RH blood group system and molecular basis of Rh-deficiency

BaillieÁre's Clinical Haematology Vol. 12, No. 4, pp. 655±689, 1999

4 RH blood group system and molecular basis of Rh-de®ciency Jean-Pierre Cartron

DSc

Director of Research, Head INSERM Unite U76, Institut National de la Transfusion Sanguine, 6, rue Alexandre Cabanel, 75015 Paris, France

Rhesus (Rh) antigens are de®ned by a complex association of membrane polypeptides that are missing or severely de®cient from the red cells of rare Rhnull individuals who su€er a clinical syndrome of varying severity characterized by abnormalities of the red cell shape, cation transport and membrane phospholipid organization. The Rhnull phenotype is an inherited condition that may arise from homozygosity either for a `suppressor' gene unrelated to the RH locus (`regulator type') or for a silent allele at the RH locus itself (`amorph type'). A current model suggests that the proteins of the Rh complex (Rh, RhAG, CD47, LW, GPB) are assembled by non-covalent bonds and that it is not assembled or transported to the cell surface when one subunit is missing. Rh and RhAG proteins belong to the same protein family and are quantitatively the major components that form the core of the complex, which is ®rmly linked to the membrane skeleton. Molecular analysis of Rhnull individuals has revealed that abnormalities occur only at the RHAG and RH loci, without alteration of the genes encoding the accessory chains. Mutations of the RHAG gene, but not of RH, occur in all Rhnull individuals of the regulator type (including Rhmod) investigated so far (13 cases), strongly suggesting that RHAG mutants act as `suppressors' and not as transcriptional regulators of the RH genes and that variable expression of the RHAG alleles may account for the Rhmod phenotypes (exhibiting weak expression of Rh antigens). Conversely, mutations of the RHCE gene, but not of RHAG, occur in two unrelated Rhnull individuals of the amorph type, supporting the view that RH mutants result from a `silent' allele at the RH locus. These ®ndings strongly support the Rh complex model since when either the Rh or RhAG protein is missing, the assembly and/or transport of the Rh complex is defective. Transcriptional as well as post-transcriptional mechanisms may account for the molecular abnormalities, but experimental evidence based on expression models is required to test these hypotheses, in the hope that they may help to clarify the biological role of the Rh proteins in the red cell membrane. Key words: Rhesus; Rhnull ; blood groups; red cells; membrane defect; gene alteration; membrane complex; protein tracking.

Since their discovery about 50 years ago, constant progress has been made in terms of the clinical signi®cance, serology and molecular genetics of Rhesus (Rh) blood group antigens. One remarkable step was the description of haemolytic disease of the newborn1±3, and the outstanding studies that followed represented a major achievement in transfusion medicine, allowing the disease to be successfully recognized, diagnosed, then treated and now prevented.4 The RH system is the most complex blood group system in humans. The D, C/c and E/e antigens are the major 1521±6926/99/040655+35 $12.00/0

c 1999 Harcourt Publishers Ltd *

656 J.-P. Cartron

Rh speci®cities, but many others have been serologically de®ned in human populations worldwide, all being potentially involved in haemolytic reactions of immune origin following transfusions or pregnancies as well as in autoimmune haemolytic anaemias.5± 7 After the discovery of rare individuals, called Rhnull , who lack all Rh antigens on their red cells, it was realized that this phenotype is associated with a chronic haemolytic anaemia of non-immune origin and with multiple phenotypic abnormalities.8,9 This was the ®rst indication that a blood group antigen could play a role in the physiology of the red cell membrane. THE RH GENE FAMILY Our knowledge of the biochemistry and molecular genetics of the RH system is relatively recent. Preliminary investigations pointed to the role of sulphydryl groups and membrane lipids for Rh antigen expression.10±13 Key information was provided even more recently, when it was shown that Rh antigens are carried by nonglycosylated hydrophobic membrane proteins of 30±32 kDa (collectively called Rh proteins hereafter and not Rh30, to avoid confusion with the low frequency antigen Rh30 or Goa)14, presumably linked to the membrane skeleton.15±18 Another key observation was the demonstration that Rh antigens are associated with a di€usely migrating glycosylated membrane component of approximately 45±75 kDa, carrying ABH blood group antigens19 onpolyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), an observation that explains why Rh antigens, although not glycosylated, could be puri®ed on lectin±Sepharose columns.20 The glycoprotein component formerly named `Rh50 glycoprotein', is now called `Rh associated glycoprotein' or RhAG, since it could be confused with the low frequency antigen Rh50 also referred to as FPTT.14 The RhAG glycoprotein can be detected using murine monoclonal antibodies that react with all red cells except those from Rhnull individuals, although those typed as Uweak react to a certain extent.21,22 These basic observations have been con®rmed and further explored using the tools of molecular genetics, as detailed in several reviews.23±29 RH locus and the genetic basis of D, C/c and E/e speci®cities The structure of the RH locus was ®rst established by studying blood samples collected from the Caucasian population.30 In D-positive individuals (85%), the RH haplotype is composed of two genes, RHD and RHCE, whereas in D-negative individuals (15%) the RHD gene is missing, most probably following a deletion event. In the Black population, however, and in other populations (Japanese for instance) where the D-negative phenotype is rare, the RHD gene may either be intact or rearranged by partial deletion, recombination via unidirectional segmental exchange between the RHD and RHCE genes (gene conversion) or by single point mutation.31±37 Moreover, an acquired D-negative phenotype occurring by somatic mutation (del G600 in the RHD gene) in the myeloid lineage of a chronic myelocytic leukaemia (LMC) patient has also been identi®ed recently.38 The RHD and RHCE genes are highly homologous and show only 3.5% divergence over the coding region (intronic regions are also highly conserved) suggesting that they are derived from the duplication of a common ancestor gene. The two genes are closely linked and tandemly organized on chromosome 1 (Figure 1). Both are composed of 10 exons and share a similar exon/intron organization. Although introns

Blood group system 657

A

Trypsin K42, 43G p34-p36

1

S

A

103

226

Bromelain/Papain G353, 354A

D

Palmitate RHD

RH

SC, RD

NH2 (1) Trypsin K42, 43G FY CROM KN

Trypsin?

COOH (417)

C/c (S/P)

E/e (P/A)

103

226

CE

Palmitate RHCE

NH2 (1)

B

Trypsin?

COOH (417)

ABO; li 6 p11-p21

V8 E34, 35Q N-37

MHC/CH:RG

Bromelain T39, 40K

RHAG

NH2

Trypsin K196, 197G

Trypsin R323, 324I

Trypsin K384, 385I

COOH (409)

Figure 1. RH and RHAG locus and their protein products. A. Schematic representation of the RH locus on chromosome 1 (with the approximative localization of other blood group loci) showing the tandem organization of the genes, the gene structure (10 exons each) and the predicted membrane topology of Rh proteins (417 residues). The 35 amino acid residues that distinguished D from non-D proteins are indicated and numbered on the D protein. Amino acid residues are given in the one-letter code. Among the cysteine (C) residues (open squares) some that are placed in a C-L-P context are palmitoylated62,63 as indicated. Because of a C ! Y substitution at position 311, the D polypeptide is not palmitoylated at this position. The role of the fatty acids in the translocation, folding or antigenic properties of the Rh proteins is unknown. Rh proteins have a unique exofacial cysteine at position 285, which however is apparently not required for antigen expression (see the text). Positions 103 and 226 on the CE protein, which determine the C/c and E/e polymorphisms (see the text), respectively, are shown. Although Ser103 and Ala226 are present on the D polypeptide, it is unreactive with anti-C or anti-e, because the C/c and E/e epitopes are not linear but conformation dependent structures. Trypsin cleavage sites on the ®rst extracellular loop (K42, 43G) and on the fourth cytoplasmic domain loop (at K189, K198 or R201) are depicted.65 B. Schematic representation of the RHAG locus on chromosome 6, showing the gene structure (10 exons) and the predicted membrane topology of the RhAG protein (409 residues). The N-glycan (carrying ABO and Ii speci®cities) at position 37 is indicated, as well as the trypsin cleavage sites currently identi®ed.65,66

658 J.-P. Cartron

of each gene have not been fully sequenced yet, some useful di€erences were noted, particularly a 654 base-pair (bp) and a 288 bp deletion within intron 4 and intron 3, respectively, of the RHD gene.39,40 Early genetic studies predicted that RHD lay on the 50 side of the RHCE gene41,42, but recent analysis of an RH yeast arti®cial chromosome clone suggested that RHD might be located 30 of RHCE.43 However, more recent studies based on short sequence repeat polymorphisms in intron 8 of both genes support the 50 -D-CE-30 theory.44 Moreover, polymerase chain reaction (PCR) analysis of the spacer region between the RHD and RHCE genes45 indicated that the 12 kb fragment separating the two genes could be ampli®ed only when using a forward primer designed from exon 10 of the RHD gene and a reverse primer in exon 1 of the RHCE gene. The spacer region contains Alu consensus sequences and a putative CpG island and might be involved in recombination events between RH genes. Transcript and gene analyses have shown that the basis of C/c speci®city is complex and results from four amino acid di€erences: C16W, encoded by exon 1 of the RHCE gene, and I60L, S68N and S103P encoded by exon 2.46 However, only the S103P substitution is strictly correlated with C/c antigen expression. Nevertheless, the presence of a cysteine at position 16 could modulate the C reactivity.47 Similar studies have shown that the E/e polymorphism results from a single amino acid substitution, P226A, encoded by exon 5 of the RHCE gene.46 Expression studies in human erythroleukaemic cells (K562) transduced using retroviral constructs containing fulllength cDNA from various RH alleles have con®rmed the molecular basis of the common RH alleles and have shown that the product of the RHD gene is a polypeptide carrying the D antigen only, whereas the product of the RHCE gene is a single polypeptide carrying both C/c and E/e antigens.48,49 Although the RhD polypeptide is well characterized, the molecular basis of the D speci®city is not clearly established, since the role of the 35 amino acid residue substitutions that distinguish the D and non-D polypeptides remains largely unknown.39,50 This was ®rst explored through the study of `partial D' individuals who are serotyped as D-positive but may produce anti-D antibodies by transfusion or pregnancy. Indeed, the D antigen is considered to be a `mosaic' composed of several epitopes, some of which are missing in partial D individuals (for review, see Tippett et al51), and it was assumed that the molecular characterization of these variants could provide detailed information on the di€erent D epitopes (epD). Based on serological51 and molecular analysis of the partial Ds (for reviews, see Cartron26, Huang28, Cartron et al29, Faas et al52), working models that correlated D polymorphic positions on external loops of the RhD protein and the individual D epitopes de®ned by cluster analysis using monoclonal antibodies (MAbs) anti-D have been proposed. A simpli®ed version was built on a nine epitope pattern53, and a more complex one on a 30 epitope pattern.54,55 In these models, individual epitopes represent discrete but overlapping motifs on the RhD polypeptide. The epitope models are an oversimpli®cation of a complex situation, since they only point to critical amino acid substitutions exposed on external loops of the D protein that are available for anti-D binding. However, it was pointed out that Rh antigens are quite sensitive to conformation and the amino acid substitutions in intramembranous or intracytoplasmic domains may also be important in the modulation of D protein conformation over long distances.26,29 More recently, expression studies following site-directed mutagenesis of the known D polymorphic positions were tentatively used to better de®ne the molecular basis of D epitopes.56,57 This is a complex problem to solve, however, since D epitopes represent the regions where anti-D antibodies bind and they may not be identical to

Blood group system 659

the 35 polymorphic positions in the RhD polypeptide, since these structural variations may induce conformation changes in the three-dimensional surface of the D antigen, as suggested above. Site-directed mutagenesis probes critical positions for the expression of D epitopes but further studies are required to de®ne the region of contact between anti-Ds and the Rh polypeptide. Additional information was obtained by studying D epitopes from the antibody and at the same time analysing human Fab fragments of anti-Ds produced by an immunized RhD-negative individual using phage-display technology. With this method, Chang and Siegel58 found that these antibodies bound to four distinct D epitopes and were made from a restricted combination of immunoglobulin genes, and that several Fabs that di€er in epitope speci®city used identical heavy chains but di€erent light chains. Moreover, they have also shown by sitedirected mutagenesis of the variable region of anti-D light chains that single amino acid substitutions resulted in a change in epitope speci®city of the antibody.59 These ®ndings suggest that the epitope speci®city of an antibody might change during the course of somatic mutation (a process that was called `epitope migration'). From these ®ndings, these authors postulated that most, if not all anti-Ds bind to the D polypeptide through an essentially identical `footprint' (or a `basic framework of residues'), and that it is the number and arrangement of contact residues on the D polypeptides that de®ne the so-called `D epitopes'. According to this view, a reliable mapping of D epitopes should combine site-directed mutagenesis and a tridimensional determination of the RhD protein structure.

Recognition of the Rh protein family The Rh and RhAG protein sequences (417 and 409 residues, respectively) were deduced from cDNA cloning (for reviews, see Agre & Cartron23, Cartron & Agre24, Anstee & Tanner25, Cartron26). The RhD and non-D (RhCE) proteins are highly homologous (92% identity), and share a substantial homology with the RhAG glycoprotein (36% identity), suggesting that they all belong to the same protein family. Moreover, these proteins show a similar membrane topology for their 12 putative transmembrane (TM) domains (Figure 1). Interestingly, four acidic residues (E21, D95, E146 and E340) are located in the TM segments 1, 3, 5 and 11 of Rh proteins, among which two (E13, E148) are conserved in RhAG. This observation and the membrane topology of Rh and RhAG suggest that these proteins might be involved in transport functions. The concept of protein family was further substantiated by the ®nding that the RH and RHAG genes, which map on chromosomes 1p34.3-p36.1 and 6p11-p21.1, respectively, both consist of 10 exons and exhibit strikingly similar exon±intron organization.60,61 The Rh and RhAG proteins are both erythroid speci®c, but di€er in that RhAG is N-glycosylated (at N37 in the ®rst extracellular loop, Figure 1) while Rh proteins are not, and Rh carries the blood group antigens, while RhAG does not. Both Rh and RhAG proteins contain cysteine residues but their positions are not conserved and none of those from RhAG are in the C-L-P context, which may represent palmitoylation signals used in Rh proteins.62,63 Site-directed mutagenesis demonstrated recently that the unique exofacial cysteine (C285) of Rh proteins is not required for D antigen expression64, as was previously supposed.10,11 Studies of the membrane topology of Rh and RhAG proteins using antibodies directed against the N- and/or C-ter of each protein65,66 suggested that the N- and C-terminal halves of each protein might comprise domains within the lipid bilayer, separated by the trypsin cleavage sites on the intracellular domain 4, each containing six transmembrane helices (Figure 1).

660 J.-P. Cartron

Evolution of the RH gene family Our knowledge of the evolutionary pathway of the RH gene family has made signi®cant progress recently following gene studies in primates and primary sequencing information obtained from a number of living species, including mammals, sponges and nematodes. The two major events that shaped the evolution of the RH gene family were the split between the RHAG and RH evolutionary pathways and the subsequent duplication event that gave rise to the RHCE and RHD genes.67,68 The evolutionary time at which the RHAG and RH genes separated from each other has been estimated at 250±350 million years ago (Mya). This suggests that RH-like sequences might be present in birds (who appeared in evolution about 310 Mya), as they are consistent with Southern blot analyses carried out on chicken DNA.69 The RHAG and RH genes then physically separated, most probably following a translocation of the RH gene to chromosome 1. Indeed, since the RHAG and RH loci are embedded in di€erent compositional genomic contexts characterized by a GC-poor and GC-rich content (L and H1 isochores), respectively, a translocation event of the RH gene towards a GC-rich genomic region (or alternatively to a region that progressively increased its GC content to become an H1 isochore), might have taken place, thereby creating the genomic environment that drove on the evolution of the RH genes.60 The second duplication event was dated at about 8.5 Mya26,43, in the common ancestor of humans, chimpanzees and gorillas, in agreement with classical serological data indicating that chimpanzees and gorillas are the only non-human primates expressing a D-like antigen on their red blood cells (RBCs).70,71 In addition, and most importantly, it was calculated from the number of synonymous and non-synonymous codon positions in the RHAG and RH genes that after their duplication from the common ancestor, the RhAG protein evolved at a rate that is about 2.6 times slower than that of Rh.67,68 Altogether, these results indicate that the RH gene family currently found in mammals originated from a RHAG-like sequence and that the RhAG protein experienced a more conservative mode of evolution. The presence of RHAG-like sequences in simpler organisms such as the nematode Caenorhabditis elegans72 and the sponge Geodia cydonium73, is also suggestive of an evolutionary founding role for a RHAG-like gene. Consistent with this view, the human RhAG protein was found to share a certain degree of homology (20±27% identity) with the Mep/Amt family of NH4‡ transporters60,74 that have been identi®ed from bacteria to yeast and plants, but not in animals. Among the three members of the Mep protein family in yeast, the human RhAG glycoprotein shares the highest similarity with Mep2, particularly in the transmembrane domains 7±11 (24% identity, 40% similarity) (B AndreÂ, pers. comm.). Interestingly Mep2 may also act both as transporter and ammonium sensor in yeast.75 These ®ndings deserve future research studies on the potential function of these molecules.

THE Rh DEFICIENCY SYNDROME Genetic background and haematological features In 1961, Vos et al76 reported the ®rst example of red cells that lacked all Rh antigens known at that time (D, C/c, E/e, Cw, Cx, V, VS etc) in a Australian aboriginal woman, but in the absence of close relatives the inheritance of this phenotype could not be established. When other samples were described, it was discovered that Rhnull RBCs also lack LW antigens and exhibit an aberrant expression of the Ss and U antigens (see

Blood group system 661

below). Moreover, family studies indicated that the Rhnull phenotype arises from two distinct genetic backgrounds (for a review, see Race & Sanger77). The `amorph type' is caused by homozygosity for a silent allele at the RH locus, whereas the more common `regulator type' is caused by homozygosity for an autosomal suppressor gene called X0r, a mutant form of a common X1r gene, which is genetically independent of the RH locus.78,79 Another gene called XQ, which may or may not be allelic to X0r, was thought to be responsible for the Rhmod phenotype, in which residual amounts of Rh antigens are expressed on RBCs.80 Rhnull phenotypes of the `amorph type' and of the `regulator type', including Rhmod , exhibit similar clinical and haematological features to those originally described by Schmidt et al8,81,82, as the `Rhnull disease', but it is now referred as the `Rh-de®ciency syndrome' (for reviews, see Agre & Cartron23, Schmidt & Holland82, Seidl et al83, Sturgeon9). Typically, there is a chronic haemolytic anaemia of varying severity and a persistent moderate reticulocytosis (3±20%). Haematocrit is in the lower range, serum haptoglobin is low and fetal haemoglobin elevated. The red cell osmotic fragility without incubation is often slightly abnormal but becomes markedly increased after 24 hours incubation at 378C. Increased autohaemolyis is correctable by the addition of glucose or ATP. One hallmark of the syndrome is the abnormal red cell morphology, notably stomatocytosis and spherocytosis, as seen using electron scan microscopy (Figure 2). These similarities between Rh de®ciency and hereditary spherocytosis were noted previously.9 Some Rhnull patients may present with signs of accelerated rate of in vivo red cell destruction only. In vivo half-life of autotransfused Rhnull cells has been investigated on only a few occasions and found to be between 7±17 days (normal 24±28 days using the 51Cr method).82±84 In severe cases (®ve have been published),

Figure 2. Scanning electron microscopy of glutaraldehyde-®xed Rhnull RBCs showing stomatocytosis and spherocytosis. (Courtesy of W. Marsh and C. Redman, New York).

662 J.-P. Cartron

clinical improvement was observed after splenectomy with a normalization of autologous life-span in most cases83, but residual haemolysis may occasionally persist9, suggesting that spleen sequestration may not be the only factor responsible for the anaemia, as postulated before.85 In hereditary spherocytosis, splenectomy cures almost all patients, although the red cell life-span remains slightly shortened when carefully measured.86 Biological features Rhnull cells of the amorph type (from patient BR, sister of patient DR, see below) also exhibited increased rates of passive and active cation transport (Na, K) and of Na±K membrane ATPase activity.87 Further studies of an Rhnull patient of the regulator type (KM) con®rmed the cation transport abnormalities, which resulted in red cell dehydration and an abnormal reduction of red cell surface area as detected by osmotic gradient ektacytometry, presumably explaining the increased osmotic fragility and suggesting membrane instability in vivo.88 Moreover, there was a relative de®ciency of membrane cholesterol.88 Examination of two Rhnull samples of the regulator type (patients AL and YT, see below) indicated these RBCs have an increased amount of phosphatidylethanolamine (PE) accessible to hydrolysis by exogenous phospholipases and that phosphatidylcholine (PC) can be exchanged with exogenous PC from rat liver microsomal membranes, suggesting an altered phospholipid transbilayer asymmetry and an enhanced passive phospholipid transmembrane ¯ip-¯op.89 The red cell enzymes of anaerobic and aerobic glycolytic pathways are normal. However, as discussed below, Rhnull cells exhibit multiple antigen and membrane protein defects, including the lack, or severe decrease, of Rh and RhAG proteins. Rhnull cells di€er from the cells of patients with hereditary stomatocytosis, since the latter carry normal Rh polypeptides of 30±32 kDa but are defective in the membrane protein 7.2b (stomatin), whereas the former lack Rh proteins but have a normal amount of protein 7.2b.90 Recent studies indicate that RBCs from stomatin-de®cient mice show no sign of stomatocytosis, suggesting that protein 7.2b plays no role in the aetiology of this disorder91 (see also Chapter 1 of this issue). All the alterations of the Rhnull cells suggest that this phenotype results from a genetic disorder causing a basic membrane defect with pleiotropic e€ects on red cell properties and function. Antigen and protein abnormalities of Rhnull cells The study of Rhnull phenotypes has revealed that Rh antigens are unexpectedly complex. Indeed, Rhnull erythrocytes not only lack all Rh antigens, but they lack also the LW and Fy5 blood group antigens (but the Fya/b and Fy3 antigens are present) and they have a reduced expression of blood group Ss, U and Duclos antigens.77,92,93 In some cases, an elevation of the i antigen, attributed to the bone marrow stress caused by the associated anaemia, was noted. The genetic background of Rhnull samples, however, cannot be distinguished on the basis of serological studies. Initial studies using a membrane impermeable maleimide have shown that Rhnull RBCs lack the Rh polypeptides of 32 and 34 kDa containing extracellular thiols.94 Further studies from di€erent laboratories, based on immunoprecipitation and Western blot analyses, using polyclonal and monoclonal antibodies, have con®rmed and extended these observations and revealed that other proteins, such as RhAG and CD47, are also missing or severely reduced on Rhnull erythrocytes (Figure 3). Neither

Figure 3. Protein abnormalities of Rhnull erythrocytes. Red cell membrane proteins from unrelated Rhnull individuals (investigated in our laboratory) were separated using SDS-PAGE, transferred on nitrocellulose sheets and immunostained with rabbit polyclonal antibodies directed against the Rh polypeptides (MPC4, MPC8) and with murine MAbs directed against the RhAG protein (clone 2D10), CD47 (clone B6H12) and the blood group LW protein (clone BS46). A rabbit antibody directed against the membrane protein p55 was used as a control to show that similar amounts of membrane proteins were present on each blot. After incubation with goat anti-mouse or antirabbit IgG conjugated to alkaline phosphatase, speci®cally bound antibodies were detected by chemiluminescence. A. Rh null of the regulator type. B. Rhnull of the Rhmod type. C. Rhnull of the amorph type. Individual patients are indicated by initials. Control RBCs were from RhD-positive (DCCee) and RhD-negative (ddccee) donors. The size (kDa) of the detected bands is indicated.

Blood group system 663

664 J.-P. Cartron

the Rh proteins detected by the polyclonal antibodies MPC-1 and MPC-8, nor the RhAG glycoprotein detected by the monoclonal antibody 2D10 could be seen on RBCs from Rhnull patients of the regulator type (Figure 3A). The RhAG protein, however, was severely reduced but not completely missing on RBCs from those Rhnull patients of the Rhmod and amorph type that were investigated (Figures 3B & 3C), which reacted with anti-U reagents (other Rhnull samples being U-negative). CD47 protein detected using the MAb B6H12 was severely reduced, whereas the LW glycoprotein detected using the MAb BS46 was missing from all RHnull cells (Figure 3). Other studies using SDS-PAGE followed by periodic acid Schi€ (PAS)-staining of red cell membrane sialoglycoproteins have shown that GPB (Ss glycoprotein) was reduced by 60±70% on Rhnull cells.93 No detectable abnormality of mobility and staining of Band 3 was noted (not shown). The proteins that are phenotypically associated with Rh and that exhibit altered expression on Rhnull cells have been characterized and their main properties are summarized in Table 1. Remarkably, all these proteins are encoded by genes located on di€erent chromosomes. Rh, RhAG and GPB proteins are quantitatively the most, and LW the least, abundant species, respectively. CD47 and Du€y proteins are of intermediate abundance. CD47 is severely reduced on Rhnull erythrocytes and the residual amount was estimated to be 10±20% of the normal level using ¯ow cytometry.95 CD47, also called integrin-associated protein (IAP), is a widely distributed multispanning membrane glycoprotein of 47±52 kDa, which is a member of the immunoglobulin (Ig) superfamily, with a N-terminal extracellular domain composed of a single Ig-like (V) domain.96,97 No blood group antigen has been found on CD47 so far. CD47 is physically associated to b3 integrins in placenta, platelets and endothelial cells and may function as an integrin-associated calcium channel in endothelial cells.98 Recent studies suggest that CD47 (IAP) may serve several functions, having a role in the transendothelial migration of neutrophils99, as a thrombospondin receptor100, and a co-stimulatory role in T cell activation.101 The function of CD47 on RBCs (which lack integrins) is not known, but it has been postulated that the decreased expression of CD47 in Rhnull cells could contribute to the ion ¯ux abnormalities found in Rh de®ciency.102 This is uncertain, however, since CD47 is decreased to a similar extent on other Rh variants (D - -, DCw-, D. .) which express RhAG normally103 (and unpublished results). Since CD47 is normally expressed on non-erythroid haematopoietic cells from Rh-de®cient individuals97 (and unpublished results), the low expression of this protein on Rhnull erythrocytes does not result from a gene alteration but more probably from defective assembly or transport to the cell surface in the absence of Rh proteins. The LW and Rh antigens are phenotypically related, since LW antigens are absent from Rhnull cells77±79 (see Figure 3). Moreover, the level of LW antigen expression is greater in RhD-positive than RhD-negative adult RBCs (4400 versus 2800 copies/cell), suggesting that the RhD protein might facilitate the cell surface expression of LW. LW is a single spanning transmembrane glycoprotein of 37±47 kDa restricted to erythrocytes, which is also a member of the Ig superfamily, with a N-terminal extracellular domain containing two Ig-like domains (C2 , C2).104 The LW protein exhibits strong sequence similarities (overall identities within a range of 30%) with the intercellular adhesion molecules, ICAM-1, -2 and -3, which are the counter-receptors for the lymphocyte function-related antigens, LFA-1. Although some critical residues involved in the binding of LFA-1 to ICAMs were only partially conserved in LW, recent studies have shown that LW still binds to the b2 integrins LFA-1 and Mac-1, and it was renamed ICAM-4.105 Preliminary studies suggest that LW might also bind b1

ICAM-4

Ss glycoprotein

DARC

LW

GPB

Du€y (Fy5)

35±45

20

37±47

47±52

45±75

30±32

Mr [10ÿ3]

{ Other antigens absent but not characterized: U, Duclos.

Rh50 glycoprotein

IAP

RhAG

Rh30 protein(s)

Rh (D & CE)

CD47

Synonym

Subunit

12±17

80±300

3±5

10±50

100±200

100±200

copies/cell [10 ÿ3]

1q23

4q28-q31

19p13

3q13

6p11-p21

1p34-p36

Gene localization

U01839

JO2982

L27671 X93093

X69398

X64594

X63097 (D) LO8429 (D) M34015 (cE) X54534 (cE)

Absent (Fya/b, Fy3, Fy6 present)

Reduced by about 70%

Absent

Reduced by about 80±90%

Absent (reduced on Rhnull Uweak)

Absent (weak on Rhmod)

Accession number Expression on Rhnull RBCs

Table 1. Protein subunits phenotypically associated with Rh.{

Blood group system 665

666 J.-P. Cartron

integrins106 suggesting a wider speci®city and a potential role in a variety of adhesion events or cell interactions, but its biological role in vivo, if any, is as yet unknown. Glycuphorin B (GPB) is a well-characterized single membrane spanning protein carrying Ss antigens, which belongs to a small gene family located on chromosome 4q28q31 that includes glycophorin A (a carrier of MN antigens) and glycophorin E (for reviews, see Cartron & Rahuel107, Fukuda108). There is a phenotypic relationship between the expression of Ss and U antigens, since most RBCs typed as S-s- lack GPB and are also Uÿ 77, but the biochemical nature of the U antigen is unknown. Rhnull RBCs contain a reduced amount of GPB93, which might explain the aberrant expression of SsU antigens on these cells, since most (mainly of the regulator type) were found to be Ss-positive but U-negative, whereas amorph-type Rhnull are Ss-positive and U‡ or Uweak.8,81,82 Dahr et al93 also postulated that Rh and GPB proteins might form a complex, and that Rh proteins facilitate the membrane incorporation of GPB and contribute to the complete expression of U (and Duclos antigens) on normal cells. Recent studies suggest that such a complex exists (see below) but involves an interaction between GPB and RhAG.109 Indeed, Western blot analysis with MAbs against RhAG indicated ®rst that this glycoprotein was present in U‡ but not in Uÿ Rhnull RBCs, suggesting that the U antigen may result from the interaction between GPB and RhAG.21,22 This is reminiscent of the Wrb antigen, which results from an interaction between GPA and Band 3.110,111 Further studies using Western blot with a RhAG anti-Cter antibody have shown that RhAG from U‡ Rhnull RBCs has a faster mobility than RhAG from wildtype U‡ RBCs and that RhAG from S-s-Uÿ and S-s-U‡ RBCs migrated slower than RhAG from wild-type RBCs that carry GPB.108 These di€erences in mobility, attributed to di€erences in glycosylation, suggested that the transit time of RhAG in the Golgi is modi®ed according to the presence or absence of the GPB and Rh proteins. Accordingly, GPB might accelerate and Rh retard, respectively, the movement of RhAG to the cell surface. Further studies based on stable K562 transfectants expressing recombinant Rh protein with or without Band 3 have shown that Band 3 enhances the cell surface reactivity of Rh antigens and of the endogenous RhAG protein, thus suggesting some interaction between Band 3 and Rh components.111 Rhnull cells do not react with anti-Fy5 antibodies (Table 1), which like anti-Fy3 do not react with Fy(a-b-) RBCs. However, Rhnull cells are not Fy3-negative and express normal levels of other Fy antigens77, presumably indicating that the Fy5 epitope might result from an interaction between Rh and Du€y proteins. Thus the Fy protein is present but cannot interact with Rh on Rhnull RBCs. The protein carrying the Fy antigens is a multispanning transmembrane glycoprotein that has a dual biological role as receptor for the Plasmodium vivax malarial parasites and for a family of proin¯ammatory cytokines named chemokines (for a review, see Hadley & Peiper112). Accordingly, the Fy protein is also called the Du€y antigen receptor for chemokines (DARC). Recent studies suggest that DARC-positive RBCs bind HIV-1 particles, suggesting that RBCs may function as a virus reservoir and as a receptor for entry of HIV-1 in some cells.113 THE Rh COMPLEX HYPOTHESIS The Rh complex It is well established that Rhnull red cells exhibit multiple protein abnormalities (Figure 3; Table 1). Because the genes encoding these proteins are located on di€erent chromosomes, the most likely hypothesis is that Rh proteins exist in the erythrocyte membrane in the form of a non-covalent complex with other proteins (RhAG, CD47, LW, GPB),

Blood group system 667

which are all collectively absent (or greatly reduced) in Rhnull individuals. That Rh proteins form a complex with GPB (the carrier of Ss antigens) was ®rst proposed more than 10 years ago93 and this concept, further extended to other membrane proteins missing on Rhnull cells, has become a widely accepted working model.23±26,114 A schematic representation of the Rh complex is shown in Figure 4. The core of this complex is thought to be a tetramer composed of two Rh and two RhAg subunits66, as suggested previously.23 This is consistent with Rh antigen size estimations of 170 and 174 kDa deduced from the hydrodynamic analysis of Triton X-100-solubilized membranes63 and from radiation inactivation115, respectively. Accessory chains CD47, LW and GPB are associated by non-covalent linkages to the core of the complex. In situ proteolysis of intact and leaky ghosts followed by immune precipitation with MAbs anti-Rh also suggested that the Rh protein might interact via a N-terminal region of RhAG located between an extracellular Staphylococcus aureus V8 protease cleavage site at E34 and a cytoplasmic trypsin cleavage site at K19666 (see Figure 1).

Figure 4. The Rh membrane complex. The diagram illustrates the putative organization of the Rh membrane complex. It is assumed that the core is a tetramer composed of two Rh proteins (dark grey) and RhAG glycoprotein (white) subunits. Accessory chains CD47 (hatched), LW (light grey) and GPB (black), encoded by independent genes, are also associated with the complex. To date, no blood group antigen has been identi®ed on CD47 and RhAG.

It is assumed that when one protein subunit is missing the complex is not assembled and/or transported to the cell surface, thus leading to the abnormalities seen on Rhnull cells. A similar mechanism is well documented for other biological models such as the T cell receptor, platelet receptors gpIIb-IIIa, the gpIb-IX-V complex, the dystrophin complex, etc (see Agre & Cartron23 and references therein). Experimental evidence supporting the existence of the Rh complex has been provided above. Other evidence includes (i) the isolation of Rh antigens (not glycosylated) on lectin±Sepharose columns20; (ii) the co-precipitation of RhAG with anti-Rh antibodies19; (iii) the co-precipitation of Rh with murine MAbs directed against the LW116 and RhAG22 proteins. Because of these protein associations, it is reasonable to assume that some antigenic determinants might be de®ned by an interaction between the Rh protein subunits, in particular between Rh and RhAG which make up the core of the complex. Further studies are required to clarify this question, either by expression analysis of Rh and RhAG in eukaryotic cells or by the puri®cation of individual proteins and their reconstitution in arti®cial vesicles. Preliminary studies enabling the immunopuri®cation of Rh and RhAG proteins should provide key information soon.117 The Rh complex might play some role in membrane architecture, possibly by interacting with the skeleton and/or by contributing to ion transport properties

668 J.-P. Cartron

and/or controlling directly or indirectly the phospholipid asymmetry (functions that are altered in the Rhnull syndrome). A study of the structure, assembly and function of the Rh complex is therefore essential for the overall understanding of the Rhnull syndrome and for correlating the molecular abnormalities of those cells with dysfunctions.

Interaction of the Rh complex with the membrane skeleton Indirect evidence based on solubilization by non-ionic detergent suggests that Rh proteins are linked to the membrane skeleton.16,17,118 Recently, more direct evidence was provided by ¯uorescence imaged microdeformation (FIMD), in a collaborative study between two groups (J.-P. Cartron in Paris, N. Mohandas in San Francisco, unpublished results). The FIMD method119 quanti®es the redistribution of ¯uorescently labelled red cell membrane (RCM) proteins during a mechanically induced membrane deformation, following cell aspiration into a micropipette (Figure 5). Consequently, the spectrinbased membrane skeleton deforms elastically, producing a constant gradient during deformation, leading schematically to a sorting of skeletal-linked and skeletal non-linked membrane components. When the ratio of protein density at the entrance and at the cap of the aspirated projection is plotted against the distance along the deformation axis (Figure 5), a positive correlation is seen for a skeletal protein such as actin (because the protein density at the cap decreases).119 Previous studies have shown a negative correlation of this ratio for the glycosyl phosphatidyl inositol (GPI)-linked CD59, which is free to move in the phospholipid bilayer, since the density increased at the cap with time.120 Band 3, which is known to have a restricted mobility within the lipid bilayer, has a behaviour that is intermediate between actin and CD59.119 When FIMD technique was used to determine the redistribution of RhD and RhAG, it was shown that the gradients were steeper than those found for Band 3 and very close to those of actin (our unpublished data). These ®ndings provide direct evidence that the Rh complex is linked to the spectrin-based membrane skeleton. However, which protein(s) of the Rh complex interacts and which skeletal protein(s) is involved are so far unknown. Although some evidence suggests that Band 3 may interact with the Rh complex111 (and see above), the di€erence in behaviour of the Rh/RhAG proteins and Band 3 in FIMD experiments suggests that the Rh complex probably interacts directly or indirectly with another (skeletal ?) membrane component. Interestingly, transgenic mice that lack Band 3 exhibit an hereditary spherocytosis that results in a very severe anaemia, but surprisingly RBCs from these animals assemble an essentially normal membrane skeleton in the absence of Band 3121,122, although previous speculation had suggested that Band 3 was required to form the membrane skeleton.123 It is likely therefore that other ecient membrane attachment sites between the skeleton and the bilayer exist. One that already has been recognized is the glycophorin C±protein 4.1±p55 ternary complex at the junctional complex124, and another might be the Rh complex. Evidence for a putative interaction between Rh and Band 3 should come from studies indicating whether or not RBCs from Band 3-de®cient animals express normal levels of Rh-like polypeptide. These ®ndings suggest that in the absence of the Rh complex normal interactions with the membrane skeleton and the phospholipid bilayer are altered, resulting in the morphological and functional abnormalities seen in Rhnull cells.

Blood group system 669

Figure 5. Fluorescence-imaged microdeformation (FIMD) of human RBCs. A. Schematic representation of a hypotonically swollen RBC aspirated into a micropipette (diameter ˆ 2  Rp) following a hydrostatic pressure (DP ˆ 0.02 atmospheres). The extent of deformation is set by the projection length (L) inside the pipette, which is controlled by prior osmotic adjustment of the cell volume.120 B. Fluorescence image showing the distribution of the skeletal protein actin (revealed using rhodamin±phalloidin labelling) at the entrance and at the cap of the aspirated membrane projection.

Critical proteins of the Rh complex An important aspect of the model shown in Figure 4 is to determine which protein(s) plays a critical role in the formation of the Rh complex. Obviously, from the known genetic background of amorph-type Rhnull variants, it is expected that Rh polypeptides have an important role since they carry the Rh antigens and the Rh complex is absent or very severely reduced when these proteins are missing (Figure 3). Neither the RhD protein itself, nor the RhCc/Ee protein by itself, has a critical role since they are absent from RhD-negative and Dÿ ÿ erythrocytes, respectively, and these exhibit no membrane abnormality and express RhAG normally. Likewise, erythrocytes lacking GPB (phenotype (S-s-U-)), the LW glycoprotein (phenotype LW(a-b-)), or the Du€y protein (phenotype Fy(a-b-)) are phenotypically normal and not de®cient in Rh or RhAG proteins. It is not known, however, whether or not human erythrocytes that are selectively de®cient in the RhAG or CD47 proteins, which have not been described, may express Rh antigens. Recently, gene-targeted mice de®cient in CD47 have been generated using homologous recombination.125 The lack of CD47 did not compromise the function of RBCs, but the animals exhibited a decreased resistance to bacterial infection and defects in granulocyte functions such as b3 integrin-dependent ligand binding, activation of oxidative burst and Fc-receptor mediated phagocytosis. Western blot analyses of CD47de®cient RBCs using MAbs have shown recently that these cells carry a normal amount of Rh and RhAG proteins, thus suggesting that CD47 is not strictly required for Rh expression, at least in mice (our unpublished results).

MOLECULAR GENETIC ANALYSIS OF Rhnull PATIENTS The Rhnull phenotype is very rare and fewer than 50 unrelated individuals have been described.6,7,77,84 Currently 15 unrelated patients of di€erent ethnic origins have been examined in di€erent laboratories, including ten Rhnull of the regulator type, three Rhmod and two Rhnull of the amorph type (Tables 2 and 3), a number to be compared

Spanish

Japanese

Japanese

White South African White South African Swiss White Australian Japanese

AC

±

TT

SF

D, CE

D, CE D, CE D, CE

CE D, CE D, CE

D, CE

D, CE

D, CE

D, CE

D, CE

D, CE

RH locus

G!T

A deletion G!A G ! A, G!A G!T G!A G!T

CCTC ! GA

e8

e9 e2 e1

e8 e6 e6

e1

e1

i7

i6

i6

i1

Intron or exon

RHAG

CCTC ! GA

G!A

G!T

G!A

G!A

Mutation

{ ‡1 is A of ATG initiation codon of the RHAG cDNA. {Frameshift after the indicated codon. } The size of the normal RhAG glycoprotein is 409 residues. kComposite heterozygote (see text for the second null allele).

CB (mod)k

WO VL (mod) SM (mod)

TBk YTk HT

Japanese White American Jewish Russian origin French

White American

AL

JL

Race

Rhnull individuals

1195

1139 236 3

1086 836 808, 838

154±157

intron 1, 50 splice-site intron 6, 30 splice-site intron 6, 30 splice site intron 7, 50 splice site 154±157

Position on RHAG{

Missense D399Y

Missense G380V Missense, S79N Missense, M1I

Frameshift codon 362 Missense, G279E Missense, V270I, G280R

Frameshift codon 51

Skipping of exon 7 Frameshift codon 315 Skipping exon 7 Frameshift codon 315 Skipping exon 7 Frameshift codon 315 Frameshift codon 51

No RhAG transcript

E€ect of RHAG mutation{

Table 2. Mutations of RHAG gene in Rhnull individuals (reg and mod types).

409

409 409

376 409 409

107

107

351

351

351

Predicted RhAG protein size}

our unpublished results

135 95 136

95 132±134 135

95

95

61

131

130

95,130

Reference

670 J.-P. Cartron

Spanish

German

DAA

DR

RHCe

RHce

RH locus

TCA ! C

G!T

RHCE mutation

{ ‡1 is A of ATG initiation codon of the RHCE cDNA. { Frameshift after the indicated codon. } The size of the normal Rh protein is 417 residues.

Race

Rhnull individuals

966±968

intron 4, 50 splice site

Position on RHCE{

Frameshift, codon 322

Aberrant transcripts

E€ect of RHCE mutation{

Table 3. Mutations of RH gene in Rhnull individuals (amorph type).

398

(231±312)

Predicted Rh protein size}

152, 153

152

Reference

Blood group system 671

672 J.-P. Cartron

to the fewer than 50 cases published so far. To establish the molecular basis of these phenotypes, the transcript and the genomic DNA encoding the protein subunits of the Rh membrane complex have been analyzed. mRNAs were ampli®ed using reverse transcriptase polymerase chain reaction (RT-PCR) from reticulocytes of the peripheral blood, then cloned and sequenced. In some cases, cDNA was introduced in appropriate vectors for transcription-coupled translation analysis. The mutation detected in the transcript was often con®rmed by genomic DNA analysis and on several occasions the exon/intron junctions of the RHAG or RH genes were ampli®ed using PCR and sequenced. Finally, where possible, the inheritance of the mutation in family members was established. These studies revealed that abnormalities occurred only at the RHAG and RH loci, without alteration of the genes encoding the accessory chains CD47 and LW.

Alteration of the RHAG gene in Rhnull individuals Currently, three categories of mutations within the RHAG gene (splice-site mutations, nucleotide deletions and missense mutations) that resulted in the total absence, or the severe reduction in some cases, of the RhAG glycoprotein on RBCs have been identi®ed. All mutation events occurred either in Rhnull individuals classi®ed as `regulator type' or in Rhmod . The RH gene transcripts, however, have a normal nucleotide sequence, but the Rh antigens are not present (Rhnull reg) or are very weakly expressed (Rhmod) on RBCs. All patients have at least one copy of a RH haplotype carrying the RHD and RHCE genes, except in one case in which the RHD gene is lacking. Table 2 summarizes the available information and Figure 6 illustrates the position of the RhAG mutations showing that they occur either in the N-ter or C-ter regions of the protein.

A

B

RhAG glycoprotein Patients SF & JL Frameshift Y51

Rh protein

Patients AC, TT, Jap Frameshift T315 Patients TB# Frameshift A362

N 37

1 Patients SM** Missense M1I

Patient DR Frameshift I322

1 Patient HT Missenses V27OI, G280R

Patient VL** Missense S79N

409

Patient YT# Missense G279E

Patient WO Missense G380V

Patient DAA Frameshift L211

417

Patient CB**# Missense D399Y

Figure 6. Topology models of the Rh and RhAG proteins showing the distribution of the mutations occurring in Rhnull individuals. Detailed descriptions of the mutants are given in Tables 2 and 3. A. Mutations within the RhAG protein. Patients presenting frameshift and missense mutations are indicated above and below the protein model, respectively. Patient AL (see Table 2) presenting with a splice-site mutation of intron 1 was not represented, since no transcript could be detected. Patient HT carries two mutations in cis (V270I and G280R). #, Composite heterozygote; **Rhmod. B. Mutations within the Rh protein. Both occurred in a RhD-negative background (absence of the RHD gene) and a€ected the RHCE gene.

Blood group system 673

Splice-site mutations Splice-site mutations cause a shift in the translational reading frame of the mRNA and result in the premature termination of translation. The spliced transcripts are translated into putative shortened protein form(s), often with a new C-terminal extension not found in the wild-type protein. In some cases, however, the mutation causes a complete inactivation of the RNA processing. A reduction in the levels of mRNA associated with splice mutations and premature termination codons has been reported for many mutant alleles from a variety of genes.126±130 Patient AL. Preliminary investigations have shown that the RHAG transcript could not be detected using PCR in reticulocyte preparations, although normal RH, LW and CD47 messenger RNAs were ampli®ed and sequenced.95 After sequencing a genomic fragment spanning exon 1 and part of intron 1, a g ! a transition (gt ! at) at the invariant g residue of the 50 donor splice site of intron 1 was discovered130, creating a novel Rca I restriction site polymorphism. PCR-restriction fragment length polymorphism (PCR-RFLP) assay based on this polymorphism showed that patient AL was homozygous for this mutation. Ribonuclease protection assay (RPA) con®rmed that the RHAG mRNA was not detectable, indicating that the mutation caused a complete inactivation of RNA processing. This may be accounted for by the instability and the consequent degradation of the misspliced RNA in the nucleus due either to the inclusion of intron 1 (about 17.7 kb) and/or to the appearance of a premature stop codon. Patient AC. Sequencing of the RHAG cDNA, ampli®ed, from reticulocyte mRNAs using RT-PCR revealed a loss of 122 bp, from position 946±1067, as compared to the wild-type RHAG sequence, corresponding to the entire transcribed region of exon 7 (encoding the TM11 segment). These results suggest that an exon had been skipped during the processing of the pre-mRNA, which may well be the result of a 30 acceptor splice-site mutation localized upstream from nucleotide position 946.130 Genomic analysis con®rmed this hypothesis since a g ! a transition at the g invariant residue of the 30 acceptor splice-site (ag ! aa) of intron 6 was identi®ed, and direct sequencing indicated that patient AC was homozygous for this mutation. This mutation led to the skipping of exon 7 and introduced a shift in the reading frame after the codon for threonine-315 that resulted in a premature stop codon, 108 bp downstream, located in the exon 9 region of the RHAG gene (Table 2; Figure 6). The predicted translated protein has ten putative transmembrane domains and is 351 residues long (instead of 409) including a stretch of 36 novel residues at the C-terminus.130 The mutant protein, however, was not found on RBCs since no reactivity of the 2D10 MAb could be detected using ¯ow cytometry analysis or Western blot analysis (Figure 3A). RPA analysis indicated that the AC transcript was present in a reduced amount, compared to controls, and might be unstable. Japanese patients. A splice-site mutation very similar to that identi®ed in patient AC (Spanish origin) was identi®ed in a young Japanese boy who was apparently healthy but showing a mild chronic haemolysis.131 Indeed, a g ! t transition at the g invariant residue of the 30 acceptor splice-site (ag ! at) of intron 6 was identi®ed, resulting in the skipping of exon 7. Recent investigation of another Japanese patient (TT) revealed a g ! a transition in the invariant gt motif of the 50 donor splice-site (gt ! at) of intron 7, also resulting in the skipping of exon 7.61 Direct sequence analysis131 and

674 J.-P. Cartron

PCR-RFLP polymorphism of a Pml I restriction site61 indicated that these patients were homozygous for their mutations. The spliced transcript and the predicted mutant protein of both Japanese patients were identical to those from patient AC (see above) indicating that distinct molecular defects may result in the same Rhnull phenotype (Table 2). Nucleotide deletions Nucleotide deletions may also cause a shift in the translational reading frame of mRNAs, which is often associated with a premature termination of translation. Patients SF and JL. Both patients are white individuals originating from South Africa and are apparently unrelated. Sequence analysis of reticulocyte RNAs reverse transcribed to cDNA revealed the same nucleotide (nt) alterations (nt 154±157), which consisted of two nucleotide changes and a 2 bp deletion (CCTC ! GA) at the end of exon 1 (Table 2). The molecular basis of these changes is not understood. These mutations introduced a frameshift after the codon for tyrosine-51 (®rst extracellular loop; Figure 6) and resulted in a premature stop codon at nt 323±325.95 The deletion was con®rmed by sequencing a genomic fragment encompassing nt 154±157. Moreover, no alteration of the 50 and 30 splice-site consensus sequences of intron 1 was detected. The mutation destroys a Mnl I restriction site and PCR-RFLP assay using this polymorphism showed that both patients were homozygous for the mutation. The predicted translation product would be a very short peptide of only 107 residues including a new C-ter extension of 56 residues (Figure 6), but is not detected on RBCs using Western blot analysis (Figure 3A). Patient TB. Examination of the RHAG transcript from patient TB revealed a single base deletion of nt A1086 (exon 8), which introduced a frameshift after the codon for alanine-362 and resulted in a premature stop codon at nt 1130±1132.95 The deduced RhAG protein would be 376 residues long (versus 409), including 14 new residues at the C-terminus. The A1086 deletion abolishes a Pvu II restriction site and PCR-RFLP using this polymorphism indicated that TB was heterozygous for this mutation. Attempts to amplify the product of the second silent RHAG allele of TB have been unsuccessful to date, strongly suggesting that this transcript was either absent or poorly represented in reticulocytes, and the molecular basis of the second silent allele remains unknown. The RhAG mutant protein encoded by the A1086 allele was not detected on RBCs using Western blot analysis with the MAb 2D10 (Figure 3A). Missense mutations Most interestingly, in several patients missense mutations that changed a single amino acid in the RhAG protein were identi®ed, but the red cell expression of the protein detected using serological tests and/or Western blot analysis with the MAb 2D10 was either severely reduced in some cases or undetectable. Patient YT. This patient, originating from Australia, is a composite heterozygote carrying two mutations in trans, and was identi®ed independently by two groups.132±134 The ®rst allele arose by a single nucleotide exchange, G836A (exon 6), yielding a missense non-conservative mutation, G279E, located in the TM9 segment of the RhAG protein (Figure 6). This allele was inherited from the mother propositus and could be detected

Blood group system 675

using PCR-RFLP after Mnl I endorestriction cleavage. The second mutant allele resulted from a 50 donor splice-site mutation of intron 1 (gt ! at), detectable using PCR-RFLP after Rca I cleavage, and was inherited from the father of YT. This mutation is identical to that found in patient AL (see above) and completely abolished RNA processing. Both mutations were con®rmed by genomic analysis. Japanese patients. Three new missense mutations have been described recently in two Rhnull Japanese patients.135 In patient HT, the RHAG gene carries two cis G ! A transitions at nt 808 and 838 (exon 6), leading to V2701 and G280R substitutions, located in the ®fth intracellular region and the TM9 segment of the RhAG protein, respectively (Table 2; Figure 6). It is currently unknown whether or not each of these mutations is deleterious when individually expressed. In patient WO, there is a single G1139T transversion (exon 9) that resulted in a G380V substitution in the TM12 segment of the RhAG polypeptide (Figure 6). The G ! T transversion, which is located at the ‡1 position of exon 9, also a€ected the pre-mRNA splicing and caused partial exon 9 skipping (which resulted in a frameshift and premature chain termination leading to a putative C-ter truncated product of 381 residues). Agglutination and immunoblotting analysis indicated that neither the Rh proteins nor the RhAG glycoproteins could be detected on the patient's RBCs. Rhmod patients VL, SM and CB. The ®rst missense mutation in the RHAG gene was described in the Rhmod patient VL95, where transcript analysis revealed a G236A transition (exon 2) that resulted in a S79N substitution located in the second intracellular region of the RhAG polypeptide (Table 2; Figure 6). The mutation was found on the genomic DNA and did not correspond to a common polymorphism of the RHAG gene. A trace amount of RhAG protein was detected on RBCs (Figure 3B). Another missense mutation caused by a single transversion G3T (exon 1) at the initiating codon and resulting in a M1I substitution in the RhAG protein136 was found in patient SM (Table 2). Sequence analysis of a genomic fragment spanning exon 1 revealed that SM was homozygous for this mutation. In vitro transcription coupledtranslation assays of the RHAG transcript from patient SM also indicated that truncated RhAG protein(s) could be translated from downstream AUGs (Met8 and 16) by a leaky translation mechanism, as has been found for translation of the GPC and GPD glycoproteins from the unique transcript produced by the GE (Gerbich) blood group gene.137,138 Because translation initiation at Met8 or Met16 would alter TM1139, the `truncated proteins' might be misfolded, and/or unable to be assembled correctly with the Rh protein, and therefore SM's RBCs contain only traces of RhAG protein (with a truncated N-ter ?), but no Rh protein. The third example of a missense mutation was identi®ed more recently in a Rhmod patient (CB) of French origin (B. CheÂrif-Zahar & J.-P. Cartron, unpublished results). A G1195T transversion (exon 8) caused a D399Y substitution in the intracellular C-terminal domain of the RhAG protein (Table 2; Figure 6), and a trace amount of the RhAG protein was present on the RBCs (Figure 3B). Patient CB was heterozygous for this mutation, as revealed by direct sequencing, but the second silent allele has not been characterized. X1r, X0r and XQ are alleles at the RHAG locus Pioneer studies by Cherif-Zahar et al95 have shown for the ®rst time that Rhnull individuals of the `regulator type' and of the Rhmod type exhibited mutations of the RHAG gene, but not of the other genes encoding proteins of the Rh complex. These

676 J.-P. Cartron

®ndings suggested that the RHAG gene is the most likely candidate to be the `regulator' or `suppressor' gene not linked to the RH locus ®rst described by Levine et al.78,79 Nonlinkage with the RH locus, which is mapped to chromosome 1p34, is obvious since the RHAG gene has been assigned to chromosome 6p21-p1195,139, and Rhnull patients (reg or mod type) do not express any Rh antigens but pass functional RH genes to their progeny. Accordingly, RH transcripts with a normal sequence are present in these patients. Further analysis of new Rhnull and Rhmod individuals have largely con®rmed these ®ndings (Table 2, and references therein). Obviously, the RHAG gene does not `regulate' the transcription of the RH locus, but encodes a protein subunit of the Rh complex, thus providing strong support for the model postulating that when one polypeptide chain is missing the complex is not expressed at the cell surface. The wild-type form of the RHAG gene corresponds to the common X1r gene (see above), which produces a functional RhAG protein and RHAG gene mutations may account for the X0r alleles causing the Rhnull phenotype of the regulator type. Moreover, since RHAG mutations occur in the Rhmod phenotype, the XQ genes (see above) are also alleles at the RHAG locus and not the product of another independent gene. A variable `expressivity' of the RHAG mutations may be responsible for the varying degree of Rh antigen expression between Rhnull regulator and Rhmod phenotypes, but the factors that control such variations are unknown. The list of RHAG mutations causing Rhnull phenotypes (reg or mod) will probably grow as more cases are investigated. The possibility remains, however, that some cases of Rhnull (reg or mod) might be caused by the defect of another gene independent of RH, particularly if a new protein component that is critical for the membrane expression of the Rh complex is discovered. Since the X1r, X0r and XQ genes all represent alleles at the RHAG locus, the former nomenclature should be abandoned. Rhnull variants (reg or mod types) could be designated using the symbol RHAG.name or RhAG.name for the gene and protein, respectively, of a given variant: for instance, RHAG.YT and RHAG.TT would describe the RHAG gene from patients YT and TT, respectively (Table 2). Molecular mechanisms involved in Rhnull of the regulator type (reg and mod) Although extensive studies of the RHAG polymorphism have not been reported, it is striking that all RHAG mutations described so far are deleterious and have been identi®ed in Rhnull patients. As a result, the RhAG protein, as well as other members of the Rh complex, is absent or severely reduced on RBCs. In all cases examined, the RH genes were structurally intact and normally transcribed, but the Rh proteins were not present on RBCs. However, some evidence for the presence of a structurally homologous Rh-like protein was reported in one Rhnull patient of the regulator type (patient AL: J. Moulds, pers. comm.), using a sensitive technique of cell surface labelling with a 125 I-labelled cysteine-speci®c probe.140 The Rh-like component of patient AL was not detected using the less sensitive Western blot analysis performed with anti-Rh antibodies (Figure 3A). The RHAG mutations may a€ect the transcription of the RHAG gene and the translation of the RhAG protein in several ways, which, in turn, may alter the formation of the Rh complex. This is illustrated in Figure 7: 1. In some cases (as with patient AL), as a result of an aberrant splicing, the RHAG transcript may be absent or unstable and the RhAG protein is not produced. 2. In other instances, there is a frameshift mutation caused by a splicing defect or a small gene deletion, resulting in a premature termination codon. Frequently, nonsense

Blood group system 677 Mutation

Genes

D

RHAG (6p11-p21)

CE

RH (1p34)

no transcript (A)n

(A)n

mRNAs

unstable ?

Proteins RhAG Instability Degradation ?

wild type protein(s)

mutant protein Rh RhAG

Rh

Defective cellular routing ? Decreased membrane insertion ?

Altered contact in N-ter or C-ter domains impaired interaction ? Complex assembly absent or decreased

RhAG

Rh

Figure 7. Hypothetical model for the defect of the Rh complex in Rhnull individuals of the regulator type. Rhnull individuals of the regulator type present a defect of the RHAG locus located on chromosome 6, but have normal RH genes on chromosome 1. Because of the mutation events occurring in these patients (see Table 2 for details), the RHAG transcript may be absent and/or unstable. Truncated proteins potentially encoded by the frameshift mutants (resulting from a splice-site mutation or deletion) may be misfolded and degraded or produced at a low level and either misrouted or unable to be assembled correctly with the Rh proteins, which are in principle normally produced by the intact RH genes. Alternatively, the abnormal complex may be degraded or ineciently transported and/or inserted in the cell membrane. Adapted from reference 136. A similar model can be put forward to explain the Rhnull phenotype of the amorph type, in which the RH but not the RHAG locus is a€ected (see the text). A variable expressivity of the RHAG or RH mutations may explain the weak level of RhAG protein detectable in some Rhnull samples (see text and Figure 3).

mutations have diminished the mRNA levels through a variety of mechanisms such as an altered eciency of transcription, pre-mRNA processing, nuclear mRNA decay, nuclear mRNA export or cytoplasmic mRNA decay.141±143 If translated, the predicted RhAG protein mutant is a truncated polypeptide that includes a new stretch of amino acid residues at the C-terminus. Truncated proteins could be rapidly degraded in the endoplasmic reticulum.144,145 However, some might be more stable, but their folding and membrane insertion might be compromised. Additionally, the RhAG mutant and the Rh protein subunits may fail to interact properly, thus preventing the Rh subunits from assembling and/or being transported to the cell surface. Indeed, formation of abnormal complexes can result in the stabilization of subunits that are sensitive to degradation by preventing either the masking of speci®c determinants for degradation or the conformation changes required for further maturation.144 Rh is thought to interact in the Nterminal region of RhAG.66 Because the truncated RhAG proteins have a di€erent C-terminus, it has been suspected that normally the C-terminus of RhAG might either stabilize this interaction or may represent another site of protein±protein interaction.95 It is currently unresolved, however, whether or not the truncated RhAG proteins are degraded intracellularly before any interaction and therefore the role of the C-terminus of RhAG remains purely speculative.

678 J.-P. Cartron

3. The absence or severe defect of the Rh complex caused by missense mutations of RhAG is very intriguing, since both the RhAG and the Rh proteins are expected to be produced. One possibility is that the single amino acid residue change may alter the protein conformation or some requirement for the assembly and/or intracellular transport of the Rh complex. Examples of such situations have been observed with other cell surface molecules. For instance, a missense mutation in the water channel aquaporin-2 (AQP-2) was shown to impair the routing of this protein to the surface of epithelial cells in kidney collecting tubules and resulted in nephrogenic diabetes.146 Similarly, some mutant versions of CFTR (cystic ®brosis transmembrane conductance regulator) leading to cystic ®brosis are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded.147 In the blood group ®eld, missense mutations responsible for the Fyx phenotype148,149 and for most Dweak phenotypes150 result in a very low level of protein expression and might occur by a similar mechanism. Some of the RhAG mutations are destabilizing and may severely a€ect protein folding and protein±protein interactions. The most typical examples are those a€ecting charged residues in the TM domains (as in patients YT and HT, see Table 2), which break the hydrophobicity of the helices, causing a conformational change or a region of local unfolding that may become the target of degradative process.144 Mutations of charged residues in the intracellular domains of RhAG, as in patient CB (Table 2), may also a€ect protein association. Other changes a€ect polar or apolar amino acid residues that are replaced by residues with a larger side-chain, which may again result in conformational changes and/or disrupted protein interaction. Future studies will be necessary to understand precisely how RHAG mutations a€ect transcription, translation and post-translation events, and in turn how the protein is transported to the cell surface. Experimental con®rmation of the causative role of these mutations in the Rhnull phenotype will be necessary when ecient expression systems of the RhAG and Rh proteins are available. RH gene alterations in Rhnull individuals of the amorph type The amorph-type of Rhnull is much less frequent than the regulator type (including Rhmod) and only four examples have been well documented.6,7,77,84 Currently, two unrelated patients of German (DR) and Spanish (DAA) origin83,151 have been investigated at the molecular level (Table 3; Figure 6). Both patients were studied in Paris152 and the German patient was independently studied in New York.153 Flow cytometry analysis using MAbs indicated that RBCs from patients DAA and DR did not react with anti-Rh(D, C, c, E, e) and anti-LW reagents, but reacted weakly with RhAG (about 25%) and with CD47 (less than 10%), compared with controls. Both samples were typed as U-positive.83,151 The absence of Rh proteins on DAA's and DR's RBCs was con®rmed using Western blot analysis, but a small amount of RhAG glycoprotein was detected with the MAb 2D10 (Figure 3C). In addition, the LW protein was absent and CD47 was drastically reduced (Figure 3C). Molecular alterations of the RHCE gene Preliminary studies of the RH locus using Southern blot analysis revealed that patients DAA and DR lack the RHD gene and were homozygous for a haplotype carrying the RHCE gene only, and molecular studies, detailed below, have shown that both patients

Blood group system 679

exhibited abnormalities of the RHCE gene without any anomalies of the RHAG gene or of the genes encoding the CD47 and LW glycoproteins. Patient DAA. Studies using RT-PCR failed to detect a mutation of the Rhce transcript suggesting that the DAA phenotype may be caused by a splice-site mutation.151 Indeed, genomic DNA analysis identi®ed a g ! t transversion (gt ! tt) at the 50 donor splicesite of intron 4 of the Rhce gene152, thus generating a shift of the translation reading frame after the codon for leucine-211 in TM7 (Figure 6). This mutation created a novel Mse I restriction site and PCR-RFLP analysis indicated that DAA was homozygous for this mutation. Analysis of Rh transcripts indicated that at least three cryptic donor splice-sites (gt) are activated as a result of this mutation, two of them located 11 and 16 nt downstream from the mutation and the third located 1 nt upstream from the mutation, at the end of exon 4. As a result, aberrant transcripts were generated (some lacking exon 4 and/or exon 5) which, because of premature termination, potentially encode truncated proteins of di€erent sizes (227±364 residues), all containing the ®rst 211 residues of the normal Rh protein (which comprises 417 residues). The activation of the third cryptic splice-site generated a transcript that di€ered from the normal Rhce cDNA by a G634 nucleotide deletion, which may account for the inconsistency with previous data describing a normal Rh transcript in DAA.151 This mutation introduced a frameshift after the codon for leucine-211 that resulted in a premature termination codon, 51 bp downstream in the exon 5 region of the RH gene. The predicted product is a truncated protein of 227 residues (7 TM), including 16 novel residues at the C-terminus. Patient DR. Sequence analysis revealed an unusual double mutation within the RhCe transcript, replacing the `TCA' nucleotides at positions 966±968 (in exon 7) by a C, possibly following a T ! C transition in codon 322 and a CA deletion in codon 323.152,153 This introduced a frameshift after the codon isoleucine-322 (Table 3; Figure 6) and resulted in a premature stop codon at nt 1197±1199. The predicted shortened protein is 398 residues long (instead of 417) and includes 76 novel residues at the Cterminus; it is organised in 10 TM (instead of 12), as deduced from hydropathy analysis. The mutations can be detected using PCR-RFLP restriction analysis since a new Bam HI site is created. PCR-RFLP and direct sequence analysis have shown that patient DR was homozygous for these mutations. Huang et al153 postulated that the double mutation targeted the two adjacent codons ATT(I322) and CAC(H323) possibly by a mechanism of microgene conversion (a recombination mechanism found in yeast, see154), which may include as a single event a T ! C transition (ATT ! ATC) and a dinucleotide deletion (CAC ! C). An alternative scheme of spontaneous mutation resulting from a non-contiguous deletion of 2 nt (ATT(I322) ! AT and CAC(H323) ! CC) cannot be ruled out. Molecular mechanisms involved in Rhnull individuals of the amorph type The two amorph-type variants occurred in the context of a `RhD-negative' haplotype, in which the RHD gene was missing and the RHCE gene was a€ected by distinct mutations, but the RHAG gene was structurally intact and normally transcribed. This is consistent with family studies indicating that the amorph-type of Rhnull resulted from a `silent' allele at the RH locus.83,151 As a result of these mutations, the Rh proteins and those of the Rh complex were absent or severely reduced on RBCs, although a small

680 J.-P. Cartron

amount of RhAG protein was detected (discussed below). Again, these ®ndings bring strong support to the Rh complex model (Figure 4). Although only splice-site mutations have been currently described as the basis of the amorph-type of Rhnull , other molecular defects of the RH gene(s) that result in the same phenotype may be expected. The molecular mechanisms involved are probably similar to those discussed above for the Rhnull of the regulator (and Rhmod) type and illustrated in Figure 7, except that the RHAG gene would be normal and the RH genes would bear the mutation. The reported splice-site mutations generated aberrant RHCE transcripts potentially encoding truncated proteins with a new C-terminal extension. Ribonuclease protection assays indicated a lower level of Rh transcripts in patient DAA but a rather more normal level in patient DR, compared to the RhD-negative control, suggesting that DAA's transcripts are probably less stable.152 In general, truncated proteins encoded by spliceoforms of the RHD and RHCE genes detected using RT-PCR155,156 have never been found on RBCs, perhaps with one exception157, suggesting that the predicted Rh protein mutants from patients DAA and DR are not expressed either, as con®rmed by Western blot analysis (Figure 3C). As discussed above, if translated, the mutant proteins might have a di€erent folding compared to wild-type Rh proteins and be degraded within the cells or, alternatively, the intracellular routing is altered, either by faulty membrane incorporation or defective protein interaction (via the C-terminal end ?) with the RhAG glycoprotein. All these possibilities are speculative and should be examined in experimental expression systems. CONCLUSION AND FUTURE PROSPECTS Molecular analyses of unrelated Rhnull phenotypes strongly support the Rh complex model, since they demonstrate that when either the RHAG or the RH genes are altered, the Rh complex is not expressed at the red cell surface, thus emphasizing the critical role of the Rh and RhAG proteins, which make up the core of the complex. Both molecular studies and rare de®cient phenotypes con®rm that there is no strict requirement for the accessory protein subunits LW, CD47 or GPB to form the Rh complex. However, some of these proteins may play a role as chaperone molecules by facilitating the transit and the maturation of the core to the cell surface. For instance, in vitro studies indicate that the transit time of RhAG in the Golgi is modi®ed acccording to the presence or absence of GPB.109 Another membrane protein, such as Band 3, may also enhance Rh antigen expression.111 Biogenesis of Rh protein subunits Obviously, much more needs to be learnt about the biogenesis of the Rh complex proteins, particularly by developing expression systems in which the normal trac of these proteins, alone or together, can be investigated. It is known that recombinant LW, CD47 or GPB proteins may be expressed in nonerythroid cell lines such as COS or CHO158,159 (and our unpublished results) in the absence of Rh or RhAG proteins. It is only recently that erythroid cell lines (K562 or KU812) that express endogenous RhAG were successfully transduced to express cell surface Rh(D, cE, etc) antigens using retroviral constructs carrying RH cDNAs48,160 although the same result was obtained more recently using conventional transfection.161 However, cell surface expression of Rh antigens was not detected when non-erythroid

Blood group system 681

cell lines (COS-1 or 293) were transduced with retroviral RH and RHAG cDNAs, alone or together, despite the presence of a high level of transcripts160, but intracellular Rh and RhAG proteins were immunoprecipitated from the cell lysates.162 Therefore, it was suggested that another `cofactor', in addition to RhAG, is needed for cell surface expression of Rh proteins.160 Although Rh proteins (endogenous or recombinant) have never been found or expressed in the absence of RhAG, current studies suggest that Rh proteins may be partially dispensable for the routing of RhAG to the membrane: (i) a low level of RhAG protein was detected on Rhnull RBCs typed as U‡ (see above), which lack detectable Rh proteins; (ii) K562 cells express about 60±90  103 copies of RhAG protein152, compared to the 180±220  103 copies on adult red cells95, but they either lack163, or express a very low quantity of, Rh proteins detectable using some selected anti-D only164 (and our unpublished data); (iii) the RhAG protein is expressed before Rh proteins during the di€erentiation of erythroid progenitors in vitro.165,166 Optimal assembly and/or transport of the Rh complex, however, may require the presence of both proteins (in equivalent amounts ?), and perhaps a cofactor present in erythroid cell lines, at least. Although permanent cell lines of erythroid and non-erythroid origin are very convenient for expression studies, the results obtained with this model should be considered with some caution, since they may not re¯ect the physiological situation that tells us, for instance, that the absence or severe reduction of the Rh antigens in Rhnull RBCs results from a defect of Rh or RhAG proteins. Recently, it was shown that forced expression of recombinant Kell167 or Kx168 protein in COS transfectants resulted in the cell surface transport (and antigenic expression) of each protein in the absence of the other, suggesting that formation of the disulphide-linked Kell±Kx complex seen on human RBCs is not obligatory for cell surface expression. This is in apparent contradiction with the lack of Kx and the very weak expression of Kell in McLeod RBCs, or with the reduced expression of the Kx protein (but not the Kx antigen) in K0 RBCs that lack Kell.169,170 Again, optimal expression of the Kell±Kx complex may require both proteins or these proteins may follow a di€erent line of assembly by interacting with di€erent components of the secretory pathways in erythroid and non-erythroid cells. Another recent study shows that the RhD protein fused to GFP (green ¯uorescent protein) in the N- or C-ter position is eciently expressed at the surface of human HEK and HeLa cells, which lack RhAG171, suggesting that the RhD polypeptide does not require co-expression with RhAG. This is not necessarily contradictory to the current Rh complex model, however, since the GFP protein may well serve as a `chaperone' in this non-physiological system, forcing the cell surface expression of the RhD protein. A fusion product between the Du€y and the RhD polypeptide was also expressed previously in K56257, but these cells have the RhAG protein. Once the biosynthetic pathways of Rh and Rh-associated proteins are clari®ed and expression systems are available, the contribution of each component of the Rh complex to Rh antigen expression might be addressed. This may also be determined by reconstitution of the Rh complex using puri®ed molecules in arti®cial liposomes, a system that may indicate whether or not the Rh proteins could express Rh antigens in the absence of RhAG, and to study further the passive modulation of these antigens by membrane lipids.172 Another important issue will be the experimental demonstration of a direct correlation between the RH and RHAG mutations found in Rhnull individuals, particularly the missense mutations, and the Rh-de®cient phenotype.

682 J.-P. Cartron

Function of Rh Although the basic molecular defects responsible for the complete or partial lack of Rh blood group antigen expression in Rh-de®cient individuals have been elucidated to some extent, these studies have not clari®ed the physiological role of the Rh complex on RBCs. One major role of the Rh proteins might be to contribute to the membrane structure and stability of RBCs, possibly through skeletal protein and/or membrane phospholipid interactions, but this requires further studies to better de®ne the membrane partners involved and to correlate the pleiotropic defects of Rhnull cells with the multiple abnormalities seen in the Rh-de®ciency syndrome. Animal models in which the RH and/ or RHAG genes are invalidated by gene targeting will certainly be useful to this purpose. Based on the observation that Rhnull RBCs exhibit an altered phospholipid organization (see above), it was postulated that the Rh proteins may be identical to the ATPdependent aminophospholipid translocase that controls membrane phospholipid asymmetry.173 Rhnull cells however, have a normal translocase activity173±175, although a low exposure of phosphatidylserine at the cell surface, detected by measurement of prothrombinase activity, was found in two out of six unrelated samples of Rhnull , but this was not accompanied by any adverse e€ect.175 Recent studies have shown that the translocase is a P-type ATPase II of 115 kDa, the structure of which is clearly distinct from Rh proteins.176,177 Whether or not the Rh proteins may associate with the translocase173,178 or may passively in¯uence the organization of surrounding phospholipids23,174 is not known. Finally, a new area of research on the potential function of Rh proteins was begun recently with the ®nding that the well conserved RhAG protein shares some homology with NH4‡ transporters of the Mep/Amt family present in micro-organisms that use NH4‡ as a source of nitrogen.74 Ammonium transporters have not been identi®ed in higher animal species, but secretion and reabsorption of NH4‡ occurs in renal epithelial cells.179 Whether RhAG may function as a NH4‡ transporter or perform a related function is currently under investigation. Acknowledgements The author would like to thank B. CheÂrif-Zahar, I. Mouro, Y. Colin (Unite INSERM U76, Paris) and the group of N. Mohandas (Lawrence Berkeley Laboratory, Berkeley) for sharing unpublished observations.

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