Transfusion clinique et biologique 13 (2006) 4–12 http://france.elsevier.com/direct/TRACLI/
Molecular genetics of RH and its clinical application☆ Génétique moléculaire sur système RH et ses applications cliniques W.A. Flegel * Institut für Klinische Transfusionsmedizin und Immungenetik Ulm, Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, Ulm, Germany Available online 24 March 2006
Abstract Background. – The RH genes RHD and RHCE encode two proteins that represent the clinically most important blood group system defined by the sequences of red cell membrane proteins. In the last five years the field has been moving from defining the underlying molecular genetics to applying the molecular genetics in clinical practice. Materials and methods. – The state of the current knowledge is briefly summarized using recent reviews and original work since 2000. Results. – The RHD and RHCE genes are strongly homologous and located closely adjacent at the human chromosomal position 1p36.11. Part of the genetic complexity is explained by the clustered orientation of both genes with their tail ends facing each other. The SMP1 gene is located interspersed between both RH genes. Using additional genetic features of the RH gene locus, RHCE was shown to represent the ancestral RH position, while RHD is the duplicated gene. More than 150 alleles have been defined for RHD alone. They were classified based on antigenic and clinical properties into phenotypes like partial D, weak D and DEL. Among the D negative phenotype a large variety of non-functional alleles were found. The frequencies of these distinct alleles vary widely among human populations, which has consequences for clinical practice. Conclusion. – Rhesus is a model system for the correlation of genotype and phenotype, facilitating the understanding of underlying genetic mechanisms in clustered genes. With regard to clinical practice, the genetic diagnostics of blood group antigens will advance the cost-effective development of transfusion medicine. © 2006 Elsevier SAS. All rights reserved. Keywords: Rhesus; Blood group; Molecular genetic; Molecular diagnostic; Transfusion Mots clés : Rhésus ; Groupes sanguins ; Génétique moléculaire ; Diagnostic moléculaire ; Transfusion
1. Introduction The field of RH molecular genetics has been moving from defining the underlying molecular genetics to applying the molecular genetics in clinical practice. Since the 26th Congress of the ISBT in Vienna 2000, the number of published RHD alleles has more than doubled (Fig. 1) [1,2].
☆ This review was supported by DRK-Blutspendedienst Baden-Württemberg–Hessen, Mannheim; and Deutsche Gesellschaft für Transfusionsmedizin und Immunhämatologie (grant DGTI/fle/03-01). * Corresponding author. Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, Helmholtzstraße 10, 89081 Ulm, Germany. Tel.: +49 0731 150 600; fax: +49 0731 150 602. E-mail address:
[email protected] (W.A. Flegel).
1246-7820/$ - see front matter © 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.tracli.2006.02.011
Fifteen years after the cloning of the first RH gene [3], which proved to be RHCE [4,5], the polymorphism has been characterized in great detail. Major progress has been achieved during the last 5 years. The molecular break point of the RHD deletion that is associated with the D negative phenotype in Europe was localized in the hybrid Rhesus box [6]. The variability of the Rhesus boxes was found [7] and explored by several groups [8]. A large number of random population screens were performed or are ongoing at the molecular level to establish allele frequencies [9,10]. These studies are instrumental in establishing the feasibility and clinical relevance of clinical RH genotyping. Other foci in the recent years were the investigation of the 3D structure and the function of the Rh proteins, which will be addressed in other reviews of this conference. Because functions are known for most blood group proteins, it may be sur-
W.A. Flegel / Transfusion clinique et biologique 13 (2006) 4–12
Fig. 1. Known RHD alleles. The approximate number of published RHD alleles are grouped according to the variant D antigen that they are encoding. Following the original description of the standard RHD allele in 1992, the number of known alleles has increased steadily and new groups were recognized. For instance, the first examples of weak D and DEL were discovered in 1999 and 2001, respectively.
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Fig. 2. Duplication of the RH gene and loss of the RHD gene. The ancestral configuration is shown as represented by the RH gene locus in mouse. The single RH gene is in close proximity to the three genes SMP1, P29associated protein (P) and NPD014 (N). A duplication event introduced a second RH gene in reverse orientation between N and SMP1. At the two break points in front and behind the RHD gene DNA segments of approximately 9000 base pairs (bp) occur. Both DNA segments are flanking the RHD gene and dubbed “upstream Rhesus box” and “downstream Rhesus box”. In the RHD positive haplotype the RHD gene may have been lost by a recombination event (see Fig. 3).
prising to note that no definite function could be attributed to Rh proteins beyond their early recognition as being structurally important for red blood cell (RBC) membrane integrity [11]. The Rhesus associated antigen RhAG transports ammonium [12–15], which could not be shown for the Rh proteins themselves [16–19]. They may be involved in the exchange of gases like CO2 [20]. Based on the 3D structure of AmtB [21] the previously available models of the Rh protein could be adapted by protein threading [22,23]. Work on the human Rhesus proteins was instrumental in defining the Rh protein superfamily whose proteins exhibit a wide tissue and species distribution and are likely involved in a variety of functions [24–26]. 2. Molecular basis of RH alleles Comparison of the human RH gene locus with preliminary data from the mouse genome project allowed to delineate the molecular events leading to the RH duplication in humans (Fig. 2) [27]. Most mammals possess one RH gene only, whose position equals the human RHCE gene. The RHD gene occurred by a duplication of the original RH gene during mammal evolution. In hominids the RHD gene was lost in some extant haplotypes (Fig. 3) [6]. The heterogeneity in the Rhesus boxes indicates several independent RHD deletion events [28,29]. The more than 150 RHD alleles may be grouped according to their phenotypes and molecular features. Most alleles harbor single nucleotide polymorphisms (SNP) or represent RHD-CED hybrid alleles caused by gene conversions. Representative molecular features and their phenotypic relevance were summarized (Table 1). The correlation of the molecular basis with the phenotype often facilitated genetic diagnostics.
Fig. 3. RHD deletion. An unequal crossing over event between an upstream Rhesus box and a downstream Rhesus box caused the RHD deletion. If one of the two crossed over chromosomal threads are resolved, an RH gene locus results lacking the RHD gene completely and harboring a hybrid Rhesus box.
3. Molecular basis of Rh phenotypes The RhD and RhCE proteins are highly homologous and vary at 36 amino acid positions only. Both comprise 12 transmembraneous protein segments and six extracellular protein loops (Fig. 4). Besides the RHD deletion causing a D negative phenotype, a host of RhD protein variants express altered D antigens. There is no absolute correlation among molecular structures, the phenotypes and clinical relevance of RHD alleles. To provide some degree of order to the large number of aberrant D antigens, the RHD alleles were classified according to their phenotype and molecular variation in partial D, weak D and DEL [30,31].
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Table 1 Representative molecular changes in RHD alleles expressing distinct phenotypes of the D antigen Classification of antigen variation
D antigen phenotype
Partial D
Qualitative change
Weak D
Quantitative change
DEL
Major quantitative change
D negative
D negative
Antithetical antigens of the RhCE protein
Expression of antigen E or antigen e
Molecular basis Protein variation Mechanisms
Representative example RHD allele Trivial name
Amino acid substitution on the RBC surface Protein segment exchange on the RBC surface Amino acid substitution in the membrane or intracellularly Grossly reduced translation or protein expression Lack of protein expression
Missense mutation
RHD(G355S)
DNB
Novel Rhesus antigen Unknown
Gene conversion (hybrid protein)
RHD-CE(3–6)-D
DVI type 3
BARC
Missense mutation
RHD(V270G)
Weak D type 1
Unknown
Missense mutation Mutation at splice site Gene deletion Nonsense mutation Frame shift mutation Modifying gene Gene conversion (hybrid protein)
RHD(M295I) in CDe RHD(K409K)
Not applicable Not applicable
Unknown Unknown
RHD-Deletion RHD(Y330X) RHD(488del4) defect of RHAG gene RHD-CE(3-7)-D
D negative Not applicable Not applicable Rhnull Cdes
Unknown Unknown Unknown Unknown Unknown
RHCE allele: Ala226 coding antigen e Pro226 coding antigen E
Not applicable
E versus e
Protein segment exchange on the RBC surface Amino acid substitution on the RBC surface
Missense mutation at amino acid position 226 in RHCE
Fig. 4. Model of Rhesus proteins in the red blood cell membrane. Both Rhesus proteins consist of 417 amino acids symbolized by circles. The first amino acid is lacking from the mature proteins in the membrane. Amino acid positions differing between the RhCE and RhD proteins are marked in light gray. All known amino acid substations encoding partial D are labeled in gray and weak D in black.
3.1. Partial D Because RhD is a multi-pass transmembrane protein, only certain protein segments are exposed at the erythrocyte surface. If an amino acid substitution was located in an extracellular loop (Fig. 4), a partial D phenotype may result. Many new partial D were found since 2000 [32–35] and probably many more still will be discovered. Partial D is of clinical relevance because carriers may produce anti-D upon exposure to the normal D antigen.
D categories (DII to DVII) are a subgroup of partial D. DIII, DIV, DV and DVI are usually caused by RHD-CE-D hybrid alleles [36–39]. 3.2. Weak D If an amino acid substitution was located in the transmembraneous or intracellular segments of the RhD protein (Fig. 4), a weak D phenotype may result [40]. Such substitutions may impede the protein integration in the membrane or its anchor-
W.A. Flegel / Transfusion clinique et biologique 13 (2006) 4–12
ing to the erythrocyte’s cytoskeleton [41]. The expressed D antigen is quantitatively reduced, but remains qualitatively unchanged, which usually excludes any anti-D immunization in carriers [40,42–48]. There are exceptions, however, like weak D type 15 [49], weak D type 4.2, also known as DAR [50], and weak D type 7, whose carriers were shown or likely capable to produce anti-D [49,51,52]. 3.3. DEL A very weakly expressed D antigen is called DEL, formerly Del, because it was originally detected by elution techniques. The underlying molecular change has more pronounced effects than in weak D, which strongly impedes but does not abridge membrane integration. All DEL are rare in Europeans [53]. However, up to 30% of seemingly D negative blood donors in East Asia carry the DEL RHD(K409K) [43]. DEL was intensively studied [54–56], because it is clinically relevant in donors [57] and for genotyping methods [46,47,58, 59]. 3.4. D negative The most frequent D negative haplotype in all human populations is due to the RHD deletion and characterized by a hybrid Rhesus box (Fig. 3). Other important D negative alleles are the RHD pseudogene RHDψ and the RHD-CE-D hybrid allele Cdes (Table 1) occurring rather frequently in African populations [60,61]. Less frequent D negative alleles harbor a host of different RHD-CE-D hybrid alleles or nonsense and frame shift mutations [53,62]. The discrimination between the D negative
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and the DEL phenotypes by serologic cut-offs may be arbitrary. Using molecular techniques the alleles are clearly defined avoiding any ambiguity. 3.5. Rhnull The lack of RhD and RhCE proteins may be caused by mutations rendering the expression both Rh proteins mute (amorph type). Because both proteins require the presence of the RhAG protein for their expression in the membrane, defects in RHAG alleles cause the Rhnull phenotype (regulator type) [63]. 3.6. RhCE variants Partial antigens were also reported for the antigens C, c, E and e [23,64–74]. Similar to partial D, the carrier of, for instance, a partial C antigen may be immunized to produce anti-C. Because a carrier of such a partial Rh antigen may be less frequent and much less prone to immunization than observed with partial D, the clinical relevance is rather limited. Some long-standing serologic conundrums were resolved by molecular methods, which contributed to the further development of widely used serologic reagents [73,74]. 4. RHD phylogeny A phylogenic tree of RH alleles was delineated and 4 clusters are defined (Fig. 5) [75,76]. For establishing this phylogeny the RHCE allele polymorphism was not considered and
Fig. 5. D clusters and recombinations. The phylogenic tree of RHD alleles is shown. The four D clusters are represented by their primordial alleles: DVI cluster (DVIa), weak D type 4 cluster (weak D type 4.0), DAU cluster (DAU-0) and Eurasian D cluster (standard RHD in the cDe haplotype).
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the actual phylogeny may be even more complex. In several DV types [32] parts of RHD exon 5 are replaced by the RHCE exon 5 homologues of different lengths. Most of these DV types represent independent gene conversion events within the Eurasian D cluster (Fig. 5) [35]. Clusters encompass alleles that are related (like DV), and that have developed from each other in a sequential manner (unlike DV) [75]. Because DV alleles have probably arisen from independent events, they should hence not be defined as a separate D cluster [72]. 5. Population studies The analysis of allele distributions in human populations was a major research topic during the last 5 years. As expected the allele variety is largest by far in Africans [60,61,65,66,72, 77]. A significant variation between African populations like West [77] and South Africans [60] is evident but has not been fully explored. Europeans and East Asians share a small and overlapping subset of the African alleles. The alleles of the Eurasian D cluster [75] diversify much further with, for instance, weak D types 15 and 17 being rare in Europeans but prevalent in East Asians [42,43,54]. Subdivisions of the most prevalent aberrant alleles have already been encountered, like weak D type 1.1 [78], DVI type 3 [79] and DVI type 4 in Europeans [39]. Random surveys revealed a greater variety of RHD alleles than anticipated for the European population [35]. These studies were extended to non-coding DNA stretches of the RH gene locus like the Rhesus boxes [7,28,29,80,81], because of their diagnostic relevance. Little is known about the large Arabian and Indian populations, ethnic minorities in the Americas and populations living in extreme environmental conditions, like at the Arctic circle or at high altitude. Population studies advanced the cataloguing of RH alleles in the human population [82], supplement human genomic diversity projects and may eventually result in a significant contribution to the understanding of Rh protein structure and function. An in-depth analysis of any major population is likely to reveal a large segment of all human RH alleles, which may be accomplished by mass scale genotyping. Improving the knowledge of alleles including their phylogenic relationship [52,78] and clinical significance will be instrumental in refining the genotyping strategies that have been established for various clinical applications. 6. Clinical application Today the accrued molecular knowledge on RH can be applied to patient care in a cost-efficient way and in a variety of clinical problems. The genetic techniques used for determining alleles are usually PCR-based with detection of the amplicons in gels [83], by real-time PCR [84], by hybridization to microarrays like biochips [85–87] or by nucleotide sequencing [23, 40].
6.1. Anti-D in patients Clinical problems are most often associated with a restricted number of frequent RHD alleles. They represent partial D or some rare weak D types, whose carriers can be immunized by the normal D antigen [48]. Among these the D category VI (DVI) is the most important in Europe. Therefore we proposed [88] the use of monoclonal anti-D reagents for routine typing that do not detect DVI. This had been introduced as the recommended procedure in the German transfusion guidelines since 1996 and has since been implemented in clinical practice and guidelines of other European countries, like the UK, the Netherlands and France. Carriers of DVI are deliberately typed “false negative” to avoid transfusion with D positive blood and their likely anti-D immunization [89]. The clinically important and most frequent partial D among Europeans, DNB, was published not before 2002 [90]. It was realized in an ongoing surveillance of anti-D immunization in D positive recipients [91]. In contrast to the alleles described in the previous paragraph, allo-anti-D immunizations have never been observed in weak D type 1, type 2 and type 3. From a clinical perspective it is fortunate that exactly these weak D alleles are the most frequent of all, and they comprise together more then 90% of weak D types in central European populations. Their carriers may safely be transfused with D positive blood and any unnecessary use of D negative blood, which is often in short supply, may be avoided [49,51,92]. 6.2. RhIg in pregnancy [93] Pregnant women with the frequent weak D type 1 to type 3 may be transfused with D positive blood and will therefore not benefit from RhIg prophylaxis. RHD genotyping performed once may spare each pregnant woman from several RhIg shots [9,92]. Also any potential side effect of this medication that is entirely unnecessary for this woman may be avoided. Utilizing RHD genotyping would ensure that women with rare weak D types who are prone to anti-D immunization would receive the required RhIg shots, which is not assured under the current transfusion medicine guidelines in Europe and elsewhere. The sensitivity of real-time PCR allows us to utilize cellfree fetal DNA in maternal peripheral blood [94,95]. This fetal DNA represents rather small DNA fragments [96,97] that are essentially cleared from maternal blood within hours after birth [98]. Many laboratories throughout Europe are collaborating to implement this technology [99–102]. If the fetus were determined D positive from maternal plasma, no RhIg shot would be required. 6.3. Prenatal diagnostics RHD genotyping is routinely performed during management of hemolytic disease of the newborn. Most of these cases are still caused by anti-D in D negative women although it may
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occur in women with partial D as well [103,104]. The prediction of D and other blood group antigens by genetic diagnostics from amniotic fluid or trophoblastic cells is so reliable, that it has been the method of first choice for many years [1]. This approach avoids taking blood from fetal cord for blood group testing. Even amniocentesis may soon be abandoned in favor of testing maternal peripheral blood [105,106]. 6.4. Anti-D and family planning For decades RHD heterozygous could not be distinguished from RHD homozygous, because all serologic methods were unsuitable. This limitation was overcome by the genetic diagnostics of Rhesus boxes [6]. If the father is tested for the RHD deletion with suitable techniques and determined to be RHD heterozygous, a mother with an allo-anti-D has a 50% chance of conceiving a D negative fetus with a pregnancy of virtually no immunohematologic risk [107]. 6.5. Genetic diagnostics in distinct diseases The benefit of genetic diagnostics in transfused patients and with auto- and alloimmunohemolytic anemias is well established, if standard serology failed [1,108–110]. 6.6. Blood donors RHD genotyping revealed weak D and DEL donors among blood donors who are typed and considered D negative according to the current serology standards [53]. Without such genetic diagnostics transfusion recipients of DEL positive blood units have been anti-D immunized [46,55,59,111]. This issue is a major current research topic of significant practical relevance [58,92]. Besides weak D and DEL donors, a potentially serious risk is posed by D negative donors who are D positive/ D negative chimeras. Every transfusion of such RBCs is capable of causing an anti-D immunization, because they may contain several milliliters of RBCs of normal D positive phenotype [53] impossible to detect with routine serology methods [58].
cilitate genotyping of large donor/patient cohorts and the selection of compatible blood units. The match of donors and patients could be much improved by computer over the currently applied serologic methods. 7. Future perspectives Molecular genetics of blood groups and in particular of RH has become a reality in practical transfusion medicine since about 2000 [83,114–117]. If RH genotyping would be attempted on a massive scale, no legal or ethical issues would be violated, which are rightfully of public concern [1]. Pregnant women carrying weak D types or D negative fetuses that can be specifically detected by RHD genotyping may be spared from receiving unnecessary RhIg shots, which could lower their overall health care bill while avoiding any potential side effects of RhIg [49,106]. Such a fortunate combination may not always be expected and we should be prepared to accept initially some additional costs for optimizing patient care by blood group genetic testing. Lessons from other industries imply that improving safety by blood group genotyping at reasonable incremental cost is a feasibility. Acknowledgments I am asking the reader to refer to the cited excellent reviews for the older original work. I apologize to the colleagues in the field for not having duly cited most publications before 2000 because of space limitations. The former and the current chairman of the Institut für Klinische Transfusionsmedizin und Immungenetik Ulm continuously supported my work on Rhesus during the last 12 years. I am grateful to the colleagues who provided unpublished data and to my 10 years’ colleague in my laboratory, Franz F. Wagner/Springe, for his 2D Rhesus protein model [23,118] and for collating RHD alleles in The RhesusBase [119]. References [1]
6.7. Mass scale genotyping Rhesus is the most complex blood group gene locus and represents a model system for clustered genes. Its molecular genetics is particularly challenging with the objective to identify all clinically important RH alleles. The encoded phenotypes were studied for decades by serology. Not many human gene loci and their phenotypes share this set of features. It has been argued that mass scale genotyping in a highly parallel approach may be the solution. [8,58,92,112]. Developments toward this end are under way in North America [86,87] and Europe [85] including the EU-funded BloodGen project [113]. These high-throughput technologies may eventually fa-
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