Springer

Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wagner, F. F.
Right arrow Articles by Flegel, W. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wagner, F. F.
Right arrow Articles by Flegel, W. A.
Related Collections
Right arrow Transfusion Medicine
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

arrow to previous article Previous Article  |  Table of Contents  |  Next Article next article arrow

Blood, Vol. 93 No. 1 (January 1), 1999: pp. 385-393

Molecular Basis of Weak D Phenotypes

By Franz F. Wagner, Christoph Gassner, Thomas H. Müller, Diether Schönitzer, Friedrich Schunter, and Willy A. Flegel

From Abteilung Transfusionsmedizin, Universitätsklinikum Ulm and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm, Ulm, Germany; Zentralinstitut für Bluttransfusion und Immunologische Abteilung Innsbruck, Innsbruck, Austria; and Institut Oldenburg, DRK-Blutspendedienst Niedersachsen-Oldenburg, Oldenburg, Germany.



    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

A Rhesus D (RhD) red blood cell phenotype with a weak expression of the D antigen occurs in 0.2% to 1% of whites and is called weak D, formerly Du. Red blood cells of weak D phenotype have a much reduced number of presumably complete D antigens that were repeatedly reported to carry the amino acid sequence of the regular RhD protein. The molecular cause of weak D was unknown. To evaluate the molecular cause of weak D, we devised a method to sequence all 10 RHD exons. Among weak D samples, we found a total of 16 different molecular weak D types plus two alleles characteristic of partial D. The amino acid substitutions of weak D types were located in intracellular and transmembraneous protein segments and clustered in four regions of the protein (amino acid positions 2 to 13, around 149, 179 to 225, and 267 to 397). Based on sequencing, polymerase chain reaction-restriction fragment length polymorphism and polymerase chain reaction using sequence-specific priming, none of 161 weak D samples investigated showed a normal RHD exon sequence. We concluded, that in contrast to the current published dogma most, if not all, weak D phenotypes carry altered RhD proteins, suggesting a causal relationship. Our results showed means to specifically detect and to classify weak D. The genotyping of weak D may guide Rhesus negative transfusion policy for such molecular weak D types that were prone to develop anti-D.
© 1999 by The American Society of Hematology.



    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

THE RHESUS D (RhD) antigen (ISBT 004.001; RH1) carried by the RhD protein is the most important blood group antigen determined by a protein. It is still the leading cause of hemolytic disease of the newborn.1 About 0.2% to 1% of whites have red blood cells with a reduced expression of the D antigen (weak D, formerly Du).2-4 A small fraction of weak D samples are explained by qualitatively altered RhD proteins, called partial D,5 and frequently caused by RHD/RHCE hybrid alleles, a flurry of which was recently published (reviewed in Huang6). Another fraction is caused by the suppressive effects of Cde haplotypes in trans position.7 These weak D likely possess the normal RHD allele, because the carriers' parents and children often express a normal RhD antigen density. Such weak D show only a minor reduction of RhD antigen expression, were loosely called high grade Du, and often typed today as normal RhD, because of the increased sensitivity of monoclonal anti-D.8

The majority of weak D phenotypes is caused by genotype(s) located either at the Rhesus genes' locus itself or in its proximity, because the weak D expression is inherited along with the RhD phenotype.2 Besides the mere quantitative reduction, no qualitative differences could be discerned in the RhD antigen of this group. Two recent studies addressed the molecular cause of the prevalent weak D phenotypes. Both groups, Rouillac et al9 and Beckers et al,10 performed reverse-transcriptase-polymerase chain reaction (RT-PCR) and claimed unanimously that their sequencing of RHD cDNA in weak D samples showed a normal RHD coding sequence. However, no definite molecular cause of the weak D expression was established and the proposed mechanisms differed. Using semiquantitative RT-PCR, Rouillac et al9 reported reduced steady-state levels of RHD transcripts and claimed that their observations provided direct evidence of a quantitative difference in RhD between normal and weak D red blood cells. In contrast, Beckers et al10,11 found no differences in the amounts of RHD transcripts, further excluded an excess of splice variants,10,12 and concluded that weak D is not caused by regulatory defects of the transcription process.

Screening of random weak D samples by PCR for RHD specific polymorphisms confirmed PCR amplification patterns representative for a normal RHD allele.13,14 However, evidence was accumulating that the underlying molecular basis can be heterogeneous,13 and some weak D may carry structurally abnormal RHD alleles. In four of 44 English weak D, no RHD specific intron 4 PCR amplicons13 were detected, and in one of 90 Northern German weak D, no RHD specific exon 5 PCR amplicons14 were detected. In a similar more extensive molecular screen by PCR-SSP,15 we found about 2.5% structural abnormalities in more than 600 weak D samples (Gassner et al, manuscript submitted).

These observations prompted us to search for further allelic variation among weak D samples by sequencing. In contrast to the commonly accepted knowledge,6,13,16-19 we found that all weak D samples tested had mutations in RHD exon sequences.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Blood samples.   EDTA- or citrate-anticoagulated blood samples were collected from white blood donors and characterized as weak D during donor typing in accordance with published standards (Du-test)20 as described.3 The donors were not known to be related. D category VI samples were excluded from this study.

Sequencing of the 10 RHD exons from genomic DNA.   DNA was prepared as described previously.15 Nucleotide sequencing was performed with a DNA sequencing unit (Prism dye terminator cycle-sequencing kit with AmpliTaq FS DNA polymerase; ABI 373A, Applied Biosystems, Weiterstadt, Germany). Nucleotide sequencing of genomic DNA stretches representative for all 10 RHD exons and parts of the promoter was accomplished using primers (Table 1) and amplification procedures (Table 2) that obviated the need for subcloning steps.


                              
View this table:
[in this window]
[in a new window]
  		Table 1. Primers Used


                              
View this table:
[in this window]
[in a new window]
  		Table 2. Sequencing Method for all 10 RHD Exons From Genomic DNA

Control of RHD specificity.   RHD exons 3 to 7 and 9 carry at least one RHD-specific nucleotide, which was used to verify the RHD origin of the sequences. For exon 1, characteristic nucleotides in the adjacent parts of intron 1 were used.21 For exon 8, the RHD specificity of the PCR amplification was checked by RHD nonspecific sequencing of the informative exon 9, because exons 8 and 9 were amplified as a single PCR amplicon (Table 2). Exon 2 and 10 were amplified in an RHD- specific way (Table 2) based on published RHD-specific nucleotide sequences (EMBL nucleotide sequence data base accession numbers U66340 and U66341)22,23; no PCR amplicons were obtained in RhD- controls (data not shown). All normal D and weak D samples showed a G at position 65424 and a C at position 1036,22 supporting the notion25 that the alternatively described C22 and T,24 respectively, were sequencing errors.

Detection of weak D specific mutations by PCR-restriction fragment length polymorphism (RFLP) and PCR using sequence-specific priming (PCR-SSP).   PCR-RFLP methods were developed to characterize distinct nucleotide substitutions detected in five RHD alleles: the C to G substitution at position 8 led to the loss of a Sac I restriction site in amplicons obtained with re01 and re11d (G to A at 29, loss of Msp I site, re01/re11d; C to A at 446, loss of Alu I site, rb20d/rb21d; T to G at 809, loss of Alw44 I site, rf51/re71; G to C at 1154, introduction of Alu I site, re82/re93). Conditions for the rf51/re71 PCR reaction were as shown in Table 2. The rb20d/rb21d reaction was done with nonproofreading Taq-polymerase (Boehringer Mannheim, Mannheim, Germany or Qiagen, Hilden, Germany) with 20 seconds denaturation at 94°C, 30 seconds annealing at 60°C, and 30 seconds extension at 72°C. The other PCR reactions were performed with nonproofreading Taq-polymerase with 20 seconds denaturation at 94°C, 30 seconds annealing at 55°C, and 1 minute extension at 72°C.

Another four RHD alleles were detected by a standard RH PCR-SSP15: the RHD(T201R,F223V) and RHD(S182T,K198N,T201R) alleles lacked specific amplicons for RHD exon 4, the RHD(G307R) and RHD(A276P) alleles lacked those for RHD exon 6. For all other weak D types, the authenticity of the point mutations was checked by nucleotide sequencing of independent PCR amplicons.

Sequencing of the RHD promoter.   To check for mutations in the RHD promoter, we amplified a 675-bp region using primer pair rb13 and rb11d (Table 2). The promoter region was sequenced using primers re02 and re01 starting at nucleotide position -545 relative to the first nucleotide of the start codon.

Characterization of RHD allele polymorphisms in introns 3 and 6.   In RHD intron 3, there was a G/C polymorphism that determined a Hae III-RFLP at position -371 relative to the intron 3/exon 4 junction. To check this polymorphism, we amplified the 3' part of intron 3 using the RHD specific primer pair rb46 and rb12 and digested the PCR products with Hae III. In RHD intron 6, there was a variable length TATT tandem repeat starting 1,915 bp 3' of exon 6. To examine this tandem repeat, we amplified the full-length intron 6 using the RHD-specific primer pair rf51 and re71 and used primer rg62 for sequencing. The haplotype association of the Hae III site was tested in 10 CCDee, 8 ccDEE, 10 ccDee (W16C+), and 10 ccDee (W16C-) samples. The haplotype association of the TATT repeat was tested in 3 CCDee, 3 ccDEE, 1ccDEe, 2 ccDee (W16C+), and 2 ccDee (W16C-) samples. In control samples, the presence and absence of the Hae III site was linked to 9 and 8 TATT repeats, respectively. The Hae III site was present in the 20 CDe haplotypes and 18 of 20 cDe haplotypes tested, but absent in 15 of 16 cDE haplotypes (CDe v cDE, P < .001; cDe v cDE, P < .001; CDe v cDe, not significant; 2 × 2 contingency tables, Fisher's exact test).

Sequencing of intron 5 and exons 6 to 9 in DIV type III.   In DIV type III exons 6 to 9 were amplified and sequenced using primers that were specific for RHCE and RHD. Therefore, primer re71 was substituted by primer rb7; primer re621 by rb26; and primer re52 by re74. To demonstrate an RHD-RHCE hybrid allele, we amplified intron 5 in a RHD-specific way using the exon 5 PCR reaction (Table 2) and sequenced the breakpoint region using primer rb15.

cDNA sequencing.   RNA was prepared and reverse-transcribed as published.21 cDNA was amplified in a nested PCR reaction (High Fidelity PCR system, Boehringer Mannheim, Mannheim, Germany) with external primers RR1 and RR4 and internal primers Rh5 and RR3 and subcloned into pMos (pMos-T-kit; United States Biochemical, Cleveland, OH).

Expected proportion of silent mutations.   In the 416 codons of the RHD gene excluding the start codon, 2,766 missense and 919 silent mutations can occur yielding an expected proportion of 0.254 of silent mutations. A total of 782 of the missense mutations and 437 of the silent mutations are transitions resulting in an expected proportion of 0.36 of silent transitions. A total of 41 missense and 30 silent mutations are located in CpG doublets resulting in a proportion of silent mutations in CpG doublets of 0.42. Independent of any possible excess of transitions or mutations in CpG doublets, random nucleotide changes should therefore lead to a minimal frequency of silent mutations of 0.254. Nonsense mutations and mutations in the start codon were assumed to prevent RhD expression13 and are excluded from the calculations.

Population frequency and haplotype association of weak D types.   The phenotype frequencies of weak D types among weak D samples were calculated separately for each serologic (CcDEe) weak D phenotype and combined according to the frequencies of the serologic weak D phenotypes.3 ccDEE weak D samples were assumed to be cDE/cdE. Phenotype frequencies in the population were calculated from the population frequency of the weak D phenotype in Southwestern Germany.3 These data are minimal estimates, because some samples with only moderately weakened D expression may have been inadvertantly grouped to normal strength D. Haplotype frequencies were calculated using a haplotype frequency of 0.411 for RhD- haplotypes3 assuming that all weak D samples were heterozygous. For weak D types 1 to 5, 9, 13, and 15, more than one proband was observed rendering the haplotype association trivial. Weak D type 11 was observed in a ccDee phenotype implying a cDe haplotype. Weak D types 6 to 8 and 12 were observed in single CcDee samples and assumed to be CDe/cde, which is correct in more than 96% of samples according to the haplotype frequencies in the population investigated.3 Weak D types 10, 14, and 16 were observed in single ccDEe samples and assumed to be cDE/cde, which is correct in more than 98% of samples.3 Hence, the probability that an cDE versus cDe misassignment corrupted the analysis of haplotype-specific polymorphisms was less than 0.05.

RhD topology prediction.   The position of the transmembraneous helices was based on an analysis of RhD by the PredictProtein server prediction of transmembrane helices (http://www.embl-heidelberg.de/predictprotein/predictprotein.html,26 helix 1 to 11) and TMpred (http://ulrec3.unil.ch/software/TMPRED_form.html,27 helix 12) with minor modifications localizing position 110 and 226 on the cell surface and 12 in the membrane in accordance with other published models.28,29 There is experimental evidence30,31 for intracellular positions of the amino and carboxytermini.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Coding sequence of RHD in weak D phenotypes.   A method for RHD-specific sequencing of the 10 RHD exons and their splice sites was developed. In a sequential analysis strategy, blood samples with weak expression of antigen D, including a random survey of 161 samples from blood donors in Southwestern Germany, were checked by this method, PCR-RFLP (Fig 1), and RHD PCR-SSP.15 We found 18 RHD alleles with distinct nucleotide changes coding for amino acid substitutions (Table 3). One allele lacked RHD exons 6 to 9 concordant with a RHD-CE-D hybrid allele dubbed hereby DIV type III. Another allele was DHMi.32 Of the remaining 16 alleles, 14 showed single, but distinct previously unknown missense mutations. None of the encoded variant amino acids occurred at the corresponding positions in the RhCE proteins. Two alleles exhibited multiple nucleotide changes typical for the RHCE gene, which were interspersed by RHD-specific sequences.



View larger version (43K):
[in this window]
[in a new window]
  		Fig 1. Detection of weak D types by PCR-RFLP. Four weak D types harbored point mutations that obliterated restriction sites: weak D type 1 lacks an Alw44 I site (A); weak D type 3, a Sac I site (C); weak D type 5, an Alu I site (D); and weak D type 6, a Msp I site (E). In a fifth weak D type, a point mutation introduced a restriction site: weak D type 2 gained an Alu I site (B). On the left side of the gels, 100 bp ladders are shown; the position of the 500 bp and 100 bp fragments are indicated on the right side of the panels. For the PCR reaction of (A), the largest restriction fragment approximation 3,000 bp is not shown.


                              
View this table:
[in this window]
[in a new window]
  		Table 3. Proposed Nomenclature, Molecular Basis and Minimal Population Frequencies of RHD Alleles Coding for Weak D Phenotypes

Distribution of weak D alleles in whites.   A set of 161 samples with weak expression of antigen D was gathered from random blood donors in Southwestern Germany. D category VI samples, but no other partial D, were excluded by serologic methods. Two samples represented known partial D (DHMi32 and D category IV33). Without any exception, all samples could be assigned to distinct RHD alleles with aberrant RHD exon sequences (Table 3). We propose that the new molecular weak D types should be referred to by trivial names, eg, weak D type 1, or by their molecular structures, eg, RHD(V270G). The weak D type 1 was the most frequent known RHD allele (f = 1:277) with aberrant coding sequence, exceeding even the DVII allele frequency.34

Amino acid substitutions in weak D alleles are clustered.   The amino acid substitutions observed in weak D types with single missense mutations were not evenly distributed in the RhD protein (Fig 2). The majority of substitutions occurred in the region of amino acid positions 267 to 397. Single and multiple amino acid substitutions in smaller portions of the RhD protein around positions 2 to 13, 149, and 179 to 225 (weak D type 4 and 14) were also found in weak D alleles. According to the current RhD loop model, the involved amino acids were positioned in the transmembraneous and intracellular protein segments (Fig 3).



View larger version (7K):
[in this window]
[in a new window]
  		Fig 2. Schematic representation of the amino acid variations observed in weak D types with single missense mutations. The affected amino acids of the prevalent normal RhD protein and their positions are shown on top. Their substitutions occurring in the weak D types are shown below the bar. The distribution of the variant positions deviated from the uniform distribution (P < .05, Kolmogoroff-Smirnov-test).



View larger version (40K):
[in this window]
[in a new window]
  		Fig 3. Localization of the amino acid substitutions of the weak D types. A predicted topology of the RhD protein with respect to the plane of the red blood cells' membrane is presented. Amino acid substitutions are shown for single events (black circles) and multiple events (gray circles). The amino acid substitutions of all weak D types were located in intracellular and transmembraneous protein segments.

Normal RhD phenotype controls and RHD promoter.   RHD specific sequencing of the 10 RHD exons predicted regular RhD protein sequences in six control samples with a normal antigen D expression; 545 bp 5' of the start codon comprising part of the RHD promoter were sequenced in one sample of each weak D type, DHMi, and DIV type III. No deviation from the normal RHD promoter sequence35 was found.

Statistical evidence that missense mutations can cause weak D phenotypes.   The frequency of altered RhD proteins in weak D (159 of 159) and normal D samples (0 of 6) was statistically significantly different (P < .0001, 2 × 2 contingency table, Fisher's exact test). A normal RhD coding sequence in the weak D phenotype was expected to occur in less than 1.9% (upper limit of 95% confidence interval, Poisson distribution). These amino acid substitutions are unlikely to reflect random nucleotide changes, because a random mechanism would lead to a frequency of silent mutations of at least 0.254, while we observed only one silent mutation among a total of 18 mutations in weak D alleles (P = .037, binomial distribution).

Haplotype-specific RHD polymorphisms.   In intron 3 and intron 6, we detected polymorphic RHD sequences that differed between the prevalent RHD alleles of the CDe and cDE haplotypes (Table 4). Weak D alleles were identical to the prevalent alleles of the same RH haplotype in regard to these polymorphisms, with the single exception of weak D type 4 displaying a unique intron 6 repeat sequence. The conservation of these haplotype-specific RHD polymorphisms suggested that weak D alleles evolved independently.


                              
View this table:
[in this window]
[in a new window]
  		Table 4. RHD Polymorphisms in Weak D Types of Various RH Haplotypes


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The weak D phenotype is represented by a group of RHD+ genotypes that code in their vast majority for altered RhD proteins associated with a reduced RhD expression on the red blood cells' surface. Our population-based study dismissed unequivocally the possibility of one distinct antigen Du and showed in contrast to previous conjectures, that weak D alleles do generally possess mutations in RHD exon sequences. We provided statistical evidence that the missense mutations observed in the alleles of all weak D types are the probable cause for the reduced antigen D expression.

We suggest a causal relation of missense mutations and reduced RhD protein integration: (1) weak D alleles evolved independently in the different haplotypes, each distinct event being associated with a change in the RhD protein sequence; (2) no sample occurred with a normal RHD sequence despite observation of 13 different alleles in 161 samples; (3) type and distribution of the observed nucleotide substitutions was not compatible with the null hypothesis of random changes; (4) missense mutations causing reduced RhD expression fit nicely into the current model of RhD membrane integration.

Rh proteins occur in a complex with the Rh50 protein, and the expression of the Rh/Rh50 complex depends on the presence of both intact Rh5036 and Rh37,38 proteins. Missense mutations in the Rh50 protein are known to cause reduced expression of the Rh complex.36,39 Amino acid substitutions in the RhD protein might hence affect the expression of the RhD/Rh50 complex. A formal experimental proof of causality would involve expression systems. The only currently available system40 has, so far, not been shown to predict expression in a quantitative way.

Based on the distribution and kind of amino acid substitutions, a general picture of the relationship of RhD structure and RhD expression arose: all amino acid substitutions in weak D were located in the intracellular or transmembraneous parts of the RhD protein. Known RhD alleles with exofacial substitutions32,41-43 were discovered by virtue of their partial D antigen, but may display discrete (DNU and DVII) to moderate (DII, DHR, and DHMi) reductions in RhD expression.43-45 Most substitutions reported in this study were nonconservative and the introduced amino acids, in particular proline, likely disrupted the secondary or tertiary structure. Two weak D alleles (type 2 and 11) were associated with conservative substitutions indicating that the involved amino acid regions at positions 295 and 385 may be particularly important for an optimal RhD membrane integration. In two alleles (type 4 and type 14), parts of exon 4 and 5 were substituted by the corresponding parts of the RHCE gene. Similar exchanges occurred in DVI type I and DVI type II that exhibited a considerably reduced RhD protein expression45 also. Previous paradoxical observations can be explained, if the N152T substitution in exon 3 is considered to facilitate the membrane integration: (1) DIIIa,46 differing from weak D type 4 by the N152T substitution only, has a normal RhD antigen density,45 and (2) DIIIc, DIVa, and DVI type III harboring the N152T substitution have enhanced antigen densities21,45 compared with their appropriate controls (normal RhD and DVI type II).

Our genomic sequencing method was optimized to detect missense mutations. The approach obviated the laborious need to obtain full-length cDNA and to differentiate missense mutations from misincorporated nucleotides introduced during PCR and subcloning steps. The demonstration of missense mutations by genomic RHD sequencing definitively refuted the current dogma9,10 of a normal RHD allele in weak D phenotypes. The possibility of additional aberrations, eg, multiple hybrid genes with complementary RHD exon patterns, was formally excluded for the most frequent weak D type 1 representing about 0.3% of all RHD alleles by sequencing the full-length cDNA. Our results are incongruous with two earlier reports asserting normal RHD coding sequences in three weak D samples from France9 and in an unspecified number of weak D samples from the Netherlands.10 Possibly, these investigators missed the mutations because of technical problems of the traditional approach based on cDNA. Alternative explanations are very unlikely: (1) There might be regional variations in the causes of weak D, although other Rh alleles as frequent as the prevalent weak D alleles do not differ much in whites. (2) Both groups may have inadvertently investigated high grade Du that are due to a suppressive effect of Cde and expected to possess a normal coding sequence. We excluded the possibility that we investigated some rare, previously uncharacterized partial D instead of weak D, because the phenotypes described by us occurred with a cumulative frequency of 0.41% and, therefore, accounted for the majority of weak D phenotypes. Weak D type 4 was partially characterized by Legler et al.14 These investigators performed seven RHD-specific PCR reactions that were not affected by most mutations in weak D types. Their approach was not designed to exclude the kind of genetic diversity that is actually present in our weak D phenotypes.

Our findings implied that there is no well-defined borderline between weak D and partial D that has an aberrant RHD coding sequence, lacks specific D epitopes, and may be associated with an allo-anti-D. The observation of anti-D production in Rhesus positive recipients is too rare, even for some D categories, to base a classification on this event. Serologic discrimination of qualitative and quantitative abnormalities is prone to mistakes in samples with strongly reduced D antigen densities. A normal exofacial Rh sequence may be accompanied by alloimmunization, as exemplified by the VS antigen of the RhCE protein caused by the transmembraneous L245V substitution.47

Although multiple publication13,14,45,48-56 suggested a heterogeneity of weak D, a serologic classification of the majority of weak D phenotypes has not been successful. There was even no defined borderline between normal D and weak D.50,51,57,58 Our findings made the classification of weak D phenotypes feasible and their classification will allow us to correlate distinct alleles with clinical data. In the case that patients carrying certain molecular weak D types were prone to develop anti-D, our classification might guide a Rhesus negative transfusion policy. The availability of weak D samples that are characterized in regard to molecular structure and RhD antigen densities will promote the quality assurance of anti-D reagents. They should reliably type probands as RhD+, whose RhD proteins are not prone to frequent anti-D immunization.3 Therefore, the use of RhD- red blood cell units for transfusions to weak D patients, which has been justified by a presumed potential for anti-D immunization, can finally be reduced to a minimum, which can be scientifically deduced.

Some RHD alleles described in the present study were more prevalent than previously known RHD alleles. The potential for a broader relevance in research was opened, as it became feasible to determine in a massive way the frequencies of molecularly defined rare alleles in natural populations. Poisson-like allele distributions were predicted by mathematical models59 that could so far not be checked in any real population. We observed two alleles (type 4 and 14) with multiple nucleotide substitutions in the RHD gene that were characteristic for the RHCE gene, but may not be explained by a single gene conversion event. This observation pointed to more complicated mechanisms shaping the allele polymorphism of homologous genes, which are frequent throughout the genomes.


    ACKNOWLEDGMENT

We thank Olga Zarupski and Katharina Schmid for expert technical assistance.


    FOOTNOTES

   Submitted June 22, 1998; accepted September 1, 1998.
   Supported by the DRK-Blutspendedienst Baden-Württemberg, Stuttgart, Germany.
   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

Presented at the 25th Congress of the International Society of Blood Transfusion held in Oslo on June 29, 1998 and published in abstract form in Vox Sang 74:55, 1998 (suppl).

Address reprint requests to Willy A. Flegel, Priv-Doz, MD, Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, and DRK-Blutspendedienst Baden-Württemberg, Institut Ulm, Helmholtzstrasse 10, D-89081 Ulm, Germany; e-mail: waf{at}ucsd.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

1. Mollison PL, Engelfriet CP, Contreras M: Blood Transfusion in Clinical Medicine. New York, NY, Blackwell, 1993.

2. Stratton F: A new Rh allelomorph. Nature 158:25, 1946

3. Wagner FF, Kasulke D, Kerowgan M, Flegel WA: Frequencies of the blood groups ABO, Rhesus, D category VI, Kell, and of clinically relevant high-frequency antigens in South-Western Germany. Infusionsther Transfusionsmed 22:285, 1995[Medline] [Order article via Infotrieve]

4. Mourant AE, Kopec AC, Domaniewska-Sobczak K: The distribution of the human blood groups and other polymorphisms. New York, NY, Oxford, 1976.

5. Salmon C, Cartron J-P, Rouger P: The Human Blood Groups. New York, NY, Masson, 1984.

6. Huang CH: Molecular insights into the Rh protein family and associated antigens. Curr Opin Hematol 4:94, 1997[Medline] [Order article via Infotrieve]

7. Ceppellini R, Dunn LC, Turry M: An interaction between alleles at the Rh locus in man which weakens the reactivity of the Rho factor (Du). Proc Natl Acad Sci USA 41:283, 1955[Free Full Text]

8. Mollison PL, Engelfriet CP, Contreras M: Blood Transfusion in Clinical Medicine. New York, NY, Blackwell, 1987.

9. Rouillac C, Gane P, Cartron J-P, Le Pennec PY, Colin Y: Molecular basis of the altered antigenic expression of RhD in weak D (Du) and RhC/e in RN phenotypes. Blood 87:4853, 1996[Abstract/Free Full Text]

10. Beckers EA, Faas BHW, Ligthart P, Overbeeke MA, von dem Borne AE, van der Schoot CE, van Rhenen DJ: Lower antigen site density and weak D immunogenicity cannot be explained by structural genomic abnormalities or regulatory defects of the RHD gene. Transfusion 37:616, 1997[Medline] [Order article via Infotrieve]

11. Beckers EAM, Faas BHW, Overbeeke MAM, von dem Borne AEGK, van Rhenen DJ, van der Schoot CE: Molecular aspects of the weak-D phenotype. Transfusion 35:50S, 1995

12. Kajii E, Umenishi F, Omi T, Ikemoto S: Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells. Hum Genet 95:657, 1995[Medline] [Order article via Infotrieve]

13. Avent ND, Martin PG, Armstrong-Fisher SS, Liu W, Finning KM, Maddocks D, Urbaniak SJ: Evidence of genetic diversity underlying Rh D negative, weak D (Du) and partial D phenotypes as determined by multiplex PCR analysis of the RHD gene. Blood 89:2568, 1997[Abstract/Free Full Text]

14. Legler TJ, Maas JH, Blaschke V, Malekan M, Ohto H, Lynen R, Bustami N, Schwartz DW, Mayr WR, Kohler M, Panzer S: RHD genotyping in weak D phenotypes by multiple polymerase chain reactions. Transfusion 38:434, 1998[Medline] [Order article via Infotrieve]

15. Gassner C, Schmarda A, Kilga-Nogler S, Jenny-Feldkircher B, Rainer E, Müller TH, Wagner FF, Flegel WA, Schönitzer D: RhesusD/CE typing by polymerase chain reaction using sequence-specific primers. Transfusion 37:1020, 1997[Medline] [Order article via Infotrieve]

16. Issitt PD, Telen MJ: D, weak D (Du), and partial D: The molecular story unfolds. Transfusion 36:97, 1996[Medline] [Order article via Infotrieve]

17. Roubinet F, Apoil PA, Blancher A: Frequency of partial D phenotypes in the south western region of France. Transfus Clin Biol 3:247, 1996[Medline] [Order article via Infotrieve]

18. Fukumori Y, Hori Y, Ohnoki S, Nagao N, Shibata H, Okubo Y, Yamaguchi H: Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers. Transfus Med 7:227, 1997[Medline] [Order article via Infotrieve]

19. Aubin JT, Le Van Kim C, Mouro I, Colin Y, Bignozzi C, Brossard Y, Cartron J-P: Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification. Br J Haematol 98:356, 1997[Medline] [Order article via Infotrieve]

20. Wissenschaftlicher Beirat der Bundesärztekammer, Bundesgesundheitsamt: Richtlinien zur Blutgruppenbestimmung und Bluttransfusion. Köln, Deutscher Ärzte-Verlag, 1992.

21. Wagner FF, Gassner C, Müller TH, Schönitzer D, Schunter F, Flegel WA: Three molecular structures cause Rhesus D category VI phenotypes with distinct immunohematologic features. Blood 91:2157, 1998[Abstract/Free Full Text]

22. Le Van Kim C, Mouro I, Cherif-Zahar B, Raynal V, Cherrier C, Cartron J-P, Colin Y: Molecular cloning and primary structure of the human blood group RhD polypeptide. Proc Natl Acad Sci USA 89:10925, 1992[Abstract/Free Full Text]

23. Kemp TJ, Poulter M, Carritt B: A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D---phenotype. Am J Hum Genet 59:1066, 1996[Medline] [Order article via Infotrieve]

24. Arce MA, Thompson ES, Wagner S, Coyne KE, Ferdman BA, Lublin DM: Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhD-negative individuals. Blood 82:651, 1993[Abstract/Free Full Text]

25. Cartron J-P: Rh DNA --- coordinator's report. Transfus Clin Biol 3:491, 1996[Medline] [Order article via Infotrieve]

26. Rost B, Casadio R, Fariselli P, Sander C: Transmembrane helices predicted at 95% accuracy. Protein Sci 4:521, 1995[Abstract]

27. Hofmann K, Stoffel W: TMbase --- a database of membrane spanning proteins segments. Biol Chem Hoppe Seyler 347:166, 1993

28. Scott ML, Voak D, Jones JW, Liu W, Avent ND, Hughes-Jones N, Sonneborn H-H: A model for RhD --- the relationship of 30 serologically defined epitopes to predicted structure. Biotest Bulletin 5:459, 1997

29. Dahr W, Schmitz G, Ernst M, Gielen W, Sonneborn H-H: Rh antigens and cysteine modification. Biotest Bulletin 5:451, 1997

30. Avent ND, Butcher SK, Liu W, Mawby WJ, Mallinson G, Parsons SF, Anstee DJ, Tanner MJA: Localisation of the C-termini of the Rh polypeptides to the cytoplasmic face of the human erythrocyte membrane. J Biol Chem 267:15134, 1992[Abstract/Free Full Text]

31. Hermand P, Mouro I, Huet M, Bloy C, Suyama K, Goldstein J, Cartron J-P, Bailly P: Immunochemical characterization of Rhesus proteins with antibodies raised against synthetic peptides. Blood 82:669, 1993[Abstract/Free Full Text]

32. Liu W, Jones JW, Scott ML, Voak D, Avent ND: Molecular analysis of two D-variants, DHMi and DHMii. Transfus Med 6:21, 1996[Medline] [Order article via Infotrieve] (suppl 2, abstr)

33. Lomas C, Tippett P, Thompson KM, Melamed MD, Hughes-Jones NC: Demonstration of seven epitopes on the Rh antigen D using human monoclonal anti-D antibodies and red cells from D categories. Vox Sang 57:261, 1989[Medline] [Order article via Infotrieve]

34. Wagner FF, Hillesheim B, Flegel WA: D-Kategorie VII beruht einheitlich auf der Aminosäuresubstitution Leu(110)Pro. Beitr Infusionsther Transfusionsmed 34:220, 1997[Medline] [Order article via Infotrieve]

35. Huang CH: Alteration of RH gene structure and expression in human dCCee and DCW-red blood cells: Phenotypic homozygosity versus genotypic heterozygosity. Blood 88:2326, 1996[Abstract/Free Full Text]

36. Cherif-Zahar B, Raynal V, Gane P, Mattei MG, Bailly P, Gibbs B, Colin Y, Cartron J-P: Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency. Nat Genet 12:168, 1996[Medline] [Order article via Infotrieve]

37. Huang CH, Chen Y, Reid ME, Seidl C: Rhnull disease: The amorph type results from a novel double mutation in RhCe gene on D-negative background. Blood 92:664, 1998[Abstract/Free Full Text]

38. Cherif-Zahar B, Matassi G, Raynal V, Gane P, Mempel W, Perez C, Cartron JP: Molecular defects of the RHCE gene in Rh-deficient individuals of the amorph type. Blood 92:639, 1998[Abstract/Free Full Text]

39. Huang CH: The human Rh50 glycoprotein gene: Structural organization and associated splicing defect resulting in Rhnull disease. J Biol Chem 273:2207, 1998[Abstract/Free Full Text]

40. Smythe JS, Avent ND, Judson PA, Parsons SF, Martin PG, Anstee DJ: Expression or RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of RH blood group antigens. Blood 87:2968, 1996[Abstract/Free Full Text]

41. Avent ND, Jones JW, Liu W, Scott ML, Voak D, Flegel WA, Wagner FF, Green C: Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein. Br J Haematol 97:366, 1997[Medline] [Order article via Infotrieve]

42. Rouillac C, Le Van Kim C, Beolet M, Cartron J-P, Colin Y: Leu110Pro substitution in the RhD polypeptide is responsible for the DVII category blood group phenotype. Am J Hematol 49:87, 1995[Medline] [Order article via Infotrieve]

43. Jones JW, Finning KM, Mattock R, Voak D, Scott ML, Avent ND: The serological profile and molecular basis of a new partial D phenotype, DHR. Vox Sang 73:252, 1997[Medline] [Order article via Infotrieve]

44. Flegel WA, Wagner FF: RHD epitope density profiles of RHD variant red cells analyzed by flow cytometry. Transfus Clin Biol 3:429, 1996[Medline] [Order article via Infotrieve]

45. Jones JW, Lloyd-Evans P, Kumpel BM: Quantitation of Rh D antigen sites on weak D and D variant red cells by flow cytometry. Vox Sang 71:176, 1996[Medline] [Order article via Infotrieve]

46. Huang CH, Chen Y, Reid M: Human DIIIa erythrocytes: RhD protein is associated with multiple dispersed amino acid variations. Am J Hematol 55:139, 1997[Medline] [Order article via Infotrieve]

47. Faas BHW, Becker EAM, Wildoer P, Ligthart PC, Overbeeke MAM, Zondervan HA, von dem Borne AEGK, van der Schoot CE: Molecular background of VS and weak C expression in blacks. Transfusion 37:38, 1997[Medline] [Order article via Infotrieve]

48. Wagner FF: Influence of Rh phenotype on the antigen density of C, c, and D: Flow cytometric study using a frozen standard red cell. Transfusion 34:671, 1994[Medline] [Order article via Infotrieve]

49. Hasekura H, Ota M, Ito S, Hasegawa Y, Ichinose A, Fukushima H, Ogata H: Flow cytometric studies of the D antigen of various Rh phenotypes with particular reference to Du and Del. Transfusion 30:236, 1990[Medline] [Order article via Infotrieve]

50. Nicholson G, Lawrence A, Ala FA, Bird GWG: Semi-quantitative assay of D antigen site density by flow cytometric analysis. Transfus Med 1:87, 1991[Medline] [Order article via Infotrieve]

51. Nelson M, Barrow LA, Popp H, Gibson J: Some observations on D antigen expression of D-positive and `weak D-positive' red cells as assessed by flow cytometry. Vox Sang 69:152, 1995[Medline] [Order article via Infotrieve]

52. Tazzari PL, Bontadini A, Belletti D, Malferrari F, Conte R: Flow cytometry: A tool in immunohematology for D+W (Du) antigen evaluation? Vox Sang 67:382, 1994[Medline] [Order article via Infotrieve]

53. Jones J, Filbey D: Selection of monoclonal antibodies for the identification of D variants: Ability to detect weak D and to split epD2, epD5 and epD6/7. Vox Sang 70:173, 1996[Medline] [Order article via Infotrieve]

54. Szymanski IO, Araszkiewicz P: Quantitative studies on the D antigen of red cells with the Du phenotype. Transfusion 29:103, 1989[Medline] [Order article via Infotrieve]

55. Sonneborn H-H, Darda C, Ernst M, Flegel WA, Helmbold W, Kasulke D, Kühnl P, Mueller-Eckhardt C, Northoff H, Poschmann A, Schönitzer D, Schütt K-H, Schunter F: Results of the DGTI workshop on the evaluation of the reactivity of monoclonal anti-D. Beitr Infusionsther Transfusionsmed 30:382, 1992

56. Gorick B, McDougall DCJ, Ouwehand WH, Overbeeke MAM, Tippett P, Hughes-Jones NC, van Rhenen DJ: Quantitation of D sites on selected `weak D' and `partial D' red cells. Vox Sang 65:136, 1993[Medline] [Order article via Infotrieve]

57. Moore BPL: Does knowledge of Du status serve a useful purpose? Vox Sang 46S1:95, 1984

58. Agre PC, Davies DM, Issitt PD, Lamy BM, Schmidt PJ, Treacy M, Vengelen-Tyler V: A proposal to standardize terminology for weak D antigen (Letter). Transfusion 32:86, 1992[Medline] [Order article via Infotrieve]

59. Joyce P, Tavare S: The distribution of rare alleles. J Math Biol 33:602, 1995[Medline] [Order article via Infotrieve]

60. Poulter M, Kemp TJ, Carritt B: DNA-based Rhesus typing: Simultaneous determination of RHC and RHD status using the polymerase chain reaction. Vox Sang 7:164, 1996

61. Mouro I, Colin Y, Cherif-Zahar B, Cartron J-P, Le Van Kim C: Molecular genetic basis of the human Rhesus blood group system. Nat Genet 5:62, 1993[Medline] [Order article via Infotrieve]

© 1999 by The American Society of Hematology.
 
0006-4971/99/9301-0024$3.00/0

Add to CiteULike CiteULike