Expression of RhCG, a new putative NH3/NH4+ transporter, along the rat nephron. J Amer Soc Nephrology 2002;13:1999–2008. 76. Weiner ID, Miller RT, Verlander JW. Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver. Gastroenterology 2003;124:1432–1440. 77. Weiner ID, Verlander JW. Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein. Acta Physiol Scand 2003;179:331–338. 78. Handlogten ME, Hong SP, Zhang L, et al. Expression of the ammonia transporter proteins Rh B glycoprotein and Rh C glycoprotein in the intestinal tract. Am J Physiol Gastrointest Liver Physiol 2005;288: G1036–G1047. 79. Liu Z, Peng J, Mo R, et al. Rh type B glycoprotein is a new member of the Rh superfamily and a putative ammonia transporter in mammals. J Biol Chem 2001;276:1424–1433. 80. Liu Z, Chen Y, Mo R, Hui C, Cheng J-F, Mohandas N, Huang C-H. Characterization of human RhCG and mouse RhCG as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis. J Biol Chem 2000;275:25641–25651. 81. Mak DO, Dang B, Weiner ID, et al. Characterization of ammonia transport by the kidney Rh glycoproteins, RhBG and RhCG. Am J Physiol Renal Physiol 2006;290:F297–F305. 82. Handlogten ME, Hong SP, Westhoff CM, Weiner ID. Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD3). Am J Physiol Renal Physiol 2005;289:F347–F358. 83. Westhoff CM. The Rh blood group system in review: a new face for the next decade. Transfusion 2004;44:1663–1673. 84. Coombs RRA, Mourant AE, Race RR. In vivo isosensitisation of red cells in babies with haemolytic disease. Lancet 1946;i:264–266. 85. Levine P, Backer M, Wigod M, Ponder R. A new human hereditary blood property (Cellano) present in 99.8% of all bloods. Science 1949;109:464–466. 86. Lee S, Zambas ED, Marsh WL, Redman CM. Molecular cloning and primary structure of Kell blood group protein. Proc Natl Acad Sci USA 1991;88:6353–6357. 87. Advani H, Zamor J, Judd WJ, Johnson CL, Marsh WL. Inactivation of Kell blood group antigens by 2-aminoethylisothiouronium bromide. Br J Haematol 1982;51:107–115. 88. Hughes-Jones NC, Gardner B. The Kell system: studies with radiolabeled anti-K. Vox Sang 1971;21:154–158.

Ch07-F039816.indd 94

89. Masouredis SP, Sudora E, Mohan LC, Victoria EJ. Immunoelectron microscopy of Kell and Cellano antigens on red cell ghosts. Haematologia 1980;13:59–64. 90. Marsh WL, Redman CM. The Kell blood group system: a review. Transfusion 1990;30:158–167. 91. Lee S. Molecular basis of Kell blood group phenotypes. Vox Sang 1997;73:1–11. 92. Lee S, Zambas ED, Marsh WL, Redman CM. The human Kell blood group gene maps to chromosome 7q33 and its expression is restricted to erythroid cells. Blood 1993;81:2804–2809. 93. Lee S, Zambas E, Green ED, Redman C. Organization of the gene encoding the human Kell blood group protein. Blood 1995;85:1364–1370. 94. Lee S, Russo DCW, Reiner AP, et al. Molecular defects underlying the Kell null phenotype. J Biol Chem 2001;276:27281–27289. 95. Yu LC, Twu YC, Chang CY, Lin M. Molecular basis of the Kell-null phenotype: a mutation at the splice site of human KEL gene abolishes the expression of Kell blood group antigens. J Biol Chem 2001;276: 10247–10252. 96. Bertelson CJ, Pogo AO, Chaudhuri A, et al. Localization of the McLeod locus (XK) within Xp21 by deletion analysis. Am J Hum Genet 1988; 42:703–711. 97. Marsh WL, Redman CM. Recent developments in the Kell blood group system. Transf Med Rev 1987;1:4–20. 98. Øyen R, Reid ME, Rubinstein P, Ralph H. A method to detect McLeod phenotype red blood cells. Immunohematology 1996;12:160–163. 99. Allen FH, Krabbe SMR, Corcoran PA. A new phenotype (McLeod) in the Kell blood group system. Vox Sang 1961;6:555–560. 100. Danek A, Rubio JP, Rampoldi L, et al. McLeod neuroacanthocytosis: genotype and phenotype. Ann Neurol 2001;50:755–764. 101. Oyen R, Powell VI, Reid ME, et al. The first non-CGD McLeod phenotype male to make anti-Kx: definition of the molecular basis (abstract). Transfusion 1999;39(Suppl 1):90S. 102. Lee S, Wu X, Son S, et al. Point mutations characterize KEL10, the KEL3, KEL4, and KEL21 alleles, and the KEL17 and KEL11 alleles. Transfusion 1996;36:490–494. 103. Lee S, Wu X, Reid ME, Zelinski T, Redman C. Molecular basis of the Kell (K1) phenotype. Blood 1995;85:912–916. 104. Lee S, Wu X, Reid ME, Redman C. Molecular basis of the K:6,-7 [Js(a+b−)] phenotype in the Kell blood group system. Transfusion 1995;35:822–825. 105. Branch DR, Muensch HA, Sy Siok Hian AL, Petz LD. Disulfide bonds are a requirement for Kell and Cartwright (Yta) blood group antigen integrity. Br J Haematol 1983;54:573–578. 106. Yazdanbakhsh K, Lee S, Yu Q, Reid ME. Identification of a defect in the intracellular trafficking of a Kell blood group variant. Blood 1999;94:310–318. 107. Allen FH Jr, Lewis SJ, Fudenberg H. Studies of anti-Kpb, a new antibody in the Kell blood group system. Vox Sang 1958;3:1–13. 108. Allen FH, Lewis SJ. Kpa (Penney), a new antigen in the Kell blood group system. Vox Sang 1957;2:81–87. 109. Øyen R, Halverson GR, Reid ME. Review: Conditions causing weak expression of Kell system antigens. Immunohematology 1997;13:75–79. 110. Anstee DJ. Blood group-active surface molecules of the human red blood cell. Vox Sang 1990;58:1–20. 111. Vaughan JI, Warwick R, Letsky E, et al. Erythropoietic suppression in fetal anemia because of Kell alloimmunization. AJOG 1994;171:247–252. 112. Redman CM, Lee S, ten Huinink D, et al. Comparison of human and chimpanzee Kell blood group systems. Transfusion 1989;29:486–490. 113. Blancher A, Reid ME, Socha WW. Cross-reactivity of antibodies to human and primate red cell antigens. Transf Med Rev 2000;14:161–179. 114. Jaber A, Loirat MJ, Willem C, et al. Characterization of murine monoclonal antibodies directed against the Kell blood group glycoprotein. Br J Haematol 1991;79:311–315. 115. Xu D, Emoto N, Giaid A, et al. ECE-1: A membrane-bound metalloprotease that catalyzes the proteolytic activation of big endothelin-1. Cell 1994;78:473–485. 116. Lee S, Lin M, Mele A, et al. Proteolytic processing of big endothelin-3 by the Kell blood group protein. Blood 1999;94:1440–1450. 117. Cutbush M, Mollison PI, Parkin DM. A new human blood group. Nature 1950;165:188. 118. Ikin EW, Mourant AE, Pettenkofer HJ, Blumenthal G. Discovery of the expected haemagglutinin anti-Fyb. Nature 1951;168:1077–1078. 119. Miller LH, Mason SJ, Dvorak JA, et al. Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants. Science 1975;189:561–563. 120. Neote K, Mak JY, Kolakowski LF Jr, Schall TJ. Functional and biochemical analysis of the cloned Duffy antigen: identity with the red blood cell chemokine receptor. Blood 1994;84:44–52.

9/1/06 8:11:04 PM

Ch07-F039816.indd 95

143. 144. 145. 146. 147.

148. 149.

150. 151. 152. 153. 154. 155. 156. 157. 158. 159.

160. 161. 162. 163. 164. 165. 166. 167.

negative individuals who lack the erythrocyte receptor. J Exp Med 1995;181:1311–1317. Chaudhuri A, Nielsen S, Elkjaer ML, et al. Detection of Duffy antigen in the plasma membranes and caveolae of vascular endothelial and epithelial cells of nonerythroid organs. Blood 1997;89:701–712. Horuk R, Martin A, Hesselgesser J, et al. The Duffy antigen receptor for chemokines: Structural analysis and expression in the brain. J Leukocyte Biol 1996;59:29–38. Horuk R, Martin AW, Wang Z, et al. Expression of chemokine receptors by subsets of neurons in the central nervous system. J Immunol 1997;158:2882–2890. Li J, Iwamoto S, Sugimoto N, et al. Dinucleotide repeat in the 3 flanking region provides a clue to the molecular evolution of the Duffy gene. Hum Genet 1997;99:573–577. Chaudhuri A, Polyakova J, Zbrzezna V, Pogo O. The coding sequence of Duffy blood group gene in humans and simians: restriction fragment length polymorphism, antibody and malarial parasite specificities, and expression in nonerythroid tissues in Duffy-negative individuals. Blood 1995;85:615–621. Luster AD. Chemokines—Chemotactic cytokines that mediate inflammation. NEJM 1998;338:436–445. Tilg H, Shapiro L, Atkins MB, et al. Induction of circulating and erythrocyte-bound IL-8 by IL-2 immunotherapy and suppression of its in vitro production by IL-1 receptor antagonist and soluble tumor necrosis factor receptor (p75) chimera. J Immunol 1993;151: 3299–3307. de Winter RJ, Manten A, de Jong YP, et al. Interleukin 8 released after acute myocardial infarction is mainly bound to erythrocytes. Heart 1997;78:598–602. Danoff TM, Hallows KR, Burns JE, et al. Influence of the Duffy blood group on renal allograft survival in African-Americans (abstract). J Amer Soc Nephrology 1998;9:670A. Liu X-H, Hadley TJ, Xu L, et al. Up-regulation of Duffy antigen receptor expression in children with renal disease. Kidney Int 1999;55:1491–1500. Allen FH, Diamond LK, Niedziela B. A new blood-group antigen. Nature 1951;167:482. Plaut G, Ikin EW, Mourant AE, et al. A new blood-group antibody, anti-Jkb. Nature 1953;171:431. Rousselet G, Ripoche P, Bailly P. Tandem sequence repeats in urea transporters: identification of an urea transporter signature sequence. Am J Physiol 1996;270:F554–F555. Olivès B, Neau P, Bailly P, et al. Cloning and functional expression of a urea transporter from human bone marrow cells. J Biol Chem 1994;269:31649–31652. Geitvik GA, Hoyheim B, Gedde-Dahl T, et al. The Kidd (JK) blood group locus assigned to chromosome 18 by close linkage to a DNARFLP. Hum Genet 1987;77:205–209. Leppert M, Ferrell R, Kambok MI, et al. Linkage of the polymorphic protein markers F13B, CIS, CIR and blood group antigen Kidd in CEPH reference families (abstract). Cytogenet Cell Genet 1987;46:647. Lucien N, Sidoux-Walter F, Olivès B, et al. Characterization of the gene encoding the human Kidd blood group/urea transporter protein: Evidence for splice site mutations in Jknull individuals. J Biol Chem 1998;273:12973–12980. Olivès B, Merriman M, Bailly P, et al. The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility. Hum Mol Genet 1997;6:1017–1020. Irshaid NM, Henry SM, Olsson ML. Genomic characterization of the Kidd blood group gene: different molecular basis of the Jk(a−b−) phenotype in Polynesians and Finns. Transfusion 2000;40:69–74. Yates J, Howell P, Overfield J, et al. IgG anti-Jka/Jkb antibodies are unlikely to fix complement. Transf Med 1998;8:133–140. Pinkerton FJ, Mermod LE, Liles BA, Jack JA, Noades J. The phenotype Jk(a−b−) in the Kidd blood group system. Vox Sang 1959;4:155–160. Heaton DC, McLoughlin K. Jk(a−b−) red blood cells resist urea lysis. Transfusion 1982;22:70–71. Macey RI, Yousef LW. Osmotic stability of red cells in renal circulation requires rapid urea transport. Am J Physiol 1988;254:C669–C674. Martial S, Olivès B, Abrami L, et al. Functional differentiation of the human red blood cell and kidney urea transporters. Am J Physiol Renal Fluid Electrolyte Physiol 1996;271:F1264–F1268. Sands JM, Gargus JJ, Frohlich O, et al. Urinary concentrating ability in patients with Jk(a−b−) blood type who lack carrier-mediated urea transport. J Amer Soc Nephrol 1992;2:1689–1696.

RH, KELL, DUFFY, AND KIDD ANTIGENS AND ANTIBODIES

121. Chaudhuri A, Zbrzezna V, Johnson C, et al. Purification and characterization of an erythrocyte membrane protein complex carrying Duffy blood group antigenicity. Possible receptor for Plasmodium vivax and Plasmodium knowlesi malaria parasite. J Biol Chem 1989; 264:13770–13774. 122. Horuk R, Colby TJ, Darbonne WC, et al. The human erythrocyte inflammatory peptide (chemokine) receptor. Biochemical characterization, solubilization, and development of a binding assay for the soluble receptor. Biochemistry 1993;32:5733–5738. 123. Chaudhuri A, Polyakova J, Zbrzezna V, et al. Cloning of glycoprotein D cDNA, which encodes the major subunit of the Duffy blood group system and the receptor for the Plasmodium vivax malaria parasite. Proc Natl Acad Sci USA 1993;90:10793–10797. 124. Mathew S, Chaudhuri A, Murty VV, Pogo AO. Confirmation of Duffy blood group antigen locus (FY) at 1q22→23 by fluorescence in situ hybridization. Cytogenet Cell Genet 1994;67:68. 125. Iwamoto S, Li J, Omi T, et al. Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium. Blood 1996;87:378–385. 126. Iwamoto S, Omi T, Kajii E, Ikemoto S. Genomic organization of the glycophorin D gene: Duffy blood group Fya/Fyb alloantigen system is associated with a polymorphism at the 44-amino acid residue. Blood 1995;85:622–626. 127. Hadley TJ, Peiper SC. From malaria to chemokine receptor: the emerging physiologic role of the Duffy blood group antigen. Blood 1997;89:3077–3091. 128. Parasol N, Reid M, Rios M, et al. A novel mutation in the coding sequence of the FY*B allele of the Duffy chemokine receptor gene is associated with an altered erythrocyte phenotype. Blood 1998;92:2237–2243. 129. Olsson ML, Smythe JS, Hansson C, et al. The Fyx phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein. Br J Haematol 1998;103:1184–1191. 130. Tournamille C, Le Van Kim C, Gane P, et al. Arg89Cys substitution results in very low membrane expression of the Duffy antigen/receptor for chemokines in Fyx individuals (erratum in 95:2753). Blood 1998;92:2147–2156. 131. Yazdanbakhsh K, Øyen R, Yu Q, et al. High level, stable expression of blood group antigens in a heterologous system. Am J Hematol 2000;63:114–124. 132. Lu ZH, Wang ZX, Horuk R, et al. The promiscuous chemokine binding profile of the Duffy antigen/receptor for chemokines is primarily localized to sequences in the amino-terminal domain. J Biol Chem 1995;270:26239–26245. 133. Wasniowska K, Blanchard D, Janvier D, et al. Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminal extracellular portion of the Duffy antigen receptor for chemokines. Mol Immunol 1996;33:917–923. 134. Tournamille C, Colin Y, Cartron JP, Le Van Kim C. Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy-negative individuals. Nature Genet 1995;10:224–228. 135. Iwamoto S, Li J, Sugimoto N, et al. Characterization of the Duffy gene promoter: evidence for tissue-specific abolishment of expression in Fy(a−b−) of black individuals. Biochem Biophys Res Commun 1996; 222:852–859. 136. Le Pennec PY, Rouger P, Klein MT, et al. Study of anti-Fy a in five black Fy(a−b−) patients. Vox Sang 1987;52:246–249. 137. Zimmerman PA, Woolley I, Masinde GL, et al. Emergence of FY * A-null in a Plasmodium vivax-endemic region of Papua New Guinea. Proc Natl Acad Sci USA 1999;96:13973–13977. 138. Mallinson G, Soo KS, Schall TJ, et al. Mutations in the erythrocyte chemokine receptor (Duffy) gene: The molecular basis of the Fya/Fyb antigens and identification of a deletion in the Duffy gene of an apparently healthy individual with the Fy(a−b−) phenotype. Br J Haematol 1995;90:823–829. 139. Rios M, Chaudhuri A, Mallinson G, et al. New genotypes in Fy(a−b−) individuals: Nonsense mutations (Trp to stop) in the coding sequence of either FY A or FY B. Br J Haematol 2000;108:448–454. 140. Masouredis SP, Sudora E, Mahan L, Victoria EJ. Quantitative immunoferritin microscopy of Fya, Fyb, Jka, U, and Dib antigen site numbers on human red cells. Blood 1980;56:969–977. 141. Hadley TJ, Lu ZH, Wasniowska K, et al. Postcapillary venule endothelial cells in kidney express a multispecific chemokine receptor that is structurally and functionally identical to the erythroid isoform, which is the Duffy blood group antigen. J Clin Invest 1994;94:985–991. 142. Peiper SC, Wang ZX, Neote K, et al. The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy

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